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Sample records for chaperone chz1p regulates

  1. Pathways of allosteric regulation in Hsp70 chaperones.

    Science.gov (United States)

    Kityk, Roman; Vogel, Markus; Schlecht, Rainer; Bukau, Bernd; Mayer, Matthias P

    2015-01-01

    Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly conserved hydrogen bond network, and define the signal transduction pathway that allows bound substrates to trigger ATP hydrolysis. We identify variants deficient in only one direction of allosteric control and demonstrate that ATP-induced substrate release is more important for chaperone activity than substrate-stimulated ATP hydrolysis. These findings provide evidence of an unexpected dichotomic allostery mechanism in Hsp70 chaperones and provide the basis for a comprehensive mechanical model of allostery in Hsp70s. PMID:26383706

  2. Pathways of allosteric regulation in Hsp70 chaperones

    OpenAIRE

    Kityk, Roman; Vogel, Markus; Schlecht, Rainer; Bukau, Bernd; Mayer, Matthias P

    2015-01-01

    Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly ...

  3. Probing molecular mechanisms of the Hsp90 chaperone: biophysical modeling identifies key regulators of functional dynamics.

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    Anshuman Dixit

    Full Text Available Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based "conformational selection" of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected

  4. Endoplasmic reticulum (ER Chaperones and Oxidoreductases: Critical Regulators of Tumor Cell Survival and Immunorecognition

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    Thomas eSimmen

    2014-10-01

    Full Text Available Endoplasmic reticulum (ER chaperones and oxidoreductases are abundant enzymes that mediate the production of fully folded secretory and transmembrane proteins. Resisting the Golgi and plasma membrane-directed bulk flow, ER chaperones and oxidoreductases enter retrograde trafficking whenever they are pulled outside of the ER. However, solid tumors are characterized by the increased production of reactive oxygen species (ROS, combined with reduced blood flow that leads to low oxygen supply and ER stress. Under these conditions, hypoxia and the unfolded protein response (UPR upregulate ER chaperones and oxidoreductases. When this occurs, ER oxidoreductases and chaperones become important regulators of tumor growth. However, under these conditions, these proteins not only promote the production of proteins, but also alter the properties of the plasma membrane and hence modulate tumor immune recognition. For instance, high levels of calreticulin serve as an eat-me signal on the surface of tumor cells. Conversely, both intracellular and surface BiP/GRP78 promotes tumor growth. Other ER folding assistants able to modulate the properties of tumor tissue include protein disulfide isomerase (PDI, Ero1α and GRP94. Understanding the roles and mechanisms of ER chaperones in regulating tumor cell functions and immunorecognition will lead to important insight for the development of novel cancer therapies.

  5. Essential role of the molecular chaperone gp96 in regulating melanogenesis

    OpenAIRE

    Zhang, Yongliang; Helke, Kristi L.; Coelho, Sergio G.; Valencia, Julio C.; Hearing, Vincent J.; SUN, SHAOLI; Liu, Bei; Li, Zihai

    2013-01-01

    Through a process known as melanogenesis, melanocyte produces melanin in specialized organelles termed melanosomes, which regulates pigmentation of the skin, eyes and hair. Gp96 is a constitutively expressed heat shock protein in the endoplasmic reticulum whose expression is further up-regulated upon ultraviolet irradiation. However, the roles and mechanisms of this chaperone in pigmentation biology are unknown. In this study, we found that knockdown of gp96 by RNA interference significantly ...

  6. Protein kinase A regulates molecular chaperone transcription and protein aggregation.

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    Yue Zhang

    Full Text Available Heat shock factor 1 (HSF1 regulates one of the major pathways of protein quality control and is essential for deterrence of protein-folding disorders, particularly in neuronal cells. However, HSF1 activity declines with age, a change that may open the door to progression of neurodegenerative disorders such as Huntington's disease. We have investigated mechanisms of HSF1 regulation that may become compromised with age. HSF1 binds stably to the catalytic domain of protein kinase A (PKAcα and becomes phosphorylated on at least one regulatory serine residue (S320. We show here that PKA is essential for effective transcription of HSP genes by HSF1. PKA triggers a cascade involving HSF1 binding to the histone acetylase p300 and positive translation elongation factor 1 (p-TEFb and phosphorylation of the c-terminal domain of RNA polymerase II, a key mechanism in the downstream steps of HSF1-mediated transcription. This cascade appears to play a key role in protein quality control in neuronal cells expressing aggregation-prone proteins with long poly-glutamine (poly-Q tracts. Such proteins formed inclusion bodies that could be resolved by HSF1 activation during heat shock. Resolution of the inclusions was inhibited by knockdown of HSF1, PKAcα, or the pTEFb component CDK9, indicating a key role for the HSF1-PKA cascade in protein quality control.

  7. Global small RNA chaperone Hfq and regulatory small RNAs are important virulence regulators in Erwinia amylovora.

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    Zeng, Quan; McNally, R Ryan; Sundin, George W

    2013-04-01

    Hfq is a global small RNA (sRNA) chaperone that interacts with Hfq-regulated sRNAs and functions in the posttranscriptional regulation of gene expression. In this work, we identified Hfq to be a virulence regulator in the Gram-negative fire blight pathogen Erwinia amylovora. Deletion of hfq in E. amylovora Ea1189 significantly reduced bacterial virulence in both immature pear fruits and apple shoots. Analysis of virulence determinants in strain Ea1189Δhfq showed that Hfq exerts pleiotropic regulation of amylovoran exopolysaccharide production, biofilm formation, motility, and the type III secretion system (T3SS). Further characterization of biofilm regulation by Hfq demonstrated that Hfq limits bacterial attachment to solid surfaces while promoting biofilm maturation. Characterization of T3SS regulation by Hfq revealed that Hfq positively regulates the translocation and secretion of the major type III effector DspE and negatively controls the secretion of the putative translocator HrpK and the type III effector Eop1. Lastly, 10 Hfq-regulated sRNAs were identified using a computational method, and two of these sRNAs, RprA and RyhA, were found to be required for the full virulence of E. amylovora.

  8. Regulation of the expression of chaperone gp96 in macrophages and dendritic cells.

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    Lutz Wolfram

    Full Text Available The chaperone function of the ER-residing heat shock protein gp96 plays an important role in protein physiology and has additionally important immunological functions due to its peptide-binding capacity. Low amounts of gp96 stimulate immunity; high quantities induce tolerance by mechanisms not fully understood. A lack of gp96 protein in intestinal macrophages (IMACs from Crohn`s disease (CD patients correlates with loss of tolerance against the host gut flora, leading to chronic inflammation. Since gp96 shows dose-dependent direction of immunological reactions, we studied primary IMACs and developed cell models to understand the regulation of gp96 expression. Induction of gp96-expression was higher in in vitro differentiated dendritic cells (i.v.DCs than in in vitro differentiated macrophages (i.v.MACs, whereas monocytes (MOs expressed only low gp96 levels. The highest levels of expression were found in IMACs. Lipopolysaccharide (LPS, muramyl dipeptide (MDP, tumour necrosis factor (TNF, and Interleukin (IL-4 induced gp96-expression, while IL12, IL-17, IL-23 and interferon (IFN-γ were not effective indicating that Th1 and Th17 cells are probably not involved in the induction of gp96. Furthermore, gp96 was able to induce its own expression. The ER-stress inducer tunicamycin increased gp96-expression in a concentration- and time-dependent manner. Both ulcerative colitis (UC and CD patients showed significantly elevated gp96 mRNA levels in intestinal biopsies which correlated positively with the degree of inflammation of the tissue. Since gp96 is highly expressed on the one hand upon stress induction as during inflammation and on the other hand possibly mediating tolerance, these results will help to understand the whether gp96 plays a role in the pathophysiology of inflammatory bowel disease (IBD.

  9. Identification of New Potential Interaction Partners for Human Cytoplasmic Copper Chaperone Atox1: Roles in Gene Regulation?

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    Helena Öhrvik

    2015-07-01

    Full Text Available The human copper (Cu chaperone Atox1 delivers Cu to P1B type ATPases in the Golgi network, for incorporation into essential Cu-dependent enzymes. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed Cys-X-X-Cys (CXXC motif. In addition to its well-documented cytoplasmic chaperone function, in 2008 Atox1 was suggested to have functionality in the nucleus. To identify new interactions partners of Atox1, we performed a yeast two-hybrid screen with a large human placenta library of cDNA fragments using Atox1 as bait. Among 98 million fragments investigated, 25 proteins were found to be confident interaction partners. Nine of these were uncharacterized proteins, and the remaining 16 proteins were analyzed by bioinformatics with respect to cell localization, tissue distribution, function, sequence motifs, three-dimensional structures and interaction networks. Several of the hits were eukaryotic-specific proteins interacting with DNA or RNA implying that Atox1 may act as a modulator of gene regulation. Notably, because many of the identified proteins contain CXXC motifs, similarly to the Cu transport reactions, interactions between these and Atox1 may be mediated by Cu.

  10. Antagonistic functions between the RNA chaperone Hfq and an sRNA regulate sensitivity to the antibiotic colicin.

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    Salvail, Hubert; Caron, Marie-Pier; Bélanger, Justine; Massé, Eric

    2013-10-16

    The RNA chaperone Hfq is a key regulator of the function of small RNAs (sRNAs). Hfq has been shown to facilitate sRNAs binding to target mRNAs and to directly regulate translation through the action of sRNAs. Here, we present evidence that Hfq acts as the repressor of cirA mRNA translation in the absence of sRNA. Hfq binding to cirA prevents translation initiation, which correlates with cirA mRNA instability. In contrast, RyhB pairing to cirA mRNA promotes changes in RNA structure that displace Hfq, thereby allowing efficient translation as well as mRNA stabilization. Because CirA is a receptor for the antibiotic colicin Ia, in addition to acting as an Fur (Ferric Uptake Regulator)-regulated siderophore transporter, translational activation of cirA mRNA by RyhB promotes colicin sensitivity under conditions of iron starvation. Altogether, these results indicate that Fur and RyhB modulate an unexpected feed-forward loop mechanism related to iron physiology and colicin sensitivity. PMID:24065131

  11. Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

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    Tommy Kaplan

    2008-11-01

    Full Text Available Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

  12. Integrating the cell stress response: a new view of molecular chaperones as immunological and physiological homeostatic regulators.

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    Henderson, Brian

    2010-01-01

    The response of cells to stress was first documented in the 1960s and 1970s and the molecular nature of the families of proteins that subserve this vital response, the molecular chaperones, were identified and subjected to critical study in the period from the late 1980s. This resulted in the rapidly advancing new field of protein folding and its role in cellular function. Emerging at the same time, but initially largely ignored, were reports that molecular chaperones could be released by cells and exist on the outer plasma membrane or in the body fluids. These secreted molecular chaperones were found to have intercellular signalling functions. There is now a growing body of evidence to support the hypothesis that molecular chaperones have properties ascribed to the Roman god Janus, the god of gates, doors, beginnings and endings, whose two faces point in different directions. Molecular chaperones appear to have one set of key functions within the cell and, potentially, a separate set of functions when they exist on the cell surface or in the various fluid phases of the body. Thus, it is a likely hypothesis that secreted molecular chaperones act as an additional level of homeostatic control possibly linking cellular stress to physiological systems such as the immune system. This review concentrates on three key molecular chaperones: Hsp10, Hsp60 and the Hsp70 family for which most information is available. An important consideration is the role that these proteins may play in human disease and in the treatment of human disease.

  13. Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90.

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    Luiz Felipe M Prota

    Full Text Available BACKGROUND: Dexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR. Our aim was to determine the effects of dexamethasone treatment on wild-type CFTR expression. METHODS AND RESULTS: Dose-response (1 nM to 10 µM and time course (3 to 48 h curves were generated for dexamethasone for mRNA expression in Calu-3 cells using a real-time PCR. Within 24 h, dexamethasone (10 nM showed a 0.3-fold decrease in CFTR mRNA expression, and a 3.2-fold increase in αENaC mRNA expression compared with control groups. Dexamethasone (10 nM induced a 1.97-fold increase in the total protein of wild-type CFTR, confirmed by inhibition by mifepristone. To access surface protein expression, biotinylation followed by Western blotting showed that dexamethasone treatment led to a 2.35-fold increase in the amount of CFTR in the cell surface compared with the untreated control groups. Once protein translation was inhibited with cycloheximide, dexamethasone could not increase the amount of CFTR protein. Protein stability was assessed by inhibition of protein synthesis with cycloheximide (50 µg/ml at different times in cells treated with dexamethasone and in untreated cells. Dexamethasone did not alter the degradation of wild-type CFTR. Assessment of the B band of CFTR within 15 min of metabolic pulse labeling showed a 1.5-fold increase in CFTR protein after treatment with dexamethasone for 24 h. Chaperone 90 (HSP90 binding to CFTR increased 1.55-fold after treatment with dexamethasone for 24 h, whereas chaperone 70 (HSP70 binding decreased 0.30 fold in an immunoprecipitation assay. CONCLUSION: Mature wild-type CFTR protein is regulated by dexamethasone post transcription, involving cotranslational mechanisms with HSP90 and HSP70, which enhances maturation and expression of wild-type CFTR.

  14. Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90.

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    Daniela Trisciuoglio

    Full Text Available BACKGROUND: Hypoxia-Inducible Factor 1 (HIF-1 is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1alpha, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF-mediated tumour angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1alpha protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1alpha protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1alpha protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1alpha stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1alpha degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1alpha protein. We also showed that bcl-2, HIF-1alpha and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1alpha protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1alpha protein during hypoxia, and in particular the isoform HSP90beta is the main player in this phenomenon. CONCLUSIONS/SIGNIFICANCE: We identified the stabilization of HIF-1alpha protein as a mechanism through which bcl-2 induces the

  15. Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

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    Trisciuoglio, Daniela; Gabellini, Chiara; Desideri, Marianna; Ziparo, Elio; Zupi, Gabriella; Del Bufalo, Donatella

    2010-01-01

    Background Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis. Methodology/Principal Findings By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon. Conclusions/Significance We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the

  16. Endoplasmic reticulum chaperone glucose regulated protein 170-Pokemon complexes elicit a robust antitumor immune response in vivo.

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    Yuan, Bangqing; Xian, Ronghua; Wu, Xianqu; Jing, Junjie; Chen, Kangning; Liu, Guojun; Zhou, Zhenhua

    2012-07-01

    Previous evidence suggested that the stress protein grp170 can function as a highly efficient molecular chaperone, binding to large protein substrates and acting as a potent vaccine against specific tumors when purified from the same tumor. In addition, Pokemon can be found in almost all malignant tumor cells and is regarded to be a promising candidate for the treatment of tumors. However, the potential of the grp170-Pokemon chaperone complex has not been well described. In the present study, the natural chaperone complex between grp170 and the Pokemon was formed by heat shock, and its immunogenicity was detected by ELISPOT and (51)Cr-release assays in vitro and by tumor bearing models in vivo. Our results demonstrated that the grp170-Pokemon chaperone complex could elicit T cell responses as determined by ELISPOT and (51)Cr-release assays. In addition, immunized C57BL/6 mice were challenged with subcutaneous (s.c.) injection of Lewis cancer cells to induce primary tumors. Treatment of mice with the grp170-Pokemon chaperone complex also significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. Our results indicated that the grp170-Pokemon chaperone complex might represent a powerful approach to tumor immunotherapy and have significant potential for clinical application. PMID:22317751

  17. The role of the molecular chaperone heat shock protein A2 (HSPA2 in regulating human sperm-egg recognition

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    Brett Nixon

    2015-01-01

    Full Text Available One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI, recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2, as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function.

  18. The role of the molecular chaperone heat shock protein A2 (HSPA2) in regulating human sperm-egg recognition.

    Science.gov (United States)

    Nixon, Brett; Bromfield, Elizabeth G; Dun, Matthew D; Redgrove, Kate A; McLaughlin, Eileen A; Aitken, R John

    2015-01-01

    One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI), recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2), as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF) success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function.

  19. Posttranslocation Chaperone PrsA2 Regulates the Maturation and Secretion of Listeria monocytogenes Proprotein Virulence Factors ▿

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    Forster, Brian M.; Zemansky, Jason; Portnoy, Daniel A.; Marquis, Hélène

    2011-01-01

    PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface. PMID:21908675

  20. Posttranslocation chaperone PrsA2 regulates the maturation and secretion of Listeria monocytogenes proprotein virulence factors.

    Science.gov (United States)

    Forster, Brian M; Zemansky, Jason; Portnoy, Daniel A; Marquis, Hélène

    2011-11-01

    PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface.

  1. A pH Switch Regulates the Inverse Relationship between Membranolytic and Chaperone-like Activities of HSP-1/2, a Major Protein of Horse Seminal Plasma.

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    Kumar, C Sudheer; Swamy, Musti J

    2016-07-01

    HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch. PMID:27292547

  2. The heat shock protein-90 co-chaperone, Cyclophilin 40, promotes ALK-positive, anaplastic large cell lymphoma viability and its expression is regulated by the NPM-ALK oncoprotein

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    Pearson Joel D

    2012-06-01

    Full Text Available Abstract Background Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL is a T cell lymphoma defined by the presence of chromosomal translocations involving the ALK tyrosine kinase gene. These translocations generate fusion proteins (e.g. NPM-ALK with constitutive tyrosine kinase activity, which activate numerous signalling pathways important for ALK+ ALCL pathogenesis. The molecular chaperone heat shock protein-90 (Hsp90 plays a critical role in allowing NPM-ALK and other signalling proteins to function in this lymphoma. Co-chaperone proteins are important for helping Hsp90 fold proteins and for directing Hsp90 to specific clients; however the importance of co-chaperone proteins in ALK+ ALCL has not been investigated. Our preliminary findings suggested that expression of the immunophilin co-chaperone, Cyclophilin 40 (Cyp40, is up-regulated in ALK+ ALCL by JunB, a transcription factor activated by NPM-ALK signalling. In this study we examined the regulation of the immunophilin family of co-chaperones by NPM-ALK and JunB, and investigated whether the immunophilin co-chaperones promote the viability of ALK+ ALCL cell lines. Methods NPM-ALK and JunB were knocked-down in ALK+ ALCL cell lines with siRNA, and the effect on the expression of the three immunophilin co-chaperones: Cyp40, FK506-binding protein (FKBP 51, and FKBP52 examined. Furthermore, the effect of knock-down of the immunophilin co-chaperones, either individually or in combination, on the viability of ALK+ ALCL cell lines and NPM-ALK levels and activity was also examined. Results We found that NPM-ALK promoted the transcription of Cyp40 and FKBP52, but only Cyp40 transcription was promoted by JunB. We also observed reduced viability of ALK+ ALCL cell lines treated with Cyp40 siRNA, but not with siRNAs directed against FKBP52 or FKBP51. Finally, we demonstrate that the decrease in the viability of ALK+ ALCL cell lines treated with Cyp40 siRNA does not appear to

  3. Experimentally guided structural modeling and dynamics analysis of Hsp90-p53 interactions: allosteric regulation of the Hsp90 chaperone by a client protein.

    Science.gov (United States)

    Blacklock, Kristin; Verkhivker, Gennady M

    2013-11-25

    A fundamental role of the Hsp90 chaperone system in mediating maturation of protein clients is essential for the integrity of signaling pathways involved in cell cycle control and organism development. Molecular characterization of Hsp90 interactions with client proteins is fundamental to understanding the activity of many tumor-inducing signaling proteins and presents an active area of structural and biochemical studies. In this work, we have probed mechanistic aspects of allosteric regulation of Hsp90 by client proteins via a detailed computational study of Hsp90 interactions with the tumor suppressor protein p53. Experimentally guided protein docking and molecular dynamics structural refinement have reconstructed the recognition-competent states of the Hsp90-p53 complexes that are consistent with the NMR studies. Protein structure network analysis has identified critical interacting networks and specific residues responsible for structural integrity and stability of the Hsp90-p53 complexes. Coarse-grained modeling was used to characterize the global dynamics of the regulatory complexes and map p53-induced changes in the conformational equilibrium of Hsp90. The variations in the functional dynamics profiles of the Hsp90-p53 complexes are consistent with the NMR studies and could explain differences in the functional role of the alternative binding sites. Despite the overall similarity of the collective movements and the same global interaction footprint, p53 binding at the C-terminal interaction site of Hsp90 may have a more significant impact on the chaperone dynamics, which is consistent with the stronger allosteric effect of these interactions revealed by the experimental studies. The results suggest that p53-induced modulation of the global dynamics and structurally stable interaction networks can target the regulatory hinge regions and facilitate stabilization of the closed Hsp90 dimer that underlies the fundamental stimulatory effect of the p53 client. PMID

  4. Inducible Hsp70 in the Regulation of Cancer Cell Survival: Analysis of Chaperone Induction, Expression and Activity

    Directory of Open Access Journals (Sweden)

    Elisa Zorzi

    2011-10-01

    Full Text Available Understanding the mechanisms that control stress is central to realize how cells respond to environmental and physiological insults. All the more important is to reveal how tumour cells withstand their harsher growth conditions and cope with drug-induced apoptosis, since resistance to chemotherapy is the foremost complication when curing cancer. Intensive research on tumour biology over the past number of years has provided significant insights into the molecular events that occur during oncogenesis, and resistance to anti-cancer drugs has been shown to often rely on stress response and expression of inducible heat shock proteins (HSPs. However, with respect to the mechanisms guarding cancer cells against proteotoxic stresses and the modulatory effects that allow their survival, much remains to be defined. Heat shock proteins are molecules responsible for folding newly synthesized polypeptides under physiological conditions and misfolded proteins under stress, but their role in maintaining the transformed phenotype often goes beyond their conventional chaperone activity. Expression of inducible HSPs is known to correlate with limited sensitivity to apoptosis induced by diverse cytotoxic agents and dismal prognosis of several tumour types, however whether cancer cells survive because of the constitutive expression of heat shock proteins or the ability to induce them when adapting to the hostile microenvironment remains to be elucidated. Clear is that tumours appear nowadays more “addicted” to heat shock proteins than previously envisaged, and targeting HSPs represents a powerful approach and a future challenge for sensitizing tumours to therapy. This review will focus on the anti-apoptotic role of heat shock 70kDa protein (Hsp70, and how regulatory factors that control inducible Hsp70 synthesis, expression and activity may be relevant for response to stress and survival of cancer cells.

  5. Inducible Hsp70 in the Regulation of Cancer Cell Survival: Analysis of Chaperone Induction, Expression and Activity

    Energy Technology Data Exchange (ETDEWEB)

    Zorzi, Elisa [OncoHematology Clinic of Pediatrics, University-Hospital of Padova, 35100 Padova (Italy); Bonvini, Paolo, E-mail: paolo.bonvini@unipd.it [OncoHematology Clinic of Pediatrics, University-Hospital of Padova, 35100 Padova (Italy); Fondazione Città della Speranza, 36030 Monte di Malo, Vicenza (Italy)

    2011-10-21

    Understanding the mechanisms that control stress is central to realize how cells respond to environmental and physiological insults. All the more important is to reveal how tumour cells withstand their harsher growth conditions and cope with drug-induced apoptosis, since resistance to chemotherapy is the foremost complication when curing cancer. Intensive research on tumour biology over the past number of years has provided significant insights into the molecular events that occur during oncogenesis, and resistance to anti-cancer drugs has been shown to often rely on stress response and expression of inducible heat shock proteins (HSPs). However, with respect to the mechanisms guarding cancer cells against proteotoxic stresses and the modulatory effects that allow their survival, much remains to be defined. Heat shock proteins are molecules responsible for folding newly synthesized polypeptides under physiological conditions and misfolded proteins under stress, but their role in maintaining the transformed phenotype often goes beyond their conventional chaperone activity. Expression of inducible HSPs is known to correlate with limited sensitivity to apoptosis induced by diverse cytotoxic agents and dismal prognosis of several tumour types, however whether cancer cells survive because of the constitutive expression of heat shock proteins or the ability to induce them when adapting to the hostile microenvironment remains to be elucidated. Clear is that tumours appear nowadays more “addicted” to heat shock proteins than previously envisaged, and targeting HSPs represents a powerful approach and a future challenge for sensitizing tumours to therapy. This review will focus on the anti-apoptotic role of heat shock 70kDa protein (Hsp70), and how regulatory factors that control inducible Hsp70 synthesis, expression and activity may be relevant for response to stress and survival of cancer cells.

  6. Gymnastics of molecular chaperones.

    Science.gov (United States)

    Mayer, Matthias P

    2010-08-13

    Molecular chaperones assist folding processes and conformational changes in many proteins. In order to do so, they progress through complex conformational cycles themselves. In this review, I discuss the diverse conformational dynamics of the ATP-dependent chaperones of the Hsp60, Hsp70, Hsp90, and Hsp100 families. PMID:20705236

  7. Histone chaperones link histone nuclear import and chromatin assembly.

    Science.gov (United States)

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

    Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  8. The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding

    Science.gov (United States)

    Woodford, Mark R.; Dunn, Diana M.; Blanden, Adam R.; Capriotti, Dante; Loiselle, David; Prodromou, Chrisostomos; Panaretou, Barry; Hughes, Philip F.; Smith, Aaron; Ackerman, Wendi; Haystead, Timothy A.; Loh, Stewart N.; Bourboulia, Dimitra; Schmidt, Laura S.; Marston Linehan, W.; Bratslavsky, Gennady; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors. PMID:27353360

  9. Molecular chaperones: The modular evolution of cellular networks

    Indian Academy of Sciences (India)

    Tamás Korcsmáros; István A Kovács; Máté S Szalay; Péter Csermely

    2007-04-01

    Molecular chaperones play a prominent role in signaling and transcriptional regulatory networks of the cell. Recent advances uncovered that chaperones act as genetic buffers stabilizing the phenotype of various cells and organisms and may serve as potential regulators of evolvability. Chaperones have weak links, connect hubs, are in the overlaps of network modules and may uncouple these modules during stress, which gives an additional protection for the cell at the network-level. Moreover, after stress chaperones are essential to re-build inter-modular contacts by their low affinity sampling of the potential interaction partners in different modules. This opens the way to the chaperone-regulated modular evolution of cellular networks, and helps us to design novel therapeutic and anti-aging strategies.

  10. The pvc operon regulates the expression of the Pseudomonas aeruginosa fimbrial chaperone/usher pathway (cup genes.

    Directory of Open Access Journals (Sweden)

    Uzma Qaisar

    Full Text Available The Pseudomonas aeruginosa fimbrial structures encoded by the cup gene clusters (cupB and cupC contribute to its attachment to abiotic surfaces and biofilm formation. The P. aeruginosa pvcABCD gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the pvc operon and the cup gene clusters in the P. aeruginosa strain MPAO1. Mutations within the pvc genes compromised biofilm development and significantly reduced the expression of cupB1-6 and cupC1-3, as well as different genes of the cupB/cupC two-component regulatory systems, roc1/roc2. Adjacent to pvc is the transcriptional regulator ptxR. A ptxR mutation in MPAO1 significantly reduced the expression of the pvc genes, the cupB/cupC genes, and the roc1/roc2 genes. Overexpression of the intact chromosomally-encoded pvc operon by a ptxR plasmid significantly enhanced cupB2, cupC2, rocS1, and rocS2 expression and biofilm development. Exogenously added paerucumarin significantly increased the expression of cupB2, cupC2, rocS1 and rocS2 in the pvcA mutant. Our results suggest that pvc influences P. aeruginosa biofilm development through the cup gene clusters in a pathway that involves paerucumarin, PtxR, and different cup regulators.

  11. The pvc operon regulates the expression of the Pseudomonas aeruginosa fimbrial chaperone/usher pathway (cup) genes.

    Science.gov (United States)

    Qaisar, Uzma; Luo, Liming; Haley, Cecily L; Brady, Sean F; Carty, Nancy L; Colmer-Hamood, Jane A; Hamood, Abdul N

    2013-01-01

    The Pseudomonas aeruginosa fimbrial structures encoded by the cup gene clusters (cupB and cupC) contribute to its attachment to abiotic surfaces and biofilm formation. The P. aeruginosa pvcABCD gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the pvc operon and the cup gene clusters in the P. aeruginosa strain MPAO1. Mutations within the pvc genes compromised biofilm development and significantly reduced the expression of cupB1-6 and cupC1-3, as well as different genes of the cupB/cupC two-component regulatory systems, roc1/roc2. Adjacent to pvc is the transcriptional regulator ptxR. A ptxR mutation in MPAO1 significantly reduced the expression of the pvc genes, the cupB/cupC genes, and the roc1/roc2 genes. Overexpression of the intact chromosomally-encoded pvc operon by a ptxR plasmid significantly enhanced cupB2, cupC2, rocS1, and rocS2 expression and biofilm development. Exogenously added paerucumarin significantly increased the expression of cupB2, cupC2, rocS1 and rocS2 in the pvcA mutant. Our results suggest that pvc influences P. aeruginosa biofilm development through the cup gene clusters in a pathway that involves paerucumarin, PtxR, and different cup regulators. PMID:23646138

  12. Mitotic regulator Mis18β interacts with and specifies the centromeric assembly of molecular chaperone holliday junction recognition protein (HJURP).

    Science.gov (United States)

    Wang, Jianyu; Liu, Xing; Dou, Zhen; Chen, Liang; Jiang, Hao; Fu, Chuanhai; Fu, Guosheng; Liu, Dan; Zhang, Jiancun; Zhu, Tongge; Fang, Jingwen; Zang, Jianye; Cheng, Jinke; Teng, Maikun; Ding, Xia; Yao, Xuebiao

    2014-03-21

    The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G1 phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437-460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction. PMID:24519934

  13. Mitotic Regulator Mis18β Interacts with and Specifies the Centromeric Assembly of Molecular Chaperone Holliday Junction Recognition Protein (HJURP)*

    Science.gov (United States)

    Wang, Jianyu; Liu, Xing; Dou, Zhen; Chen, Liang; Jiang, Hao; Fu, Chuanhai; Fu, Guosheng; Liu, Dan; Zhang, Jiancun; Zhu, Tongge; Fang, Jingwen; Zang, Jianye; Cheng, Jinke; Teng, Maikun; Ding, Xia; Yao, Xuebiao

    2014-01-01

    The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G1 phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437–460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction. PMID:24519934

  14. Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation.

    Science.gov (United States)

    Shu, Qin; Krezel, Andrzej M; Cusumano, Zachary T; Pinkner, Jerome S; Klein, Roger; Hultgren, Scott J; Frieden, Carl

    2016-06-28

    Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel β-sheet with flexible N and C termini. The structure of CsgE fits well into the cryoelectron microscopy density map of the CsgG-CsgE complex. We highlight a striking feature of the electrostatic potential surface in CsgE structure and present an assembly model of the CsgG-CsgE complex. We suggest a structural mechanism of the interaction between CsgE and CsgA. Understanding curli formation can provide the information necessary to develop treatments and therapeutic agents for biofilm-related infections and may benefit the prevention and treatment of amyloid diseases. CsgE could establish a paradigm for the regulation of amyloidogenesis because of its unique role in curli formation. PMID:27298344

  15. Chaperoning ribosome assembly

    OpenAIRE

    Karbstein, Katrin

    2010-01-01

    Chaperones help proteins fold in all cellular compartments, and many associate directly with ribosomes, capturing nascent chains to assist their folding and prevent aggregation. In this issue, new data from Koplin et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200910074) and Albanèse et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201001054) suggest that in addition to promoting protein folding, the chaperones ribosome-associated complex (RAC), nascent chain–associated complex (NAC), and Jjj1 also...

  16. Histone chaperone-mediated nucleosome assembly process.

    Science.gov (United States)

    Fan, Hsiu-Fang; Liu, Zi-Ning; Chow, Sih-Yao; Lu, Yi-Han; Li, Hsin

    2015-01-01

    A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1's specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.

  17. Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase.

    Science.gov (United States)

    Sha, Chong; Yu, Xiao-Wei; Lin, Nai-Xin; Zhang, Meng; Xu, Yan

    2013-12-10

    Pichia pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, but there is still a large room of improvement for this expression system. Two factors drastically influence the lipase r27RCL production from Rhizopus chinensis CCTCC M201021, which are gene dosage and protein folding in the endoplasmic reticulum (ER). Regarding the effect of gene dosage, the enzyme activity for recombinant strain with three copies lipase gene was 1.95-fold higher than that for recombinant strain with only one copy lipase gene. In addition, the lipase production was further improved by co-expression with chaperone PDI involved in the disulfide bond formation in the ER. Overall, the maximum enzyme activity reached 355U/mL by the recombinant strain with one copy chaperone gene PDI plus five copies lipase gene proRCL in shaking flasks, which was 2.74-fold higher than that for the control strain with only one copy lipase gene. Overall, co-expression with PDI vastly increased the capacity for processing proteins of ER in P. pastoris. PMID:24315648

  18. Cell-cycle-regulated control of VSG expression site silencing by histones and histone chaperones ASF1A and CAF-1b in Trypanosoma brucei.

    Science.gov (United States)

    Alsford, Sam; Horn, David

    2012-11-01

    Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G2/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G2/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.

  19. Histone chaperones: assisting histone traffic and nucleosome dynamics.

    Science.gov (United States)

    Gurard-Levin, Zachary A; Quivy, Jean-Pierre; Almouzni, Geneviève

    2014-01-01

    The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

  20. Engineering of recombinant crystallization chaperones

    OpenAIRE

    Koide, Shohei

    2009-01-01

    The preparation of diffraction quality crystals remains the major bottleneck in macromolecular x-ray crystallography. A crystallization chaperone is an auxiliary protein, such as fragments of monoclonal antibodies, that binds to and increases the crystallization probability of a target molecule of interest. Such chaperones reduce conformational heterogeneity, mask counterproductive surfaces while extending surfaces predisposed to forming crystal contacts, and provide phasing information. Crys...

  1. Molecular chaperones and neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Neurodegenerative diseases are characterized by the accumulation of intracellular or extracellular protein aggregates that result from conformational changes in proteins. These diseases may result from an imbalance between the production of misfolded proteins and normal chaperone capacity. Molecular chaperones provide a first line of defence against misfolded, aggregation-prone proteins and are, therefore, promising therapeutic targets for neurodegenerative diseases.

  2. 分子伴侣对酵母朊病毒[ PSI+]增殖影响的研究进展%Advances in molecular chaperones regulating yeast prion [ PSI+] propagation

    Institute of Scientific and Technical Information of China (English)

    林康伟; 连惠勇; 蔡澎

    2015-01-01

    The finding and research on yeast prion are of great values for biology and medical sciences.Research advances in molecular chaperones, especially in Hsp104p, Hsp70p and Hsp40p, regulating yeast prion [PSI+] propaga-tion,are reviewed.%酵母朊病毒( prion)的发现和研究在生物学和医学上有着极其重要的理论价值和实际应用价值。该文综述了分子伴侣对酵母prion[PSI+]增殖影响的研究进展,重点介绍了热休克蛋白Hsp104p、Hsp70p和Hsp40p在其中发挥的重要作用。

  3. Advances in molecular chaperones regulating yeast prion [ PSI+] propagation%分子伴侣对酵母朊病毒[ PSI+]增殖影响的研究进展

    Institute of Scientific and Technical Information of China (English)

    林康伟; 连惠勇; 蔡澎

    2015-01-01

    The finding and research on yeast prion are of great values for biology and medical sciences.Research advances in molecular chaperones, especially in Hsp104p, Hsp70p and Hsp40p, regulating yeast prion [PSI+] propaga-tion,are reviewed.%酵母朊病毒( prion)的发现和研究在生物学和医学上有着极其重要的理论价值和实际应用价值。该文综述了分子伴侣对酵母prion[PSI+]增殖影响的研究进展,重点介绍了热休克蛋白Hsp104p、Hsp70p和Hsp40p在其中发挥的重要作用。

  4. Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers

    NARCIS (Netherlands)

    Mannini, Benedetta; Cascella, Roberta; Zampagni, Mariagioia; Van Waarde-Verhagen, Maria; Meehan, Sarah; Roodveldt, Cintia; Campioni, Silvia; Boninsegna, Matilde; Penco, Amanda; Relini, Annalisa; Kampinga, Harm H.; Dobson, Christopher M.; Wilson, Mark R.; Cecchi, Cristina; Chiti, Fabrizio

    2012-01-01

    Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligom

  5. Chaperoning prions: the story unfolds

    OpenAIRE

    O'Connor, David; Jones, Gary

    2006-01-01

    Prions are infectious proteins that are responsible for a number of mammalian degenerative diseases. The discovery of prions in yeast has allowed detailed genetic analysis to be carried out to identify cellular factors involved in prion propagation. It is now clear that a complex relationship exists between molecular chaperones and prion propagation. Prions may actually have evolved to exploit the cell's chaperone machinery to ensure their own propaga...

  6. Identification of putative RuBisCo activase (TaRca1 ˗ the catalytic chaperone regulating carbon assimilatory pathway in wheat (Triticum aestivum under the heat stress

    Directory of Open Access Journals (Sweden)

    RANJEET RANJAN KUMAR

    2016-07-01

    Full Text Available RuBisCo activase (Rca is a catalytic chaperone involved in modulating the activity of RuBisCo (key enzyme of photosynthetic pathway. Here, we identified eight novel transcripts from wheat through data mining predicted to be Rca and cloned a transcript of 1.4 kb from cv. HD2985, named as TaRca1 (GenBank acc. no. KC776912. Single copy number of TaRca1 was observed in wheat genome. Expression analysis in diverse wheat genotypes (HD2985, Halna, PBW621 and HD2329 showed very high relative expression of TaRca1 in Halna under control and HS-treated, as compared to other cultivars at different stages of growth. TaRca1 protein was predicted to be chloroplast-localized with numerous potential phosphorylation sites. Nothern blot analysis showed maximum accumulation of TaRca1 transcript in thermotolerant cv. during mealy-ripe stage, as compared to thermosusceptible. Decrease in the photosynthetic parameters was observed in all the cultivars, except PBW621 in response to HS. We observed significant increase in the Rca activity in all the cultivars under HS at different stages of growth. HS causes decrease in the RuBisCo activity; maximum reduction was observed during pollination stage in thermosusceptible cvs. as validated through immunoblotting. We observed uniform carbon distribution in different tissues of thermotolerant cvs., as compared to thermosusceptible. Similarly, tolerance level of leaf was observed maximum in Halna having high Rca activity under HS. A positive correlation was observed between the transcript and activity of TaRca1 in HS-treated Halna. Similarly, TaRca1 enzyme showed positive correlation with the activity of RuBisCo. There is, however, need to manipulate the thermal stability of TaRca1 enzyme through protein engineering for sustaining the photosynthetic rate under HS – a novel approach towards development of ‘climate-smart’ crop.

  7. Targeting Molecular Chaperones for the Treatment of Cystic Fibrosis: Is It a Viable Approach?

    Science.gov (United States)

    Heard, Ashley; Thompson, Jake; Carver, Jessica; Bakey, Michelle; Wang, X Robert

    2015-01-01

    Cystic Fibrosis (CF) is largely caused by protein misfolding and the loss of function of a plasma membrane anion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). The most common CF-causing mutation, F508del, leads to severe conformational defect in CFTR. The cellular chaperone machinery plays an important role in CFTR biogenesis and quality control. Multiple attempts have been made to improve the cell surface functional expression of the mutant CFTR by modulating the expression of components of the cellular chaperone machinery. The efficacy of such an approach has been low largely due to the severe intrinsic folding defects of the F508del CFTR. Moreover, the impact of chaperone perturbation on the chaperone machinery itself and on other physiologically important proteins might lead to potentially severe side effects. Approaches aimed at disrupting chaperone-CFTR interactions show greater efficacy, and are compatible with small-molecule drug discovery and gene therapy. Combination between chaperone modulators and F508del correctors might further enhance potency and specificity. As molecular chaperones play important roles in regulating inflammation and immunity, they can be potential targets for controlling airway infection and inflammation in patients. If such effects can be synergized with chaperone-mediated regulation of CFTR biogenesis and quality control, more efficacious therapeutics will be developed to combat CF lung disease. PMID:25981601

  8. Do nucleic acids moonlight as molecular chaperones?

    Science.gov (United States)

    Docter, Brianne E.; Horowitz, Scott; Gray, Michael J.; Jakob, Ursula; Bardwell, James C.A.

    2016-01-01

    Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation. PMID:27105849

  9. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity

    Science.gov (United States)

    2016-01-01

    ABSTRACT Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  10. DegP Chaperone Suppresses Toxic Inner Membrane Translocation Intermediates

    Science.gov (United States)

    Braselmann, Esther; Chaney, Julie L.; Champion, Matthew M.

    2016-01-01

    The periplasm of Gram-negative bacteria includes a variety of molecular chaperones that shepherd the folding and targeting of secreted proteins. A central player of this quality control network is DegP, a protease also suggested to have a chaperone function. We serendipitously discovered that production of the Bordetella pertussis autotransporter virulence protein pertactin is lethal in Escherichia coli ΔdegP strains. We investigated specific contributions of DegP to secretion of pertactin as a model system to test the functions of DegP in vivo. The DegP chaperone activity was sufficient to restore growth during pertactin production. This chaperone dependency could be relieved by changing the pertactin signal sequence: an E. coli signal sequence leading to co-translational inner membrane (IM) translocation was sufficient to suppress lethality in the absence of DegP, whereas an E. coli post-translational signal sequence was sufficient to recapitulate the lethal phenotype. These results identify a novel connection between the DegP chaperone and the mechanism used to translocate a protein across the IM. Lethality coincided with loss of periplasmic proteins, soluble σE, and proteins regulated by this essential stress response. These results suggest post-translational IM translocation can lead to the formation of toxic periplasmic folding intermediates, which DegP can suppress. PMID:27626276

  11. DegP Chaperone Suppresses Toxic Inner Membrane Translocation Intermediates.

    Science.gov (United States)

    Braselmann, Esther; Chaney, Julie L; Champion, Matthew M; Clark, Patricia L

    2016-01-01

    The periplasm of Gram-negative bacteria includes a variety of molecular chaperones that shepherd the folding and targeting of secreted proteins. A central player of this quality control network is DegP, a protease also suggested to have a chaperone function. We serendipitously discovered that production of the Bordetella pertussis autotransporter virulence protein pertactin is lethal in Escherichia coli ΔdegP strains. We investigated specific contributions of DegP to secretion of pertactin as a model system to test the functions of DegP in vivo. The DegP chaperone activity was sufficient to restore growth during pertactin production. This chaperone dependency could be relieved by changing the pertactin signal sequence: an E. coli signal sequence leading to co-translational inner membrane (IM) translocation was sufficient to suppress lethality in the absence of DegP, whereas an E. coli post-translational signal sequence was sufficient to recapitulate the lethal phenotype. These results identify a novel connection between the DegP chaperone and the mechanism used to translocate a protein across the IM. Lethality coincided with loss of periplasmic proteins, soluble σE, and proteins regulated by this essential stress response. These results suggest post-translational IM translocation can lead to the formation of toxic periplasmic folding intermediates, which DegP can suppress.

  12. Hsp70 chaperone systems: diversity of cellular functions and mechanism of action.

    Science.gov (United States)

    Mayer, M P; Bukau, B

    1998-03-01

    Hsp70 chaperone systems play an essential role in the life cycle of many proteins not only in an hostile environment but also under normal growth conditions. In the course of evolution the diversification of functions was accompanied by an amplification of components of the Hsp70 system. Here strategies are reviewed how different Hsp70 systems work independently or cooperate with each other in a functional network to perform their housekeeping tasks even under stress conditions. We further discuss how co-chaperones which act as targeting factors regulate the cycle of substrate binding and release upon which the Hsp70 chaperone activity depends.

  13. Chaperone proteins identified from synthetic proteasome inhibitor-induced inclusions in PC12 cells by proteomic analysis

    Institute of Scientific and Technical Information of China (English)

    Xing'an Li; Yinjiu Zhang; Yihong Hu; Ming Chang; Tao Liu; Danping Wang; Yu Zhang; Lei Zhang; Linsen Hu

    2008-01-01

    Chaperone proteins are significant in Lewy bodies, but the profile of chaperone proteins is incompletely unraveled.Protcomic analysis is used to determine protein candidates for further study. Here, to identify potential chaperone proteins from agent-induced inclusions, we carried out proteomic analysis of artificially synthetic proteasome inhibitor (PSI)-induced inclusions formed in PC12 cells exposed to 10 μM PSI for 48 h. Using biochemical fractionation, 2-D electrophoresis, and identification through peptide mass fingerprints searched against multiple protein databases, we repeatedly identified eight reproducible chaperone proteins from the PSI-induced inclusions. Of these, 58 kDa glucose regulated protein, 75 kDa glucose regulated protein, and caldum-binding protein I were newly identified. The other five had been reported to be consistent components of Lewy bodies. These findings suggested that the three potential chaperone proteins might be recruited to PSI-induced inclusions in PC12 cells under proteasome inhibition.

  14. Insight into the assembly of chaperones

    Energy Technology Data Exchange (ETDEWEB)

    May, R.P. [Institut Max von Laue - Paul Langevin (ILL), 38 - Grenoble (France); Stegmann, R.; Manakova, E.; Roessle, M.; Hermann, T.; Heumann, H. [Max-Planck-Institut fuer Biochemie, Martinsried (Germany); Axmann, S.; Plueckthun, A. [Zurich Univ. (Switzerland); Wiedenmann, A. [HMI, Berlin (Germany)

    1997-04-01

    Chaperones are proteins that help other proteins (substrate proteins) to acquire a `good` conformation. The folding is a dynamic process and involves repetitive binding and release of the chaperone components and of the substrate protein. Small-angle neutron scattering is used to investigate the structural changes that appear to happen during the folding process. (author). 2 refs.

  15. The histone chaperones Nap1 and Vps75 bind histones H3 and H4 in a tetrameric conformation.

    Science.gov (United States)

    Bowman, Andrew; Ward, Richard; Wiechens, Nicola; Singh, Vijender; El-Mkami, Hassane; Norman, David George; Owen-Hughes, Tom

    2011-02-18

    Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.

  16. Modulation of the chaperone heat shock cognate 70 by embryonic (pro)insulin correlates with prevention of apoptosis

    OpenAIRE

    De La Rosa, Enrique J; Vega-Núñez, Elena; Morales, Aixa V.; Serna, José; Rubio, Eva; Pablo, Flora de

    1998-01-01

    Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA a...

  17. Oxidative stress induces monocyte necrosis with enrichment of cell-bound albumin and overexpression of endoplasmic reticulum and mitochondrial chaperones.

    Directory of Open Access Journals (Sweden)

    Haiping Tang

    Full Text Available In the present study, monocytes were treated with 5-azacytidine (azacytidine, gossypol or hydrogen peroxide to induce cell death through oxidative stress. A shift from apoptotic to necrotic cell death occurred when monocytes were treated with 100 µM azacytidine for more than 12 hours. Necrotic monocytes exhibited characteristics, including enrichment of cell-bound albumin and up-regulation of endoplasmic reticulum (ER- and mitochondrial-specific chaperones to protect mitochondrial integrity, which were not observed in other necrotic cells, including HUH-7, A2780, A549 and HOC1a. Our results show that the cell-bound albumin originates in the culture medium rather than from monocyte-derived hepatocytes, and that HSP60 is a potential binding partner of the cell-bound albumin. Proteomic analysis shows that HSP60 and protein disulfide isomerase are the most abundant up-regulated mitochondrial and ER-chaperones, and that both HSP60 and calreticulin are ubiquitinated in necrotic monocytes. In contrast, expression levels of the cytosolic chaperones HSP90 and HSP71 were down-regulated in the azacytidine-treated monocytes, concomitant with an increase in the levels of these chaperones in the cell culture medium. Collectively, our results demonstrates that chaperones from different organelles behave differently in necrotic monocytes, ER- and mitochondrial chaperones being retained and cytosolic and nuclear chaperones being released into the cell culture medium through the ruptured cell membrane. HSP60 may serve as a new target for development of myeloid leukemia treatment.

  18. Chaperone-mediated autophagy: roles in neuroprotection.

    Science.gov (United States)

    Cai, Zhibiao; Zeng, Weijun; Tao, Kai; E, Zhen; Wang, Bao; Yang, Qian

    2015-08-01

    Chaperone-mediated autophagy (CMA), one of the main pathways of lysosomal proteolysis, is characterized by the selective targeting and direct translocation into the lysosomal lumen of substrate proteins containing a targeting motif biochemically related to the pentapeptide KFERQ. Along with the other two lysosomal pathways, macro- and micro-autophagy, CMA is essential for maintaining cellular homeostasis and survival by selectively degrading misfolded, oxidized, or damaged cytosolic proteins. CMA plays an important role in pathologies such as cancer, kidney disorders, and neurodegenerative diseases. Neurons are post-mitotic and highly susceptible to dysfunction of cellular quality-control systems. Maintaining a balance between protein synthesis and degradation is critical for neuronal functions and homeostasis. Recent studies have revealed several new mechanisms by which CMA protects neurons through regulating factors critical for their viability and homeostasis. In the current review, we summarize recent advances in the understanding of the regulation and physiology of CMA with a specific focus on its possible roles in neuroprotection. PMID:26206599

  19. Lipid Chaperones and Metabolic Inflammation

    Directory of Open Access Journals (Sweden)

    Masato Furuhashi

    2011-01-01

    Full Text Available Over the past decade, a large body of evidence has emerged demonstrating an integration of metabolic and immune response pathways. It is now clear that obesity and associated disorders such as insulin resistance and type 2 diabetes are associated with a metabolically driven, low-grade, chronic inflammatory state, referred to as “metaflammation.” Several inflammatory cytokines as well as lipids and metabolic stress pathways can activate metaflammation, which targets metabolically critical organs and tissues including adipocytes and macrophages to adversely affect systemic homeostasis. On the other hand, inside the cell, fatty acid-binding proteins (FABPs, a family of lipid chaperones, as well as endoplasmic reticulum (ER stress, and reactive oxygen species derived from mitochondria play significant roles in promotion of metabolically triggered inflammation. Here, we discuss the molecular and cellular basis of the roles of FABPs, especially FABP4 and FABP5, in metaflammation and related diseases including obesity, diabetes, and atherosclerosis.

  20. CrAgDb--a database of annotated chaperone repertoire in archaeal genomes.

    Science.gov (United States)

    Rani, Shikha; Srivastava, Abhishikha; Kumar, Manish; Goel, Manisha

    2016-03-01

    Chaperones are a diverse class of ubiquitous proteins that assist other cellular proteins in folding correctly and maintaining their native structure. Many different chaperones cooperate to constitute the 'proteostasis' machinery in the cells. It has been proposed earlier that archaeal organisms could be ideal model systems for deciphering the basic functioning of the 'protein folding machinery' in higher eukaryotes. Several chaperone families have been characterized in archaea over the years but mostly one protein at a time, making it difficult to decipher the composition and mechanistics of the protein folding system as a whole. In order to deal with these lacunae, we have developed a database of all archaeal chaperone proteins, CrAgDb (Chaperone repertoire in Archaeal genomes). The data have been presented in a systematic way with intuitive browse and search facilities for easy retrieval of information. Access to these curated datasets should expedite large-scale analysis of archaeal chaperone networks and significantly advance our understanding of operation and regulation of the protein folding machinery in archaea. Researchers could then translate this knowledge to comprehend the more complex protein folding pathways in eukaryotic systems. The database is freely available at http://14.139.227.92/mkumar/cragdb/. PMID:26862144

  1. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.;

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit...

  2. Supercharging Chaperones: A Meeting Toolkit for Maximizing Learning for Youth and Chaperones

    Science.gov (United States)

    Brandt, Brian

    2016-01-01

    Trip and conference chaperones are a wonderful resource in youth development programs. These well-intended volunteers, many parents of youth participating in the event, want the best experience for the youth but are not necessarily trained in positive youth development. A consequence of this circumstance is that not all chaperones provide the best…

  3. Modulation and elimination of yeast prions by protein chaperones and co-chaperones

    OpenAIRE

    Reidy, Michael; Masison, Daniel C.

    2011-01-01

    The yeast system has provided considerable insight into the biology of amyloid and prions. Here we focus on how alterations in abundance or function of protein chaperones and co-chaperones affect propagation of yeast prions. In spite of a considerable amount of information, a clear understanding of the molecular mechanisms underlying these effects remains wanting.

  4. Sequence and domain conservation of the coelacanth Hsp40 and Hsp90 chaperones suggests conservation of function.

    Science.gov (United States)

    Bishop, Özlem Tastan; Edkins, Adrienne Lesley; Blatch, Gregory Lloyd

    2014-09-01

    Molecular chaperones and their associated co-chaperones play an important role in preserving and regulating the active conformational state of cellular proteins. The chaperone complement of the Indonesian Coelacanth, Latimeria menadoensis, was elucidated using transcriptomic sequences. Heat shock protein 90 (Hsp90) and heat shock protein 40 (Hsp40) chaperones, and associated co-chaperones were focused on, and homologous human sequences were used to search the sequence databases. Coelacanth homologs of the cytosolic, mitochondrial and endoplasmic reticulum (ER) homologs of human Hsp90 were identified, as well as all of the major co-chaperones of the cytosolic isoform. Most of the human Hsp40s were found to have coelacanth homologs, and the data suggested that all of the chaperone machinery for protein folding at the ribosome, protein translocation to cellular compartments such as the ER and protein degradation were conserved. Some interesting similarities and differences were identified when interrogating human, mouse, and zebrafish homologs. For example, DnaJB13 is predicted to be a non-functional Hsp40 in humans, mouse, and zebrafish due to a corrupted histidine-proline-aspartic acid (HPD) motif, while the coelacanth homolog has an intact HPD. These and other comparisons enabled important functional and evolutionary questions to be posed for future experimental studies.

  5. Chaperoning the Chaperone: A Role for the Co-chaperone Cpr7 in Modulating Hsp90 Function in Saccharomyces cerevisiae

    OpenAIRE

    Zuehlke, Abbey D.; Johnson, Jill L.

    2012-01-01

    Heat-shock protein 90 (Hsp90) of Saccharomyces cerevisiae is an abundant essential eukaryotic molecular chaperone involved in the activation and stabilization of client proteins, including several transcription factors and oncogenic kinases. Hsp90 undergoes a complex series of conformational changes and interacts with partner co-chaperones such as Sba1, Cpr6, Cpr7, and Cns1 as it binds and hydrolyzes ATP. In the absence of nucleotide, Hsp90 is dimerized only at the carboxy-terminus. In the pr...

  6. A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility.

    Science.gov (United States)

    Fernández-Rivero, Noelia; Franco, Aitor; Velázquez-Campoy, Adrian; Alonso, Edurne; Muga, Arturo; Prado, Adelina

    2016-01-01

    Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A-H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A-H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A-H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. PMID:27558753

  7. Inhibitors of the AAA+ Chaperone p97

    Directory of Open Access Journals (Sweden)

    Eli Chapman

    2015-02-01

    Full Text Available It is remarkable that a pathway as ubiquitous as protein quality control can be targeted to treat cancer. Bortezomib, an inhibitor of the proteasome, was first approved by the US Food and Drug Administration (FDA more than 10 years ago to treat refractory myeloma and later extended to lymphoma. Its use has increased the survival rate of myeloma patients by as much as three years. This success was followed with the recent accelerated approval of the natural product derived proteasome inhibitor carfilzomib (Kyprolis®, which is used to treat patients with bortezomib-resistant multiple myeloma. The success of these two drugs has validated protein quality control as a viable target to fight select cancers, but begs the question why are proteasome inhibitors limited to lymphoma and myeloma? More recently, these limitations have encouraged the search for additional targets within the protein quality control system that might offer heightened cancer cell specificity, enhanced clinical utility, a lower rate of resistance, reduced toxicity, and mitigated side effects. One promising target is p97, an ATPase associated with various cellular activities (AAA+ chaperone. p97 figures prominently in protein quality control as well as serving a variety of other cellular functions associated with cancer. More than a decade ago, it was determined that up-regulation of p97 in many forms of cancer correlates with a poor clinical outcome. Since these initial discoveries, a mechanistic explanation for this observation has been partially illuminated, but details are lacking. Understandably, given this clinical correlation, myriad roles within the cell, and its importance in protein quality control, p97 has emerged as a potential therapeutic target. This review provides an overview of efforts towards the discovery of small molecule inhibitors of p97, offering a synopsis of efforts that parallel the excellent reviews that currently exist on p97 structure, function, and

  8. The future of molecular chaperones and beyond.

    Science.gov (United States)

    Giffard, Rona G; Macario, Alberto J L; de Macario, Everly Conway

    2013-08-01

    Protection of hair cells by HSP70 released by supporting cells is reported by May et al. in this issue of the JCI. Their findings suggest a new way to reduce ototoxicity from therapeutic medications and raise larger questions about the role and integration of heat shock proteins in non–cell-autonomous responses to stress. Increasing evidence suggests an important role for extracellular heat shock proteins in both the nervous system and the immune system. The work also suggests that defective chaperones could cause ear disease and supports the potential use of chaperone therapeutics.

  9. Chaperone binding at the ribosomal exit tunnel

    DEFF Research Database (Denmark)

    Kristensen, Ole; Gajhede, Michael

    2003-01-01

    The exit tunnel region of the ribosome is well established as a focal point for interaction between the components that guide the fate of nascent polypeptides. One of these, the chaperone trigger factor (TF), associates with the 50S ribosomal subunit through its N-terminal domain. Targeting of TF...

  10. Degradation of AF1Q by chaperone-mediated autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Li, Huanjie; Cui, Taixing; Li Wang, Xing [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: tangdq@sdu.edu.cn [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Center for Stem Cell and Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250033 (China); Ji, Chunyan, E-mail: jichunyan@sdu.edu.cn [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China)

    2014-09-10

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components.

  11. Identification of Putative RuBisCo Activase (TaRca1)—The Catalytic Chaperone Regulating Carbon Assimilatory Pathway in Wheat (Triticum aestivum) under the Heat Stress

    Science.gov (United States)

    Goswami, Suneha; Singh, Khushboo; Dubey, Kavita; Singh, Shweta; Sharma, Renu; Verma, Neeraj; Kala, Yugal K.; Rai, Gyanendra K.; Grover, Monendra; Mishra, Dwijesh C.; Singh, Bhupinder; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly

    2016-01-01

    RuBisCo activase (Rca) is a catalytic chaperone involved in modulating the activity of RuBisCo (key enzyme of photosynthetic pathway). Here, we identified eight novel transcripts from wheat through data mining predicted to be Rca and cloned a transcript of 1.4 kb from cv. HD2985, named as TaRca1 (GenBank acc. no. KC776912). Single copy number of TaRca1 was observed in wheat genome. Expression analysis in diverse wheat genotypes (HD2985, Halna, PBW621, and HD2329) showed very high relative expression of TaRca1 in Halna under control and HS-treated, as compared to other cultivars at different stages of growth. TaRca1 protein was predicted to be chloroplast-localized with numerous potential phosphorylation sites. Northern blot analysis showed maximum accumulation of TaRca1 transcript in thermotolerant cv. during mealy-ripe stage, as compared to thermosusceptible. Decrease in the photosynthetic parameters was observed in all the cultivars, except PBW621 in response to HS. We observed significant increase in the Rca activity in all the cultivars under HS at different stages of growth. HS causes decrease in the RuBisCo activity; maximum reduction was observed during pollination stage in thermosusceptible cvs. as validated through immunoblotting. We observed uniform carbon distribution in different tissues of thermotolerant cvs., as compared to thermosusceptible. Similarly, tolerance level of leaf was observed maximum in Halna having high Rca activity under HS. A positive correlation was observed between the transcript and activity of TaRca1 in HS-treated Halna. Similarly, TaRca1 enzyme showed positive correlation with the activity of RuBisCo. There is, however, need to manipulate the thermal stability of TaRca1 enzyme through protein engineering for sustaining the photosynthetic rate under HS—a novel approach toward development of “climate-smart” crop. PMID:27462325

  12. Chaperones in hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    The hepatitis C virus (HCV) infects approximately 3% ofthe world population or more than 185 million peopleworldwide. Each year, an estimated 350000-500000deaths occur worldwide due to HCV-associated diseasesincluding cirrhosis and hepatocellular carcinoma. HCV isthe most common indication for liver transplantation inpatients with cirrhosis worldwide. HCV is an envelopedRNA virus classified in the genus Hepacivirus in theFlaviviridae family. The HCV viral life cycle in a cellcan be divided into six phases (1) binding and internalization;(2) cytoplasmic release and uncoating; (3)viral polyprotein translation and processing; (4) RNAgenome replication; (5) encapsidation (packaging) andassembly; and (6) virus morphogenesis (maturation)and secretion. Many host factors are involved in theHCV life cycle. Chaperones are an important group ofhost cytoprotective molecules that coordinate numerouscellular processes including protein folding, multimericprotein assembly, protein trafficking, and proteindegradation. All phases of the viral life cycle requirechaperone activity and the interaction of viral proteinswith chaperones. This review will present our currentknowledge and understanding of the role of chaperonesin the HCV life cycle. Analysis of chaperones in HCVinfection will provide further insights into viral/hostinteractions and potential therapeutic targets for bothHCV and other viruses.

  13. Multiple functions of the histone chaperone Jun dimerization protein 2.

    Science.gov (United States)

    Tsai, Ming-Ho; Wuputra, Kenly; Lin, Yin-Chu; Lin, Chang-Shen; Yokoyama, Kazunari K

    2016-09-30

    The Jun dimerization protein 2 (JDP2) is part of the family of stress-responsible transcription factors such as the activation protein-1, and binds the 12-O-tetradecanoylphorbol-13-acetateresponse element and the cAMP response element. It also plays a role as a histone chaperone and participates in diverse processes, such as cell-cycle arrest, cell differentiation, apoptosis, senescence, and metastatic spread, and functions as an oncogene and anti-oncogene, and as a cellular reprogramming factor. However, the molecular mechanisms underlying these multiple functions of JDP2 have not been clarified. This review summarizes the structure and function of JDP2, highlighting the specific role of JDP2 in cellular-stress regulation and prevention. PMID:27041241

  14. Molecular chaperone genes in the sugarcane expressed sequence database (SUCEST)

    OpenAIRE

    Borges, Júlio C; Maria C. Peroto; Ramos, Carlos H. I.

    2001-01-01

    Some newly synthesized proteins require the assistance of molecular chaperones for their correct folding. Chaperones are also involved in the dissolution of protein aggregates making their study significant for both biotechnology and medicine and the identification of chaperones and stress-related protein sequences in different organisms is an important task. We used bioinformatic tools to investigate the information generated by the Sugarcane Expressed Sequence Tag (SUCEST) genome project in...

  15. Differential roles of NF-Y transcription factor in ER chaperone expression and neuronal maintenance in the CNS

    Science.gov (United States)

    Yamanaka, Tomoyuki; Tosaki, Asako; Miyazaki, Haruko; Kurosawa, Masaru; Koike, Masato; Uchiyama, Yasuo; Maity, Sankar N.; Misawa, Hidemi; Takahashi, Ryosuke; Shimogori, Tomomi; Hattori, Nobutaka; Nukina, Nobuyuki

    2016-01-01

    The mammalian central nervous system (CNS) contains various types of neurons with different neuronal functions. In contrast to established roles of cell type-specific transcription factors on neuronal specification and maintenance, whether ubiquitous transcription factors have conserved or differential neuronal function remains uncertain. Here, we revealed that inactivation of a ubiquitous factor NF-Y in different sets of neurons resulted in cell type-specific neuropathologies and gene downregulation in mouse CNS. In striatal and cerebellar neurons, NF-Y inactivation led to ubiquitin/p62 pathologies with downregulation of an endoplasmic reticulum (ER) chaperone Grp94, as we previously observed by NF-Y deletion in cortical neurons. In contrast, NF-Y inactivation in motor neurons induced neuronal loss without obvious protein deposition. Detailed analysis clarified downregulation of another ER chaperone Grp78 in addition to Grp94 in motor neurons, and knockdown of both ER chaperones in motor neurons recapitulated the pathology observed after NF-Y inactivation. Finally, additional downregulation of Grp78 in striatal neurons suppressed ubiquitin accumulation induced by NF-Y inactivation, implying that selective ER chaperone downregulation mediates different neuropathologies. Our data suggest distinct roles of NF-Y in protein homeostasis and neuronal maintenance in the CNS by differential regulation of ER chaperone expression. PMID:27687130

  16. Mitochondrial chaperones may be targets for anti-cancer drugs

    Science.gov (United States)

    Scientists at NCI have found that a mitochondrial chaperone protein, TRAP1, may act indirectly as a tumor suppressor as well as a novel target for developing anti-cancer drugs. Chaperone proteins, such as TRAP1, help other proteins adapt to stress, but sc

  17. Disaggregases, molecular chaperones that resolubilize protein aggregates

    Directory of Open Access Journals (Sweden)

    David Z. Mokry

    2015-08-01

    Full Text Available The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of protein aggregates, an insoluble association of polypeptides that harm cell physiology, either by themselves or in the process of formation. Several biological processes have evolved to prevent and eliminate the existence of non-functional and amyloidogenic aggregates, as they are associated with several human pathologies. Molecular chaperones and heat shock proteins are specialized in controlling the quality of the proteins in the cell, specifically by aiding proper folding, and dissolution and clearance of already formed protein aggregates. The latter is a function of disaggregases, mainly represented by the ClpB/Hsp104 subfamily of molecular chaperones, that are ubiquitous in all organisms but, surprisingly, have no orthologs in the cytosol of metazoan cells. This review aims to describe the characteristics of disaggregases and to discuss the function of yeast Hsp104, a disaggregase that is also involved in prion propagation and inheritance.

  18. Chaperones as potential therapeutics for Krabbe disease.

    Science.gov (United States)

    Graziano, Adriana Carol Eleonora; Pannuzzo, Giovanna; Avola, Rosanna; Cardile, Venera

    2016-11-01

    Krabbe's disease (KD) is an autosomal recessive, neurodegenerative disorder. It is classified among the lysosomal storage diseases (LSDs). It was first described in , but the genetic defect for the galactocerebrosidase (GALC) gene was not discovered until the beginning of the 1970s, 20 years before the GALC cloning. Recently, in 2011, the crystal structures of the GALC enzyme and the GALC-product complex were obtained. For this, compared with other LSDs, the research on possible therapeutic interventions is much more recent. Thus, it is not surprising that some treatment options are still under preclinical investigation, whereas their relevance for other pathologies of the same group has already been tested in clinical studies. This is specifically the case for pharmacological chaperone therapy (PCT), a promising strategy for selectively correcting defective protein folding and trafficking and for enhancing enzyme activity by small molecules. These compounds bind directly to a partially folded biosynthetic intermediate, stabilize the protein, and allow completion of the folding process to yield a functional protein. Here, we review the chaperones that have demonstrated potential therapeutics during preclinical studies for KD, underscoring the requirement to invigorate research for KD-addressed PCT that will benefit from recent insights into the molecular understanding of GALC structure, drug design, and development in cellular models. © 2016 Wiley Periodicals, Inc. PMID:27638605

  19. Chaperoning Proteins for Destruction: Diverse Roles of Hsp70 Chaperones and their Co-Chaperones in Targeting Misfolded Proteins to the Proteasome

    Directory of Open Access Journals (Sweden)

    Ayala Shiber

    2014-07-01

    Full Text Available Molecular chaperones were originally discovered as heat shock-induced proteins that facilitate proper folding of proteins with non-native conformations. While the function of chaperones in protein folding has been well documented over the last four decades, more recent studies have shown that chaperones are also necessary for the clearance of terminally misfolded proteins by the Ub-proteasome system. In this capacity, chaperones protect misfolded degradation substrates from spontaneous aggregation, facilitate their recognition by the Ub ligation machinery and finally shuttle the ubiquitylated substrates to the proteasome. The physiological importance of these functions is manifested by inefficient proteasomal degradation and the accumulation of protein aggregates during ageing or in certain neurodegenerative diseases, when chaperone levels decline. In this review, we focus on the diverse roles of stress-induced chaperones in targeting misfolded proteins to the proteasome and the consequences of their compromised activity. We further discuss the implications of these findings to the identification of new therapeutic targets for the treatment of amyloid diseases.

  20. Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

    Science.gov (United States)

    Asim, Mohammad; Massie, Charles E.; Orafidiya, Folake; Pértega-Gomes, Nelma; Warren, Anne Y.; Esmaeili, Mohsen; Selth, Luke A.; Zecchini, Heather I.; Luko, Katarina; Qureshi, Arham; Baridi, Ajoeb; Menon, Suraj; Madhu, Basetti; Escriu, Carlos; Lyons, Scott; Vowler, Sarah L.; Zecchini, Vincent R.; Shaw, Greg; Hessenkemper, Wiebke; Russell, Roslin; Mohammed, Hisham; Stefanos, Niki; Lynch, Andy G.; Grigorenko, Elena; D’Santos, Clive; Taylor, Chris; Lamb, Alastair; Sriranjan, Rouchelle; Yang, Jiali; Stark, Rory; Dehm, Scott M.; Rennie, Paul S.; Carroll, Jason S.; Griffiths, John R.; Tavaré, Simon; Mills, Ian G.; McEwan, Iain J.; Baniahmad, Aria; Tilley, Wayne D.; Neal, David E.

    2016-01-01

    Background: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. Methods: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. Results: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. Conclusions: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa. PMID:26657335

  1. Stratified analysis of lectin-like chaperones in the folding disease-related metabolic syndrome rat model.

    Science.gov (United States)

    Hirano, Makoto; Imagawa, Ayami; Totani, Kiichiro

    2016-09-01

    The metabolic syndrome including obesity and diabetes mellitus is known to be a major health problem worldwide. A recent study reported that obesity causes endoplasmic reticulum (ER) stress and subsequently leads to insulin resistance and type 2 diabetes. However, little is known about the alterations in the components of the calnexin/calreticulin (CNX/CRT) cycle, which promote glycoprotein folding in obese and diabetic conditions. To understand the operating status of the lectin-like chaperones related to the CNX/CRT cycle in the metabolic syndrome, we analyzed the chaperones for the activity, protein expression, and mRNA expression levels using Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rat models for obesity and diabetes, respectively. We demonstrated that misfolded proteins were gradually increased with progression of the syndrome, obesity to diabetes. The individual chaperone activities of CNX and CRT were both decreased in the ZF rat ER and, in contrast, were increased in the ZDF rat ER. The protein quantities and mRNA expressions of CNX and CRT were decreased in the ZF rats, but increased in the ZDF rats compared with those of the healthy model. Therefore, these results indicate that obesity down-regulates CNX and CRT expressions and their activities and diabetes up-regulates the expressions and activities of CNX and CRT. Our findings clearly suggest that metabolic syndrome affects the lectin-like chaperones in the CNX/CRT cycle at both the activity and expression levels. PMID:27425249

  2. Principles of Quantitative Estimation of the Chaperone-Like Activity

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Molecular chaperones are able to interact with unfolded states of the protein molecule preventing their aggregation and facilitating folding of the polypeptide chain into the native structure. An understanding of the mechanism of protein aggregation is required to estimate the efficiency of action of chaperones in the test-systems based on the suppression of aggregation of protein substrates. The kinetic regimes of aggregation of proteins are discussed. The analysis of the aggregation kinetics of proteins shows that after passing the lag phase, aggregation follows, as a rule, first order kinetics. The quantitative characterization methods of the ability of chaperones to prevent aggregation of protein substrates have been elaborated.

  3. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  4. The histone chaperone CAF-1 safeguards somatic cell identity.

    Science.gov (United States)

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Jung, Youngsook L; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Euong Ang, Cheen; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin; Rathert, Philipp; Jude, Julian; Ferrari, Francesco; Blanco, Andres; Fellner, Michaela; Wenzel, Daniel; Zinner, Marietta; Vidal, Simon E; Bell, Oliver; Stadtfeld, Matthias; Chang, Howard Y; Almouzni, Genevieve; Lowe, Scott W; Rinn, John; Wernig, Marius; Aravin, Alexei; Shi, Yang; Park, Peter J; Penninger, Josef M; Zuber, Johannes; Hochedlinger, Konrad

    2015-12-10

    Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. PMID:26659182

  5. FKBP immunophilins and Alzheimer's disease: A chaperoned affair

    Indian Academy of Sciences (India)

    Weihuan Cao; Mary Konsolaki

    2011-08-01

    The FK506-binding protein (FKBP) family of immunophilins consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. Analysis of the functions of immunophilins has been the focus of studies in recent years and has led to the identification of various molecular pathways in which FKBPs play an active role. All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. Binding of the immunosuppressant molecule FK506 to this domain inhibits their PPIase activity while mediating immune suppression through inhibition of calcineurin. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration. This review will focus on recent studies involving FKBP immunophilins in Alzheimer’s-disease-related pathways.

  6. Chaperone-Mediated Autophagy and Mitochondrial Homeostasis in Parkinson's Disease.

    Science.gov (United States)

    Yang, Ruixin; Gao, Guodong; Mao, Zixu; Yang, Qian

    2016-01-01

    Parkinson's disease (PD), a complex neurodegenerative disorder, is pathologically characterized by the formation of Lewy bodies and loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial dysfunction is considered to be one of the most important causative mechanisms. In addition, dysfunction of chaperone-mediated autophagy (CMA), one of the lysosomal proteolytic pathways, has been shown to play an important role in the pathogenesis of PD. An exciting and important development is recent finding that CMA and mitochondrial quality control may be linked. This review summarizes the studies revealing the link between autophagy and mitochondrial function. Discussions are focused on the connections between CMA and mitochondrial failure and on the role of MEF2D, a neuronal survival factor, in mediating the regulation of mitochondria in the context of CMA. These new findings highlight the need to further explore the possibility of targeting the MEF2D-mitochondria-CMA network in both understanding the PD pathogenesis and developing novel therapeutic strategies.

  7. Chemical chaperones mitigate experimental asthma by attenuating endoplasmic reticulum stress.

    Science.gov (United States)

    Makhija, Lokesh; Krishnan, Veda; Rehman, Rakhshinda; Chakraborty, Samarpana; Maity, Shuvadeep; Mabalirajan, Ulaganathan; Chakraborty, Kausik; Ghosh, Balaram; Agrawal, Anurag

    2014-05-01

    Endoplasmic reticulum (ER) stress and consequent unfolded protein response (UPR) are important in inflammation but have been poorly explored in asthma. We used a mouse model of allergic airway inflammation (AAI) with features of asthma to understand the role of ER stress and to explore potential therapeutic effects of inhaled chemical chaperones, which are small molecules that can promote protein folding and diminish UPR. UPR markers were initially measured on alternate days during a 7-day daily allergen challenge model. UPR markers increased within 24 hours after the first allergen challenge and peaked by the third challenge, before AAI was fully established (from the fifth challenge onward). Three chemical chaperones-glycerol, trehalose, and trimethylamine-N-oxide (TMAO)-were initially administered during allergen challenge (preventive regimen). TMAO, the most effective of these chemical chaperones and 4-phenylbutyric acid, a chemical chaperone currently in clinical trials, were further tested for potential therapeutic activities after AAI was established (therapeutic regimen). Chemical chaperones showed a dose-dependent reduction in UPR markers, airway inflammation, and remodeling in both regimens. Our results indicate an early and important role of the ER stress pathway in asthma pathogenesis and show therapeutic potential for chemical chaperones.

  8. Chaperoning Roles of Macromolecules Interacting with Proteins in Vivo

    Directory of Open Access Journals (Sweden)

    Baik L. Seong

    2011-03-01

    Full Text Available The principles obtained from studies on molecular chaperones have provided explanations for the assisted protein folding in vivo. However, the majority of proteins can fold without the assistance of the known molecular chaperones, and little attention has been paid to the potential chaperoning roles of other macromolecules. During protein biogenesis and folding, newly synthesized polypeptide chains interact with a variety of macromolecules, including ribosomes, RNAs, cytoskeleton, lipid bilayer, proteolytic system, etc. In general, the hydrophobic interactions between molecular chaperones and their substrates have been widely believed to be mainly responsible for the substrate stabilization against aggregation. Emerging evidence now indicates that other features of macromolecules such as their surface charges, probably resulting in electrostatic repulsions, and steric hindrance, could play a key role in the stabilization of their linked proteins against aggregation. Such stabilizing mechanisms are expected to give new insights into our understanding of the chaperoning functions for de novo protein folding. In this review, we will discuss the possible chaperoning roles of these macromolecules in de novo folding, based on their charge and steric features.

  9. The use of a chaperone in obstetrical and gynaecological practice.

    LENUS (Irish Health Repository)

    Afaneh, I

    2010-05-01

    The aim of this study was to assess the use of a chaperone in obstetrical and gynaecological practice in Ireland and to explore patients\\' opinions. Two questionnaires were designed; one for patients and the other one was sent to 145 gynaecologists in Ireland. One hundred and fifty two women took part in this survey of whom 74 were gynaecological and 78 were obstetric patients. Ninety five (65%) patients felt no need for a chaperone during a vaginal examination (VE) by a male doctor. On the other hand 34 (23%) participating women would request a chaperone if being examined by a female doctor. Among clinicians 116 (80%) responded by returning the questionnaire. Overall 60 (52%) always used a chaperone in public practice, in contrast to 24 (27%) in private practice. The study demonstrated that most patients do not wish to have a chaperone during a VE but a small proportion would still request one regardless of the examiner\\'s gender. Patients should be offered the choice of having a chaperone and their opinion should be respected and documented.

  10. The use of a chaperone in obstetrical and gynaecological practice.

    LENUS (Irish Health Repository)

    Afaneh, I

    2012-02-01

    The aim of this study was to assess the use of a chaperone in obstetrical and gynaecological practice in Ireland and to explore patients\\' opinions. Two questionnaires were designed; one for patients and the other one was sent to 145 gynaecologists in Ireland. One hundred and fifty two women took part in this survey of whom 74 were gynaecological and 78 were obstetric patients. Ninety five (65%) patients felt no need for a chaperone during a vaginal examination (VE) by a male doctor. On the other hand 34 (23%) participating women would request a chaperone if being examined by a female doctor. Among clinicians 116 (80%) responded by returning the questionnaire. Overall 60 (52%) always used a chaperone in public practice, in contrast to 24 (27%) in private practice. The study demonstrated that most patients do not wish to have a chaperone during a VE but a small proportion would still request one regardless of the examiner\\'s gender. Patients should be offered the choice of having a chaperone and their opinion should be respected and documented.

  11. c-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Diana M. Dunn

    2015-08-01

    Full Text Available The ability of Heat Shock Protein 90 (Hsp90 to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1, promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo.

  12. Direct interplay among histones, histone chaperones, and a chromatin boundary protein in the control of histone gene expression.

    Science.gov (United States)

    Zunder, Rachel M; Rine, Jasper

    2012-11-01

    In Saccharomyces cerevisiae, the histone chaperone Rtt106 binds newly synthesized histone proteins and mediates their delivery into chromatin during transcription, replication, and silencing. Rtt106 is also recruited to histone gene regulatory regions by the HIR histone chaperone complex to ensure S-phase-specific expression. Here we showed that this Rtt106:HIR complex included Asf1 and histone proteins. Mutations in Rtt106 that reduced histone binding reduced Rtt106 enrichment at histone genes, leading to their increased transcription. Deletion of the chromatin boundary element Yta7 led to increased Rtt106:H3 binding, increased Rtt106 enrichment at histone gene regulatory regions, and decreased histone gene transcription at the HTA1-HTB1 locus. These results suggested a unique regulatory mechanism in which Rtt106 sensed the level of histone proteins to maintain the proper level of histone gene transcription. The role of these histone chaperones and Yta7 differed markedly among the histone gene loci, including the two H3-H4 histone gene pairs. Defects in silencing in rtt106 mutants could be partially accounted for by Rtt106-mediated changes in histone gene repression. These studies suggested that feedback mediated by histone chaperone complexes plays a pivotal role in regulating histone gene transcription.

  13. Heat shock protein 90: the cancer chaperone

    Indian Academy of Sciences (India)

    Len Neckers

    2007-04-01

    Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signalling proteins, as well as multiple mutated, chimeric, and/or over-expressed signalling proteins, that promote cancer cell growth and/or survival. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple cellular signalling pathways. By inhibiting nodal points in multiple overlapping survival pathways utilized by cancer cells, combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent. Hsp90 inhibitors may circumvent the characteristic genetic plasticity that has allowed cancer cells to eventually evade the toxic effects of most molecularly targeted agents. The mechanism-based use of Hsp90 inhibitors, both alone and in combination with other drugs, should be effective toward multiple forms of cancer. Further, because Hsp90 inhibitors also induce Hsf-1-dependent expression of Hsp70, and because certain mutated Hsp90 client proteins are neurotoxic, these drugs display ameliorative properties in several neurodegenerative disease models, suggesting a novel role for Hsp90 inhibitors in treating multiple pathologies involving neurodegeneration.

  14. Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding ▿

    OpenAIRE

    Jones, Christopher P.; Datta, Siddhartha A.K.; Rein, Alan; Rouzina, Ioulia; Musier-Forsyth, Karin

    2010-01-01

    Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA3Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA3Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid ...

  15. The G protein α chaperone Ric-8 as a potential therapeutic target.

    Science.gov (United States)

    Papasergi, Makaía M; Patel, Bharti R; Tall, Gregory G

    2015-01-01

    Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein α subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Gα-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein-coupled receptors (GPCRs). G protein βγ is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Gα protein-protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies. PMID:25319541

  16. Hsp70-Hsp40 chaperone complex functions in controlling polarized growth by repressing Hsf1-driven heat stress-associated transcription.

    Directory of Open Access Journals (Sweden)

    Aleksandar Vjestica

    Full Text Available How the molecular mechanisms of stress response are integrated at the cellular level remains obscure. Here we show that the cellular polarity machinery in the fission yeast Schizosaccharomyces pombe undergoes dynamic adaptation to thermal stress resulting in a period of decreased Cdc42 activity and altered, monopolar growth. Cells where the heat stress-associated transcription was genetically upregulated exhibit similar growth patterning in the absence of temperature insults. We identify the Ssa2-Mas5/Hsp70-Hsp40 chaperone complex as repressor of the heat shock transcription factor Hsf1. Cells lacking this chaperone activity constitutively activate the heat-stress-associated transcriptional program. Interestingly, they also exhibit intermittent monopolar growth within a physiological temperature range and are unable to adapt to heat stress. We propose that by negatively regulating the heat stress-associated transcription, the Ssa2-Mas5 chaperone system could optimize cellular growth under different temperature regiments.

  17. Chaperone-assisted translocation of flexible polymers in three dimensions

    CERN Document Server

    Suhonen, P M

    2016-01-01

    Polymer translocation through a nanometer-scale pore assisted by chaperones binding to the polymer is a process encountered in vivo for proteins. Studying the relevant models by computer simulations is computationally demanding. Accordingly, previous studies are either for stiff polymers in three dimensions or flexible polymers in two dimensions. Here, we study chaperone-assisted translocation of flexible polymers in three dimensions using Langevin dynamics. We show that differences in binding mechanisms, more specifically, whether a chaperone can bind to a single or multiple sites on the polymer, lead to substantial differences in translocation dynamics in three dimensions. We show that the single-binding mode leads to dynamics that is very much like that in the constant-force driven translocation and accordingly mainly determined by tension propagation on the cis side. We obtain $\\beta \\approx 1.26$ for the exponent for the scaling of the translocation time with polymer length. This fairly low value can be ...

  18. In vivo Study of the Histone Chaperone Activity of Nucleolin by FRAP

    Directory of Open Access Journals (Sweden)

    Xavier Gaume

    2011-01-01

    Full Text Available Nucleolin is a major nucleolar protein involved in various aspects of ribosome biogenesis such as regulation of polymerase I transcription, pre-RNA maturation, and ribosome assembly. Nucleolin is also present in the nucleoplasm suggesting that its functions are not restricted to nucleoli. Nucleolin possesses, in vitro, chromatin co-remodeler and histone chaperone activities which could explain numerous functions of nucleolin related to the regulation of gene expression. The goal of this report was to investigate the consequences of nucleolin depletion on the dynamics of histones in live cells. Changes in histone dynamics occurring in nucleolin silenced cells were measured by FRAP experiments on eGFP-tagged histones (H2B, H4, and macroH2A. We found that nuclear histone dynamics was impacted in nucleolin silenced cells; in particular we measured higher fluorescence recovery kinetics for macroH2A and H2B but not for H4. Interestingly, we showed that nucleolin depletion also impacted the dissociation constant rate of H2B and H4. Thus, in live cells, nucleolin could play a role in chromatin accessibility by its histone chaperone and co-remodeling activities.

  19. In vivo Study of the Histone Chaperone Activity of Nucleolin by FRAP.

    Science.gov (United States)

    Gaume, Xavier; Monier, Karine; Argoul, Françoise; Mongelard, Fabien; Bouvet, Philippe

    2011-01-01

    Nucleolin is a major nucleolar protein involved in various aspects of ribosome biogenesis such as regulation of polymerase I transcription, pre-RNA maturation, and ribosome assembly. Nucleolin is also present in the nucleoplasm suggesting that its functions are not restricted to nucleoli. Nucleolin possesses, in vitro, chromatin co-remodeler and histone chaperone activities which could explain numerous functions of nucleolin related to the regulation of gene expression. The goal of this report was to investigate the consequences of nucleolin depletion on the dynamics of histones in live cells. Changes in histone dynamics occurring in nucleolin silenced cells were measured by FRAP experiments on eGFP-tagged histones (H2B, H4, and macroH2A). We found that nuclear histone dynamics was impacted in nucleolin silenced cells; in particular we measured higher fluorescence recovery kinetics for macroH2A and H2B but not for H4. Interestingly, we showed that nucleolin depletion also impacted the dissociation constant rate of H2B and H4. Thus, in live cells, nucleolin could play a role in chromatin accessibility by its histone chaperone and co-remodeling activities. PMID:21403913

  20. Phosphorylation-mediated control of histone chaperone ASF1 levels by Tousled-like kinases.

    Directory of Open Access Journals (Sweden)

    Maxim Pilyugin

    Full Text Available Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases.

  1. Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

    Science.gov (United States)

    Chaudhury, Sukanya; Nordhues, Bryce A; Kaur, Kawaljit; Zhang, Na; De Guzman, Roberto N

    2015-11-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion.

  2. Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

    Energy Technology Data Exchange (ETDEWEB)

    Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H., E-mail: laurence.pearl@sussex.ac.uk; Prodromou, Chrisostomos, E-mail: laurence.pearl@sussex.ac.uk [University of Sussex, Falmer, Brighton BN1 9RQ (United Kingdom)

    2015-05-01

    A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90){sub 2}–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

  3. Coffee enhances the expression of chaperones and antioxidant proteins in rats with nonalcoholic fatty liver disease.

    Science.gov (United States)

    Salomone, Federico; Li Volti, Giovanni; Vitaglione, Paola; Morisco, Filomena; Fogliano, Vincenzo; Zappalà, Agata; Palmigiano, Angelo; Garozzo, Domenico; Caporaso, Nicola; D'Argenio, Giuseppe; Galvano, Fabio

    2014-06-01

    Coffee consumption is inversely related to the degree of liver injury in patients with nonalcoholic fatty liver disease (NAFLD). Molecular mediators contributing to coffee's beneficial effects in NAFLD remain to be elucidated. In this study, we administrated decaffeinated espresso coffee or vehicle to rats fed an high-fat diet (HFD) for 12 weeks and examined the effects of coffee on liver injury by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis combined with mass spectrometry. Rats fed an HFD and water developed panacinar steatosis, lobular inflammation, and mild fibrosis, whereas rats fed an HFD and coffee exhibited only mild steatosis. Coffee consumption increased liver expression of the endoplasmic reticulum chaperones glucose-related protein 78 and protein disulfide-isomerase A3; similarly, coffee drinking enhanced the expression of the mitochondrial chaperones heat stress protein 70 and DJ-1. Furthermore, in agreement with reduced hepatic levels of 8-isoprostanes and 8-hydroxy-2'-deoxyguanosine, proteomic analysis showed that coffee consumption induces the expression of master regulators of redox status (i.e., peroxiredoxin 1, glutathione S-transferase α2, and D-dopachrome tautomerase). Last, proteomics revealed an association of coffee intake with decreased expression of electron transfer flavoprotein subunit α, a component of the mitochondrial respiratory chain, involved in de novo lipogenesis. In this study, we were able to identify by proteomic analysis the stress proteins mediating the antioxidant effects of coffee; moreover, we establish for the first time the contribution of specific coffee-induced endoplasmic reticulum and mitochondrial chaperones ensuring correct protein folding and degradation in the liver. PMID:24365744

  4. Divergent tissue and sex effects of rapamycin on the proteasome-chaperone network of old mice

    Directory of Open Access Journals (Sweden)

    Karl Andrew Rodriguez

    2014-11-01

    Full Text Available Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24mg/kg (14 ppm rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS, heat shock factor 1 (HSF1, and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome.

  5. Hsp100/ClpB Chaperone Function and Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Vierling, Elizabeth [University of Massachusetts

    2015-01-27

    The supported research investigated the mechanism of action of a unique class of molecular chaperones in higher plants, the Hsp100/ClpB proteins, with the ultimate goal of defining how these chaperones influence plant growth, development, stress tolerance and productivity. Molecular chaperones are essential effectors of cellular “protein quality control”, which comprises processes that ensure the proper folding, localization, activation and turnover of proteins. Hsp100/ClpB proteins are required for temperature acclimation in plants, optimal seed yield, and proper chloroplast development. The model plant Arabidopsis thaliana and genetic and molecular approaches were used to investigate two of the three members of the Hsp100/ClpB proteins in plants, cytosolic AtHsp101 and chloroplast-localized AtClpB-p. Investigating the chaperone activity of the Hsp100/ClpB proteins addresses DOE goals in that this activity impacts how “plants generate and assemble components” as well as “allowing for their self repair”. Additionally, Hsp100/ClpB protein function in plants is directly required for optimal “utilization of biological energy” and is involved in “mechanisms that control the architecture of energy transduction systems”.

  6. Reconfiguration of the proteasome during chaperone-mediated assembly

    Science.gov (United States)

    Park, Soyeon; Li, Xueming; Kim, Ho Min; Singh, Chingakham Ranjit; Tian, Geng; Hoyt, Martin A.; Lovell, Scott; Battaile, Kevin P.; Zolkiewski, Michal; Coffino, Philip; Roelofs, Jeroen; Cheng, Yifan; Finley, Daniel

    2013-01-01

    The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt C-terminal tails inserting into pockets of the α ring1–4. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit5–10. We report that the base subassembly of the proteasome, which includes the Rpt ring, forms a high affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6, and Rpn14. Chaperone-mediated dissociation was abrogated by a nonhydrolyzable ATP analog, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3 pocket. Although the Rpt6 tail is not visualized within an α pocket in mature proteasomes2–4, it inserts into the α2/α3 pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme. PMID:23644457

  7. The co-chaperone p23 is degraded by caspases and the proteasome during apoptosis

    DEFF Research Database (Denmark)

    Mollerup, Jens; Berchtold, Martin Werner

    2005-01-01

    The heat shock protein 90 co-chaperone p23 has recently been shown to be up-regulated in cancer cells and down-regulated in atheroschlerotic plaques. We found that p23 is degraded during apoptosis induced by several stimuli, including Fas and TNFa-receptor activation as well as staurosporine...... treatment. Caspase inhibition protected p23 from degradation in several cell lines. In addition, recombinant caspase-3 and 8 cleaved p23 at Asp 142 generating a degradation product of 18 kDa as seen in apoptotic cells. Truncated p23 is further degraded in a proteasome dependent process during apoptosis....... Furthermore, we found that the anti-aggregating activity of truncated p23 was reduced compared to full length p23 indicating that caspase mediated p23 degradation contributes to protein destabilisation in apoptosis....

  8. Chaperone-interacting TPR proteins in Caenorhabditis elegans.

    Science.gov (United States)

    Haslbeck, Veronika; Eckl, Julia M; Kaiser, Christoph J O; Papsdorf, Katharina; Hessling, Martin; Richter, Klaus

    2013-08-23

    The ATP-hydrolyzing molecular chaperones Hsc70/Hsp70 and Hsp90 bind a diverse set of tetratricopeptide repeat (TPR)-containing cofactors via their C-terminal peptide motifs IEEVD and MEEVD. These cochaperones contribute to substrate turnover and confer specific activities to the chaperones. Higher eukaryotic genomes encode a large number of TPR-domain-containing proteins. The human proteome contains more than 200 TPR proteins, and that of Caenorhabditis elegans, about 80. It is unknown how many of them interact with Hsc70 or Hsp90. We systematically screened the C. elegans proteome for TPR-domain-containing proteins that likely interact with Hsc70 and Hsp90 and ranked them due to their similarity with known chaperone-interacting TPRs. We find C. elegans to encode many TPR proteins, which are not present in yeast. All of these have homologs in fruit fly or humans. Highly ranking uncharacterized open reading frames C33H5.8, C34B2.5 and ZK370.8 may encode weakly conserved homologs of the human proteins RPAP3, TTC1 and TOM70. C34B2.5 and ZK370.8 bind both Hsc70 and Hsp90 with low micromolar affinities. Mutation of amino acids involved in EEVD binding disrupts the interaction. In vivo, ZK370.8 is localized to mitochondria in tissues with known chaperone requirements, while C34B2.5 colocalizes with Hsc70 in intestinal cells. The highest-ranking open reading frame with non-conserved EEVD-interacting residues, F52H3.5, did not show any binding to Hsc70 or Hsp90, suggesting that only about 15 of the TPR-domain-containing proteins in C. elegans interact with chaperones, while the many others may have evolved to bind other ligands.

  9. Blocking the chaperone kinome pathway: Mechanistic insights into a novel dual inhibition approach for supra-additive suppression of malignant tumors

    International Nuclear Information System (INIS)

    Research highlights: → Withaferin A and 17-DMAG synergistically inhibit the Hsp90-Cdc37 chaperone pair. → Binding of WA to Cdc37 cleft suppresses its kinase binding activity. → 17-DMAG binding to the association complex results in H-bonds with 60% clustering. → The ligands' bound complex was found structurally and thermodynamically stable. -- Abstract: The chaperone Hsp90 is involved in regulating the stability and activation state of more than 200 'client' proteins and takes part in the cancer diseased states. The major clientele-protein kinases depend on Hsp90 for their proper folding and functioning. Cdc37, a kinase targeting co-chaperone of Hsp90, mediates the interactions between Hsp90 and protein kinases. Targeting of Cdc37 has the prospect of delivering predominantly kinase-selective molecular responses as compared to the current pharmacologic Hsp90 inhibitors. The present work reports a bio-computational study carried out with the aim of exploring the dual inhibition of Hsp90/Cdc37 chaperone/co-chaperone association complex by the naturally occurring drug candidates withaferin A and 17-DMAG along with their possible modes of action. Our molecular docking studies reveal that withaferin A in combination with 17-DMAG can act as potent chaperone system inhibitors. The structural and thermodynamic stability of the ligands' bound complex was also observed from molecular dynamics simulations in water. Our results suggest a novel tumor suppressive action mechanism of herbal ligands which can be looked forward for further clinical investigations for possible anticancer drug formulations.

  10. Chaperonopathies of senescence and the scrambling of interactions between the chaperoning and the immune systems.

    Science.gov (United States)

    Macario, Alberto J L; Cappello, Francesco; Zummo, Giovanni; Conway de Macario, Everly

    2010-06-01

    Aging entails progressive deterioration of molecules and supramolecular structures, including Hsp chaperones and their complexes, paralleled by functional decline. Recent research has changed our views on Hsp chaperones. They work inside and outside cells in many locations, alone or forming teams, interacting with cells, receptors, and molecules that are not chaperones, in roles that are not typically attributed to chaperones, such as protein folding. Hsp chaperones form a physiological system with a variety of functions and interactions with other systems, for example, the immune system. We propose that chaperone malfunctioning due to structural damage or gene dysregulation during aging has an impact on the immune system, creating the conditions for an overall malfunction of both systems. Pathological chaperones cannot interact with the immune system as normal ones do, and this leads to an overall readjustment of the interactions that is apparent during senescence and is likely to cause many of its manifestations.

  11. Hsp90 as a "Chaperone" of the Epigenome: Insights and Opportunities for Cancer Therapy.

    Science.gov (United States)

    Isaacs, Jennifer S

    2016-01-01

    The cellular functions of Hsp90 have historically been attributed to its ability to chaperone client proteins involved in signal transduction. Although numerous stimuli and the signaling cascades they activate contribute to cancer progression, many of these pathways ultimately require transcriptional effectors to elicit tumor-promoting effects. Despite this obvious connection, the majority of studies evaluating Hsp90 function in malignancy have focused upon its regulation of cytosolic client proteins, and particularly members of receptor and/or kinase families. However, in recent years, Hsp90 has emerged as a pivotal orchestrator of nuclear events. Discovery of an expanding repertoire of Hsp90 clients has illuminated a vital role for Hsp90 in overseeing nuclear events and influencing gene transcription. Hence, this chapter will cast a spotlight upon several regulatory themes involving Hsp90-dependent nuclear functions. Highlighted topics include a summary of chaperone-dependent regulation of key transcription factors (TFs) and epigenetic effectors in malignancy, as well as a discussion of how the complex interplay among a subset of these TFs and epigenetic regulators may generate feed-forward loops that further support cancer progression. This chapter will also highlight less recognized indirect mechanisms whereby Hsp90-supported signaling may impinge upon epigenetic regulation. Finally, the relevance of these nuclear events is discussed within the framework of Hsp90's capacity to enable phenotypic variation and drug resistance. These newly acquired insights expanding our understanding of Hsp90 function support the collective notion that nuclear clients are major beneficiaries of Hsp90 action, and their impairment is likely responsible for many of the anticancer effects elicited by Hsp90-targeted approaches.

  12. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

    Science.gov (United States)

    Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

    2012-11-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented. PMID:22728817

  13. Transporters, chaperones, and P-type ATPases controlling grapevine copper homeostasis.

    Science.gov (United States)

    Leng, Xiangpeng; Mu, Qian; Wang, Xiaomin; Li, Xiaopeng; Zhu, Xudong; Shangguan, Lingfei; Fang, Jinggui

    2015-11-01

    With more copper and copper-containing compounds used as bactericides and fungicides in viticulture, copper homeostasis in grapevine (Vitis) has become one of the serious environmental crises with great risk. To better understand the regulation of Cu homeostasis in grapevine, grapevine seedlings cultured in vitro with different levels of Cu were utilized to investigate the tolerance mechanisms of grapevine responding to copper availability at physiological and molecular levels. The results indicated that Cu contents in roots and leaves arose with increasing levels of Cu application. With copper concentration increasing, malondialdehyde (MDA) content increased in roots and leaves and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased to protect the plant itself from damage. The expression patterns of 19 genes, encoding transporters, chaperones, and P-type ATPases involved in copper homeostasis in root and leaf of grapevine seedling under various levels of Cu(2+) were further analyzed. The expression patterns indicated that CTr1, CTr2, and CTr8 transporters were significantly upregulated in response both to Cu excess and deficiency. ZIP2 was downregulated in response to Cu excess and upregulated under Cu-deficient conditions, while ZIP4 had an opposite expression pattern under similar conditions. The expression of chaperones and P-type ATPases in response to Cu availability in grapevine were also briefly studied.

  14. New concept in nutrition for the maintenance of the aging eye redox regulation and therapeutic treatment of cataract disease; synergism of natural antioxidant imidazole-containing amino acid-based compounds, chaperone, and glutathione boosting agents: a systemic perspective on aging and longevity emerged from studies in humans.

    Science.gov (United States)

    Babizhayev, Mark A

    2010-01-01

    Cataract, opacification of the lens, is one of the commonest causes of loss of useful vision during aging, with an estimated 16 million people world-wide affected. The role of nutritional supplementation in prevention of onset or progression of ocular disease is of interest to health care professionals and patients. The aging eye seems to be at considerable risk from oxidative stress. This review outlines the potential role of the new nutritional strategy on redox balance in age-related eye diseases and detail how the synergism and interaction of imidazole-containing amino acid-based compounds (nonhydrolized L-carnosine, histidine), chaperone agents (such as, L-carnosine, D-pantethine), glutathione-boosting agents (N-acetylcysteine, vitamin E, methionine), and N-acetylcarnosine eye drops plays key roles in the function and maintenance of the redox systems in the aging eye and in the treatment of human cataract disease. A novel patented oral health supplement is presented which enhances the anticataract activity of eye drops and activates functional visual acuity. The clinical data demonstrate the effectiveness and safety of a combined oral health care treatment with amino acids possessing chaperone-like activity with N-acetylcarnosine lubricant eye drops. L-carnosine and N-acetylcarnosine protected the chaperone activity of alpha-crystallin and reduced the increased posttranslational modifications of lens proteins. Biological activities of the nonhydrolyzed carnosine in the oral formulation are based on its antioxidant and antiglycating (transglycating) action that, in addition to heavy metal chelation and pH-buffering ability, makes carnosine an essential factor for preventing sight-threatening eye disorders having oxidative stress in their pathogenesis, neurodegeneration, and accumulation of senile features. The findings suggest that synergism is required between carnosine or other imidazole-containing compounds and reduced glutathione in tissues and cells for

  15. The crystal structure of the human co-chaperone P58(IPK.

    Directory of Open Access Journals (Sweden)

    Maria Svärd

    Full Text Available P58(IPK is one of the endoplasmic reticulum- (ER- localised DnaJ (ERdj proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58(IPK, expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58(IPK, and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58(IPK to 3.0 Å resolution using a combination of molecular replacement and single wavelength anomalous diffraction. The structure shows the human P58(IPK monomer to have a very elongated overall shape. In addition to the conserved J domain, P58(IPK contains nine N-terminal tetratricopeptide repeat motifs, divided into three subdomains of three motifs each. The J domain is attached to the C-terminal end via a flexible linker, and the structure shows the conserved Hsp70-binding histidine-proline-aspartate (HPD motif to be situated on the very edge of the elongated protein, 100 Å from the putative binding site for unfolded protein substrates. The residues that comprise the surface surrounding the HPD motif are highly conserved in P58(IPK from other organisms but more varied between the human ERdj proteins, supporting the view that their regulation of different BiP functions is facilitated by differences in BiP-binding.

  16. Metal chaperones: a holistic approach to the treatment of AD

    Directory of Open Access Journals (Sweden)

    Paul Anthony Adlard

    2012-03-01

    Full Text Available As the burden of proof for the role of metal ion dysregulation in the pathogenesis of multiple CNS disorders grows, it has become important to more precisely identify and differentiate the biological effects of various pharmacological modulators of metal ion homeostasis. This is particularly evident in disorders such as Alzheimer’s disease, where the use of metal chaperones (that transport metals, as opposed to chelators (which exclude metals from biological interactions, may prove to be the first truly disease modifying approach for this condition. The purpose of this mini-review is to highlight the emerging notion that metal chaperones, such as PBT2 (Prana Biotechnology, modulate a variety of critical pathways affecting key aspects of the AD cascade to provide a more holistic approach to the treatment of this disease.

  17. Crystal Structures of Cisplatin Bound to a Human Copper Chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Boal, Amie K.; Rosenzweig, Amy C.; (NWU)

    2010-08-16

    Copper trafficking proteins, including the chaperone Atox1 and the P{sub 1B}-type ATPase ATP7B, have been implicated in cellular resistance to the anticancer drug cisplatin. We have determined two crystal structures of cisplatin-Atox1 adducts that reveal platinum coordination by the conserved CXXC copper-binding motif. Direct interaction of cisplatin with this functionally relevant site has significant implications for understanding the molecular basis for resistance mediated by copper transport pathways.

  18. Site-directed mutations in the C-terminal extension of human alphaB-crystallin affect chaperone function and block amyloid fibril formation.

    Directory of Open Access Journals (Sweden)

    Teresa M Treweek

    Full Text Available BACKGROUND: Alzheimer's, Parkinson's and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including alphaB-crystallin, play a role in the prevention of protein deposition. METHODOLOGY/PRINCIPAL FINDINGS: A series of site-directed mutants of the human molecular chaperone, alphaB-crystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of alphaB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of alphaB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein. CONCLUSIONS/SIGNIFICANCE: Together, our results highlight the important role of the C-terminal region of alphaB-crystallin in regulating its secondary, tertiary and quaternary structure and conferring thermostability to the protein. The capacity to genetically modify alphaB-crystallin for improved ability to block amyloid fibril formation provides a platform for the future use of such engineered molecules in treatment of diseases caused by amyloid fibril formation.

  19. A novel C-terminal homologue of Aha1 co-chaperone binds to heat shock protein 90 and stimulates its ATPase activity in Entamoeba histolytica.

    Science.gov (United States)

    Singh, Meetali; Shah, Varun; Tatu, Utpal

    2014-04-17

    Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with Kd values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12×10(-4) min(-1) μM(-1). Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.

  20. The Role of Copper Chaperone Atox1 in Coupling Redox Homeostasis to Intracellular Copper Distribution

    Science.gov (United States)

    Hatori, Yuta; Lutsenko, Svetlana

    2016-01-01

    Human antioxidant protein 1 (Atox1) is a small cytosolic protein with an essential role in copper homeostasis. Atox1 functions as a copper carrier facilitating copper transfer to the secretory pathway. This process is required for activation of copper dependent enzymes involved in neurotransmitter biosynthesis, iron efflux, neovascularization, wound healing, and regulation of blood pressure. Recently, new cellular roles for Atox1 have emerged. Changing levels of Atox1 were shown to modulate response to cancer therapies, contribute to inflammatory response, and protect cells against various oxidative stresses. It has also become apparent that the activity of Atox1 is tightly linked to the cellular redox status. In this review, we summarize biochemical information related to a dual role of Atox1 as a copper chaperone and an antioxidant. We discuss how these two activities could be linked and contribute to establishing the intracellular copper balance and functional identity of cells during differentiation. PMID:27472369

  1. Zinc-L-carnosine binds to molecular chaperone HSP70 and inhibits the chaperone activity of the protein.

    Science.gov (United States)

    Haga, Asami; Okamoto, Tomoya; Yamada, Shintaroh; Kubota, Toshihiko; Sanpei, Ann; Takahashi, Shota; Nakayama, Masahiro; Nagai, Miki; Otaka, Michiro; Miyazaki, Toshio; Nunomura, Wataru; Grave, Ewa; Itoh, Hideaki

    2013-09-01

    In this study, we have investigated the specific binding proteins of Zinc-L-carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro. A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0-1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70. PMID:23687308

  2. Protein refolding in peroxisomes is dependent upon an HSF1-regulated function

    NARCIS (Netherlands)

    Heldens, Lonneke; van Genesen, Siebe T.; Hanssen, Lars L. P.; Hageman, Jurre; Kampinga, Harm H.; Lubsen, Nicolette H.

    2012-01-01

    Post-heat shock refolding of luciferase requires chaperones. Expression of a dominant negative HSF1 mutant (dnHSF1), which among other effects depletes cells of HSF1-regulated chaperones, blocked post-heat shock refolding of luciferase targeted to the cytoplasm, nucleus, or peroxisomes, while refold

  3. HSP-molecular chaperones in cancer biogenesis and tumor therapy: an overview.

    Science.gov (United States)

    Rappa, Francesca; Farina, Felicia; Zummo, Giovanni; David, Sabrina; Campanella, Claudia; Carini, Francesco; Tomasello, Giovanni; Damiani, Provvidenza; Cappello, Francesco; DE Macario, Everly Conway; Macario, Alberto J L

    2012-12-01

    Molecular chaperones, many of which are heat-shock proteins (HSPs), are an important class of molecules with various functions. Pathological conditions in which chaperones become etiological and/or pathogenic factors are called chaperonopathies, and are classified into by defect, by excess, and by 'mistake'. In the latter case, the chaperone is structurally and functionally normal but participates in pathways that favor disease, although in some cases the chaperone may have post-translational modifications that may lead it to change its location and function and, thus, to become pathogenic. For example, HSP-chaperones are involved in carcinogenesis in various ways, so that some forms of cancer may be considered 'chaperonopathies by mistake'. This concept suggests new strategies for anticancer therapy (chaperonotherapy), in which the primary targets or therapeutic agents are chaperones. Chaperonotherapy consists of the utilization of HSP-chaperones for treating chaperonopathies, including cancer. Negative chaperonotherapy is aimed at eliminating or blocking the action of chaperones that favor carcinogenesis or other diseases, whereas positive chaperonotherapy uses chaperones, genes or proteins, to fight against diseases, such as cancer, by stimulating the immune system or the cellular defenses against stress.

  4. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    Directory of Open Access Journals (Sweden)

    Gennady Verkhivker

    2013-11-01

    Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

  5. Modulation of Chaperone Gene Expression in Mutagenized Saccharomyces cerevisiae Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins▿

    OpenAIRE

    Payne, T.; Finnis, C.; Evans, L R; Mead, D. J.; Avery, S V; Archer, D. B.; Sleep, D.

    2008-01-01

    The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regul...

  6. Molecular characterization of two novel molecular chaperones in bacterial-challenged Apostichopus japonicus.

    Science.gov (United States)

    Wang, Haihong; Shao, Yina; Zhang, Weiwei; Li, Chenghua; Lv, Zhimeng; Jin, Chunhua

    2015-10-01

    Molecular chaperones of 78 kDa glucose-regulated protein (GRP78) and protein disulfide isomerase (PDI) are involved in protein folding and assembly in the endoplasmic reticulum (ER). Increasing evidences also suggest that these two molecules play an important role in immune response. In the present study, we cloned and characterized GRP78 and PDI genes from Apostichopus japonicus by RNA-seq and RACE approaches (designated as AjGRP78 and AjPDI, respectively). The AjGRP78 cDNA was of 2355bp including an open reading frame (ORF) of 2013 bp encoding a protein of 670 amino acids with three heat shock protein 70 (HSP70) family signatures. AjGRP78 contained a 23-amino acid signal peptide at the N-terminus and a HDEL motif at the C-terminus, which supported the location of the protein in the ER. The full length cDNA of AjPDI was of 1893 bp with a 5' untranslated region (UTR) of 153 bp, a 3' UTR of 228 bp and an ORF of 1512 bp encoding a protein of 503 amino acids. A 17-amino acid signal peptide, two thioredoxin domains with two active sites of CGHC, and KDEL retention signal were totally conserved in the deduced amino acid of AjPDI. Phylogenic analysis and multiple alignments have shown that both genes shared remarkably higher degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates, further supporting that the two proteins were novel members of molecular chaperone family. Spatial expression analysis revealed that AjGRP78 mRNA transcripts were dominantly expressed in the tentacle, while AjPDI mRNA levels were abundant in the muscle, intestine and respiratory trees. For Vibrio splendidus challenged sea cucumber, the peak expression of AjGRP78 and AjPDI mRNAs in coelomocytes were detected at 24h with 1.73-fold increase and at 6h with 1.83-fold increase compared with the control group, respectively. Similarly, a significant increase in the relative mRNA levels of AjGRP78 and AjPDI was also identified in 1 μg mL(-1

  7. Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity

    NARCIS (Netherlands)

    Eerde, André van; Hamiaux, Cyril; Pérez, Javier; Parsot, Claude; Dijkstra, Bauke W.

    2004-01-01

    Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15 f

  8. Natural products triptolide, celastrol, and withaferin A inhibit the chaperone activity of peroxiredoxin i

    NARCIS (Netherlands)

    Zhao, Qian; Ding, Yu; Deng, Zhangshuang; Lee, On Yi; Gao, Peng; Chen, Pin; Rose, Rebecca J.; Zhao, Hong; Zhang, Zhehao; Tao, Xin Pei; Heck, Albert J R; Kao, Richard; Yang, Dan

    2015-01-01

    Peroxiredoxin I (Prx I) plays an important role in cancer development and inflammation. It is a dual-functional protein which acts as both an antioxidant enzyme and a molecular chaperone. While there have been intensive studies on its peroxidase activity, Prx I's chaperone activity remains elusive,

  9. A Surveillance Function of the HSPB8-BAG3-HSP70 Chaperone Complex Ensures Stress Granule Integrity and Dynamism.

    Science.gov (United States)

    Ganassi, Massimo; Mateju, Daniel; Bigi, Ilaria; Mediani, Laura; Poser, Ina; Lee, Hyun O; Seguin, Samuel J; Morelli, Federica F; Vinet, Jonathan; Leo, Giuseppina; Pansarasa, Orietta; Cereda, Cristina; Poletti, Angelo; Alberti, Simon; Carra, Serena

    2016-09-01

    Stress granules (SGs) are ribonucleoprotein complexes induced by stress. They sequester mRNAs and disassemble when the stress subsides, allowing translation restoration. In amyotrophic lateral sclerosis (ALS), aberrant SGs cannot disassemble and therefore accumulate and are degraded by autophagy. However, the molecular events causing aberrant SG formation and the molecular players regulating this transition are largely unknown. We report that defective ribosomal products (DRiPs) accumulate in SGs and promote a transition into an aberrant state that renders SGs resistant to RNase. We show that only a minor fraction of aberrant SGs is targeted by autophagy, whereas the majority disassembles in a process that requires assistance by the HSPB8-BAG3-HSP70 chaperone complex. We further demonstrate that HSPB8-BAG3-HSP70 ensures the functionality of SGs and restores proteostasis by targeting DRiPs for degradation. We propose a system of chaperone-mediated SG surveillance, or granulostasis, which regulates SG composition and dynamics and thus may play an important role in ALS. PMID:27570075

  10. Molecular chaperone activity and biological regulatory actions of the TPR-domain immunophilins FKBP51 and FKBP52.

    Science.gov (United States)

    Erlejman, Alejandra G; Lagadari, Mariana; Harris, Diondra C; Cox, Marc B; Galigniana, Mario D

    2014-05-01

    Immunophilins comprise a family of intracellular proteins with peptidyl-prolyl-(cis/trans)-isomerase activity. These foldases are abundant, ubiquitous, and able to bind immunosuppressant drugs, from which the term immunophilin derives. Family members are found in abundance in virtually all organisms and subcellular compartments, and their amino acid sequences are conserved phylogenetically. Immunophilins possess the ability to function as molecular chaperones favoring the proper folding and biological regulation of their biological actions. Their ability to interact via their TPR domains with the 90-kDa heat-shock protein, and through this chaperone, with several signalling cascade factors is of particular importance. Among the family members, the highly homologous proteins FKBP51 and FKBP52 were first characterized due to their ability to interact with steroid hormone receptors. Since then, much progress has been made in understanding the mechanisms by which they regulate receptor signaling and the resulting roles they play not only in endocrine processes, but also in cell architecture, neurodifferentiation, and tumor progression. In this article we review the most relevant features of these two immunophilins and their potential as pharmacologic targets.

  11. Purification, crystallization and preliminary X-ray analysis of FliT, a bacterial flagellar substrate-specific export chaperone

    International Nuclear Information System (INIS)

    FliT is a cytoplasmic flagellar type III substrate-specific export chaperone; it has been expressed, purified and crystallized and the crystals have been characterized by X-ray diffraction. The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD4C2 complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3121 or P3221 and diffracted to 3.2 Å resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination

  12. Copper transporters and chaperones: Their function on angiogenesis and cellular signalling

    Indian Academy of Sciences (India)

    SR BHARATHI DEVI; DHIVYA M ALOYSIUS; KN SULOCHANA

    2016-09-01

    Copper, although known as a micronutrient, has a pivotal role in modulating the cellular metabolism. Many studieshave reported the role of copper in angiogenesis. Copper chaperones are intracellular proteins that mediate coppertrafficking to various cell organelles. However, the role and function of copper chaperones in relation to angiogenesishas to be further explored. The intracellular copper levels when in excess are deleterious and certain mutations ofcopper chaperones have been shown to induce cell death and influence various cellular metabolisms. The study ofthese chaperones will be helpful in understanding the players in the cascade of events in angiogenesis and their role incellular metabolic pathways. In this review we have briefly listed the copper chaperones associated with angiogenicand metabolic signalling and their function.

  13. Experimental Milestones in the Discovery of Molecular Chaperones as Polypeptide Unfolding Enzymes.

    Science.gov (United States)

    Finka, Andrija; Mattoo, Rayees U H; Goloubinoff, Pierre

    2016-06-01

    Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers.

  14. Small intestinal mucosa expression of putative chaperone fls485

    Directory of Open Access Journals (Sweden)

    Raupach Kerstin

    2010-03-01

    Full Text Available Abstract Background Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa. Methods fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies. Results fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c. Conclusions Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.

  15. Solution structure of histone chaperone ANP32B: interaction with core histones H3-H4 through its acidic concave domain.

    Science.gov (United States)

    Tochio, Naoya; Umehara, Takashi; Munemasa, Yoshiko; Suzuki, Toru; Sato, Shin; Tsuda, Kengo; Koshiba, Seizo; Kigawa, Takanori; Nagai, Ryozo; Yokoyama, Shigeyuki

    2010-08-01

    Eukaryotic gene expression is regulated by histone deposition onto and eviction from nucleosomes, which are mediated by several chromatin-modulating factors. Among them, histone chaperones are key factors that facilitate nucleosome assembly. Acidic nuclear phosphoprotein 32B (ANP32B) belongs to the ANP32 family, which shares N-terminal leucine-rich repeats (LRRs) and a C-terminal variable anionic region. The C-terminal region functions as an inhibitor of histone acetylation, but the functional roles of the LRR domain in chromatin regulation have remained elusive. Here, we report that the LRR domain of ANP32B possesses histone chaperone activity and forms a curved structure with a parallel beta-sheet on the concave side and mostly helical elements on the convex side. Our analyses revealed that the interaction of ANP32B with the core histones H3-H4 occurs on its concave side, and both the acidic and hydrophobic residues that compose the concave surface are critical for histone binding. These results provide a structural framework for understanding the functional mechanisms of acidic histone chaperones.

  16. The Deinococcus radiodurans DR1245 protein, a DdrB partner homologous to YbjN proteins and reminiscent of type III secretion system chaperones.

    Directory of Open Access Journals (Sweden)

    Cédric Norais

    Full Text Available The bacterium Deinococcus radiodurans exhibits an extreme resistance to ionizing radiation. A small subset of Deinococcus genus-specific genes were shown to be up-regulated upon exposure to ionizing radiation and to play a role in genome reconstitution. These genes include an SSB-like protein called DdrB. Here, we identified a novel protein encoded by the dr1245 gene as an interacting partner of DdrB. A strain devoid of the DR1245 protein is impaired in growth, exhibiting a generation time approximately threefold that of the wild type strain while radioresistance is not affected. We determined the three-dimensional structure of DR1245, revealing a relationship with type III secretion system chaperones and YbjN family proteins. Thus, DR1245 may display some chaperone activity towards DdrB and possibly other substrates.

  17. The Deinococcus radiodurans DR1245 Protein, a DdrB Partner Homologous to YbjN Proteins and Reminiscent of Type III Secretion System Chaperones

    Energy Technology Data Exchange (ETDEWEB)

    Norais, Cédric [Univ. of Wisconsin, Madison, WI (United States); Ecole Polytechnic, Paaiseau (France); Servant, Pascale [Univ. of Paris-Sud, Orsay (France); National Center of Scientific Research, Orsay (France); Bouthier-de-la-Tour, Claire [Univ. of Paris-Sud, Orsay (France); National Center of Scientific Research, Orsay (France); Coureux, Pierre-Damien [Ecole Polytechnic, Paaiseau (France); Ithurbide, Solenne [Univ. of Paris-Sud, Orsay (France); National Center of Scientific Research, Orsay (France); Vannier, Françoise [Univ. of Paris-Sud, Orsay (France); National Center of Scientific Research, Orsay (France); Guerin, Philippe P. [French Alternatives Energy Commission (France); Dulberger, Charles L. [Univ. of Wisconsin, Madison, WI (United States); Satyshur, Kenneth A. [Univ. of Wisconsin, Madison, WI (United States); Keck, James L. [Univ. of Wisconsin, Madison, WI (United States); Armengaud, Jean [French Alternatives Energy Commission (France); Cox, Michael M. [Univ. of Wisconsin, Madison, WI (United States); Sommer, Suzanne [Univ. of Paris-Sud, Orsay (France); National Center of Scientific Research, Orsay (France)

    2013-02-18

    The bacterium Deinococcus radiodurans exhibits an extreme resistance to ionizing radiation. A small subset of Deinococcus genus-specific genes were shown to be up-regulated upon exposure to ionizing radiation and to play a role in genome reconstitution. These genes include an SSB-like protein called DdrB. Here, we identified a novel protein encoded by the dr1245gene as an interacting partner of DdrB. A strain devoid of the DR1245 protein is impaired in growth, exhibiting a generation time approximately threefold that of the wild type strain while radioresistance is not affected. We determined the three-dimensional structure of DR1245, revealing a relationship with type III secretion system chaperones and YbjN family proteins. Thus, DR1245 may display some chaperone activity towards DdrB and possibly other substrates.

  18. Quercetin mediated reduction of angiogenic markers and chaperones in DLA-induced solid tumours.

    Science.gov (United States)

    Anand, Kushi; Asthana, Pallavi; Kumar, Anup; Ambasta, Rashmi K; Kumar, Pravir

    2011-01-01

    Diet-derived flavonoids, in particular quercetin, may play advantageous roles by preventing or/and inhibiting oncogenesis. Evidence suggests that quercetin can elicit various properties depending on the cell type. The aim of this study was to evaluate its effects on Dalton's lymphoma ascites (DLA) induced solid tumours and to identify the target(s) of action. We addressed this question by inducing subcutaneous solid tumours in Swiss albino mice and investigated whether the quercetin affects essential biological processes that are responsible for tumour growth, morphology, angiogenesis and apoptosis. We also studied influence on several heat shock proteins (HSPs). Our findings demonstrate that intra-tumour administration of quercetin results in decreased volume/weight. Furthermore, we demonstrate that quercetin promotes apoptosis of cancer cells by down-regulating the levels of Hsp90 and Hsp70. Depletion of these two chaperones by quercetin might result in triggering of caspase-3 in treated tumours. Moreover, it also down-regulated the expression of major key angiogenic or pro-angiogenic factors, like HIF-1α and VEGF In addition, H and E staining together with immunofluorescence of fixed tumour tissue provided evidence in support of increased cell death in quercetin-treated mice. PMID:22393949

  19. Reversible thermal unfolding of a yfdX protein with chaperone-like activity

    Science.gov (United States)

    Saha, Paramita; Manna, Camelia; Chakrabarti, Jaydeb; Ghosh, Mahua

    2016-01-01

    yfdX proteins are ubiquitously present in a large number of virulent bacteria. A member of this family of protein in E. coli is known to be up-regulated by the multidrug response regulator. Their abundance in such bacteria suggests some important yet unidentified functional role of this protein. Here, we study the thermal response and stability of yfdX protein STY3178 from Salmonella Typhi using circular dichroism, steady state fluorescence, dynamic light scattering and nuclear magnetic resonance experiments. We observe the protein to be stable up to a temperature of 45 °C. It folds back to the native conformation from unfolded state at temperature as high as 80 °C. The kinetic measurements of unfolding and refolding show Arrhenius behavior where the refolding involves less activation energy barrier than that of unfolding. We propose a homology model to understand the stability of the protein. Our molecular dynamic simulation studies on this model structure at high temperature show that the structure of this protein is quite stable. Finally, we report a possible functional role of this protein as a chaperone, capable of preventing DTT induced aggregation of insulin. Our studies will have broader implication in understanding the role of yfdX proteins in bacterial function and virulence. PMID:27404435

  20. Transcriptional activation of endoplasmic reticulum chaperone GRP78 by HCMV IE1-72 protein

    Institute of Scientific and Technical Information of China (English)

    Derick Shi-Chen Ou; Sung-Bau Lee; Chi-Shuen Chu; Liang-Hao Chang; Bon-chu Chung; Li-Jung Juan

    2011-01-01

    Glucose-regulated protein 78 (GRP78), a key regulator of endoplasmic reticulum (ER) stress, facilitates cancer cell growth and viral replication. The mechanism leading to grp78 gene activation during viral infection is largely unknown, in this study, we show that the immediate-early 1 (IE1-72) protein of the human cytomegalovirus (HCMV) is essential for HCMV-mediated GRP78 activation. IE1-72 upregulated grp 78 gene expression depending on the ATPbinding site, the zinc-finger domain and the putative leucine-zipper motif of IE1-72, as well as the ER stress response elements (ERSEs) on the grp78 promoter. The purified IE1-72 protein bound to the CCAAT box within ERSE in vitro, whereas deletion mutants of IE1-72 deficient in grp78 promoter stimulation failed to do so. Moreover, IE1-72 binding to the grp78 promoter in infected cells accompanied the recruitment of TATA box-binding protein-associated factor 1 (TAF1), a histone acetyltransferase, and the increased level of acetylated histone H4, an indicator of activestate chromatin. These results provide evidence that HCMV IE1-72 activates grp78 gene expression through direct promoter binding and modulation of the local chromatin structure, indicating an active viral mechanism of cellular chaperone induction for viral growth.

  1. In silico identification of carboxylate clamp type tetratricopeptide repeat proteins in Arabidopsis and rice as putative co-chaperones of Hsp90/Hsp70.

    Directory of Open Access Journals (Sweden)

    Bishun D Prasad

    Full Text Available The essential eukaryotic molecular chaperone Hsp90 operates with the help of different co-chaperones, which regulate its ATPase activity and serve as adaptors to recruit client proteins and other molecular chaperones, such as Hsp70, to the Hsp90 complex. Several Hsp90 and Hsp70 co-chaperones contain the tetratricopeptide repeat (TPR domain, which interacts with the highly conserved EEVD motif at the C-terminal ends of Hsp90 and Hsp70. The acidic side chains in EEVD interact with a subset of basic residues in the TPR binding pocket called a 'carboxylate clamp'. Since the carboxylate clamp residues are conserved in the TPR domains of known Hsp90/Hsp70 co-chaperones, we carried out an in silico search for TPR proteins in Arabidopsis and rice comprising of at least one three-motif TPR domain with conserved amino acid residues required for Hsp90/Hsp70 binding. This approach identified in Arabidopsis a total of 36 carboxylate clamp (CC-TPR proteins, including 24 novel proteins, with potential to interact with Hsp90/Hsp70. The newly identified CC-TPR proteins in Arabidopsis and rice contain additional protein domains such as ankyrin, SET, octicosapeptide/Phox/Bem1p (Phox/PB1, DnaJ-like, thioredoxin, FBD and F-box, and protein kinase and U-box, indicating varied functions for these proteins. To provide proof-of-concept of the newly identified CC-TPR proteins for interaction with Hsp90, we demonstrated interaction of AtTPR1 and AtTPR2 with AtHsp90 in yeast two-hybrid and in vitro pull down assays. These findings indicate that the in silico approach used here successfully identified in a genome-wide context CC-TPR proteins with potential to interact with Hsp90/Hsp70, and further suggest that the Hsp90/Hsp70 system relies on TPR co-chaperones more than it was previously realized.

  2. Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18

    Science.gov (United States)

    Nandi, Sandip Kumar; Panda, Alok Kumar; Chakraborty, Ayon; Ray, Sougata Sinha; Biswas, Ashis

    2015-01-01

    Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31–43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25–43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min-1. Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18. PMID:26098662

  3. The nucleotide exchange factors of Hsp70 molecular chaperone

    Directory of Open Access Journals (Sweden)

    Andreas eBracher

    2015-04-01

    Full Text Available Molecular chaperones of the Hsp70 family form an important hub in the cellular protein folding networks in bacteria and eukaryotes, connecting translation with the downstream machineries of protein folding and degradation. The Hsp70 folding cycle is driven by two types of cochaperones: J-domain proteins stimulate ATP hydrolysis by Hsp70, while nucleotide exchange factors (NEFs promote replacement of Hsp70-bound ADP with ATP. Bacteria and organelles of bacterial origin have only one known NEF type for Hsp70, GrpE. In contrast, a large diversity of Hsp70 NEFs has been discovered in the eukaryotic cell. These NEFs belong to the Hsp110/Grp170, HspBP1/Sil1 and BAG domain protein families. In this short review we compare the structures and molecular mechanisms of nucleotide exchange factors for Hsp70 and discuss how these cochaperones contribute to protein folding and quality control in the cell.

  4. Nicotine inhibits histone deacetylase 6 activity and chaperone-dependent activation of the glucocorticoid receptor in A549 cells

    Institute of Scientific and Technical Information of China (English)

    SUN Li-chao; LIN Jiang-tao; LI Wen; ZHANG Lan; ZHOU Tong-liang; ZHANG Xiao-yan

    2012-01-01

    Background Nicotine,a major component of tobacco,is the main cause of smoking addiction.It was found that asthmatic patients who smoke were insensitive to glucocorticoid treatment.In this paper,we investigated whether nicotine could inhibit histone deacetylase 6 activity (HDAC6) and chaperone-dependent activation of the glucocorticoid receptor (GR) in A549 cells.Furthermore,the expression level of heat shock protein 90 (HSP90) was determined.Methods Quantitative real-time polymerase chain reaction was used to detect the levels of RNA transcription,and Western blotting was applied to analyze the levels of protein expression of HDAC6,GR,and HSP90 in A549 cells.Moreover,the effects of dexamethasone and trichostatin A were observed in A549 cells.Results A549 cell proliferation was inhibited in the presence of nicotine,and the level of RNA and protein expression of HDAC6 and GR were down-regulated.Conclusions Nicotine could inhibit HDAC6 activity and chaperone-dependent activation of GR.This might be the main reason why asthmatic patients who smoke show insensitivity to the glucocorticoid treatment.

  5. Estetrol, molecular chaperones, and the epigenetics of longevity and cancer resistance.

    Science.gov (United States)

    Krøll, Jens

    2014-04-01

    Evidence is given that replicative senescence--possibly as organismal aging--constitutes epigenetic phenomena, counteracted by homeostatic factors such as, e.g., the molecular chaperones, which are housekeeping molecules essential for the folding, repair, and transport of proteins, RNA, and DNA. Weakening of the chaperone defense with age probably contributes to the frailty in senescence. The present review presents evidence that the human fetal estrogen hormone estetrol, by promotion of chaperone functions, homeostasis, and cancer resistance, may prove useful as a supplement during human senescence. PMID:23992378

  6. The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats.

    Science.gov (United States)

    Bowman, Andrew; Lercher, Lukas; Singh, Hari R; Zinne, Daria; Timinszky, Gyula; Carlomagno, Teresa; Ladurner, Andreas G

    2016-04-20

    Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from(1)H-(15)N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell.

  7. Universal Stress Protein exhibits a redox-dependent chaperone function in Arabidopsis and enhances plant tolerance to heat shock and oxidative stress

    Directory of Open Access Journals (Sweden)

    Jung eYoung Jun

    2015-12-01

    Full Text Available Although a wide range of physiological information on Universal Stress Proteins (USPs is available from many organisms, their biochemical and molecular functions remain unidentified. The biochemical function of AtUSP (At3g53990 from Arabidopsis thaliana was therefore investigated. Plants over-expressing AtUSP showed a strong resistance to heat shock and oxidative stress, compared with wild-type and Atusp knock-out plants, confirming the crucial role of AtUSP in stress tolerance. AtUSP was present in a variety of structures including monomers, dimers, trimers, and oligomeric complexes, and switched in response to external stresses from low molecular weight (LMW species to high molecular weight (HMW complexes. AtUSP exhibited a strong chaperone function under stress conditions in particular, and this activity was significantly increased by heat treatment. Chaperone activity of AtUSP was critically regulated by the redox status of cells and accompanied by structural changes to the protein. Over-expression of AtUSP conferred a strong tolerance to heat shock and oxidative stress upon Arabidopsis, primarily via its chaperone function.

  8. The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution

    Science.gov (United States)

    Bowman, Andrew; Hammond, Colin M.; Stirling, Andrew; Ward, Richard; Shang, Weifeng; El-Mkami, Hassane; Robinson, David A.; Svergun, Dmitri I.; Norman, David G.; Owen-Hughes, Tom

    2014-01-01

    NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron–electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a ‘self-chaperoning’ type mechanism. PMID:24688059

  9. Quantifying the role of chaperones in protein translocation by computational modeling

    OpenAIRE

    Assenza, Salvatore; De Los Rios, Paolo; Barducci, Alessandro

    2015-01-01

    The molecular chaperone Hsp70 plays a central role in the import of cytoplasmic proteins into organelles, driving their translocation by binding them from the organellar interior. Starting from the experimentally-determined structure of the E. coli Hsp70, we computed, by means of molecular simulations, the effective free-energy profile for substrate translocation upon chaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import process, w...

  10. Quantifying the role of chaperones in protein translocation by computational modelling

    OpenAIRE

    Assenza, Salvatore; Rios, Paolo De Los; Barducci, Alessandro

    2014-01-01

    The molecular chaperone Hsp70 plays a central role in the import of cytoplasmic proteins into organelles, driving their translocation by binding them from the organellar interior. Starting from the experimentally-determined structure of the \\textit{E. coli} Hsp70, we computed, by means of molecular simulations, the effective free-energy profile for substrate translocation upon chaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import p...

  11. The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution

    OpenAIRE

    Bowman, A.; Hammond, C. M.; Stirling, A.; Ward, R.; Shang, W.; El-Mkami, H.; Robinson, D A; Svergun, Dmitri; Norman, D. G.; Owen-Hughes, T.

    2014-01-01

    MRC [G1100021]; Wellcome Senior Fellowship [095062]. Source of open access funding: The Wellcome Trust [094090]. NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pu...

  12. Capturing a Dynamic Chaperone-Substrate Interaction Using NMR-Informed Molecular Modeling.

    Science.gov (United States)

    Salmon, Loïc; Ahlstrom, Logan S; Horowitz, Scott; Dickson, Alex; Brooks, Charles L; Bardwell, James C A

    2016-08-10

    Chaperones maintain a healthy proteome by preventing aggregation and by aiding in protein folding. Precisely how chaperones influence the conformational properties of their substrates, however, remains unclear. To achieve a detailed description of dynamic chaperone-substrate interactions, we fused site-specific NMR information with coarse-grained simulations. Our model system is the binding and folding of a chaperone substrate, immunity protein 7 (Im7), with the chaperone Spy. We first used an automated procedure in which NMR chemical shifts inform the construction of system-specific force fields that describe each partner individually. The models of the two binding partners are then combined to perform simulations on the chaperone-substrate complex. The binding simulations show excellent agreement with experimental data from multiple biophysical measurements. Upon binding, Im7 interacts with a mixture of hydrophobic and hydrophilic residues on Spy's surface, causing conformational exchange within Im7 to slow down as Im7 folds. Meanwhile, the motion of Spy's flexible loop region increases, allowing for better interaction with different substrate conformations, and helping offset losses in Im7 conformational dynamics that occur upon binding and folding. Spy then preferentially releases Im7 into a well-folded state. Our strategy has enabled a residue-level description of a dynamic chaperone-substrate interaction, improving our understanding of how chaperones facilitate substrate folding. More broadly, we validate our approach using two other binding partners, showing that this approach provides a general platform from which to investigate other flexible biomolecular complexes through the integration of NMR data with efficient computational models. PMID:27415450

  13. Capturing a Dynamic Chaperone-Substrate Interaction Using NMR-Informed Molecular Modeling.

    Science.gov (United States)

    Salmon, Loïc; Ahlstrom, Logan S; Horowitz, Scott; Dickson, Alex; Brooks, Charles L; Bardwell, James C A

    2016-08-10

    Chaperones maintain a healthy proteome by preventing aggregation and by aiding in protein folding. Precisely how chaperones influence the conformational properties of their substrates, however, remains unclear. To achieve a detailed description of dynamic chaperone-substrate interactions, we fused site-specific NMR information with coarse-grained simulations. Our model system is the binding and folding of a chaperone substrate, immunity protein 7 (Im7), with the chaperone Spy. We first used an automated procedure in which NMR chemical shifts inform the construction of system-specific force fields that describe each partner individually. The models of the two binding partners are then combined to perform simulations on the chaperone-substrate complex. The binding simulations show excellent agreement with experimental data from multiple biophysical measurements. Upon binding, Im7 interacts with a mixture of hydrophobic and hydrophilic residues on Spy's surface, causing conformational exchange within Im7 to slow down as Im7 folds. Meanwhile, the motion of Spy's flexible loop region increases, allowing for better interaction with different substrate conformations, and helping offset losses in Im7 conformational dynamics that occur upon binding and folding. Spy then preferentially releases Im7 into a well-folded state. Our strategy has enabled a residue-level description of a dynamic chaperone-substrate interaction, improving our understanding of how chaperones facilitate substrate folding. More broadly, we validate our approach using two other binding partners, showing that this approach provides a general platform from which to investigate other flexible biomolecular complexes through the integration of NMR data with efficient computational models.

  14. Chaperone-Mediated Autophagy and Mitochondrial Homeostasis in Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    Ruixin Yang

    2016-01-01

    Full Text Available Parkinson’s disease (PD, a complex neurodegenerative disorder, is pathologically characterized by the formation of Lewy bodies and loss of dopaminergic neurons in the substantia nigra pars compacta (SNc. Mitochondrial dysfunction is considered to be one of the most important causative mechanisms. In addition, dysfunction of chaperone-mediated autophagy (CMA, one of the lysosomal proteolytic pathways, has been shown to play an important role in the pathogenesis of PD. An exciting and important development is recent finding that CMA and mitochondrial quality control may be linked. This review summarizes the studies revealing the link between autophagy and mitochondrial function. Discussions are focused on the connections between CMA and mitochondrial failure and on the role of MEF2D, a neuronal survival factor, in mediating the regulation of mitochondria in the context of CMA. These new findings highlight the need to further explore the possibility of targeting the MEF2D-mitochondria-CMA network in both understanding the PD pathogenesis and developing novel therapeutic strategies.

  15. The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

    Science.gov (United States)

    Boulon, Séverine; Marmier-Gourrier, Nathalie; Pradet-Balade, Bérengère; Wurth, Laurence; Verheggen, Céline; Jády, Beáta E.; Rothé, Benjamin; Pescia, Christina; Robert, Marie-Cécile; Kiss, Tamás; Bardoni, Barbara; Krol, Alain; Branlant, Christiane; Allmang, Christine; Bertrand, Edouard; Charpentier, Bruno

    2008-01-01

    RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth. PMID:18268104

  16. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change

    Directory of Open Access Journals (Sweden)

    Seung Sik Lee

    2015-11-01

    Full Text Available AtTDX, a thioredoxin-like plant-specific protein present in Arabidospis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.

  17. Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

    Science.gov (United States)

    Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

    2016-01-01

    Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

  18. Mechanism of Amyloidogenesis of a Bacterial AAA+ Chaperone.

    Science.gov (United States)

    Chan, Sze Wah Samuel; Yau, Jason; Ing, Christopher; Liu, Kaiyin; Farber, Patrick; Won, Amy; Bhandari, Vaibhav; Kara-Yacoubian, Nareg; Seraphim, Thiago V; Chakrabarti, Nilmadhab; Kay, Lewis E; Yip, Christopher M; Pomès, Régis; Sharpe, Simon; Houry, Walid A

    2016-07-01

    Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a β-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation. PMID:27265850

  19. Expressional patterns of chaperones in ten human tumor cell lines

    Directory of Open Access Journals (Sweden)

    Slavc Irene

    2004-12-01

    Full Text Available Abstract Background Chaperones (CH play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression. Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. Results A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. Conclusions The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity.

  20. Investigation of the chaperone function of the small heat shock protein — AgsA

    Directory of Open Access Journals (Sweden)

    Nagamune Hideaki

    2010-07-01

    Full Text Available Abstract Background A small heat shock protein AgsA was originally isolated from Salmonella enterica serovar Typhimurium. We previously demonstrated that AgsA was an effective chaperone that could reduce the amount of heat-aggregated proteins in an Escherichia coli rpoH mutant. AgsA appeared to promote survival at lethal temperatures by cooperating with other chaperones in vivo. To investigate the aggregation prevention mechanisms of AgsA, we constructed N- or C-terminal truncated mutants and compared their properties with wild type AgsA. Results AgsA showed significant overall homology to wheat sHsp16.9 allowing its three-dimensional structure to be predicted. Truncations of AgsA until the N-terminal 23rd and C-terminal 11th amino acid (AA from both termini preserved its in vivo chaperone activity. Temperature-controlled gel filtration chromatography showed that purified AgsA could maintain large oligomeric complexes up to 50°C. Destabilization of oligomeric complexes was observed for N-terminal 11- and 17-AA truncated AgsA; C-terminal 11-AA truncated AgsA could not form large oligomeric complexes. AgsA prevented the aggregation of denatured lysozyme, malate dehydrogenase (MDH and citrate synthase (CS but did not prevent the aggregation of insulin at 25°C. N-terminal 17-AA truncated AgsA showed no chaperone activity towards MDH. C-terminal 11-AA truncated AgsA showed weak or no chaperone activity towards lysozyme, MDH and CS although it prevented the aggregation of insulin at 25°C. When the same amount of AgsA and C-terminal 11-AA truncated AgsA were mixed (half of respective amount required for efficient chaperone activities, good chaperone activity for all substrates and temperatures was observed. Detectable intermolecular exchanges between AgsA oligomers at 25°C were not observed using fluorescence resonance energy transfer analysis; however, significant exchanges between AgsA oligomers and C-terminal truncated AgsA were observed at 25

  1. High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK

    OpenAIRE

    Schloot Nanette C; Fingberg Waltraud; Alloza Iraide; Vandenbroeck Koen; Blasius Elias; Siegenthaler Rahel K; Burkart Volker; Christen Philipp; Kolb Hubert

    2010-01-01

    Abstract Background Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (pre)proinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK. Results Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further regi...

  2. OSU-03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood–Brain Barrier: Implications for Anti-Cancer Therapies

    OpenAIRE

    BOOTH, LAURENCE; Roberts, Jane L.; Tavallai, Mehrad; NOURBAKHSH, AIDA; CHUCKALOVCAK, JOHN; Carter, Jori; Poklepovic, Andrew; Dent, Paul

    2015-01-01

    We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a r...

  3. ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish.

    OpenAIRE

    Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi

    2013-01-01

    ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double kn...

  4. ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish

    OpenAIRE

    Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi

    2013-01-01

    ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double kn...

  5. Interactive domains in the molecular chaperone human alphaB crystallin modulate microtubule assembly and disassembly.

    Directory of Open Access Journals (Sweden)

    Joy G Ghosh

    Full Text Available Small heat shock proteins regulate microtubule assembly during cell proliferation and in response to stress through interactions that are poorly understood.Novel functions for five interactive sequences in the small heat shock protein and molecular chaperone, human alphaB crystallin, were investigated in the assembly/disassembly of microtubules and aggregation of tubulin using synthetic peptides and mutants of human alphaB crystallin.The interactive sequence (113FISREFHR(120 exposed on the surface of alphaB crystallin decreased microtubule assembly by approximately 45%. In contrast, the interactive sequences, (131LTITSSLSSDGV(142 and (156ERTIPITRE(164, corresponding to the beta8 strand and the C-terminal extension respectively, which are involved in complex formation, increased microtubule assembly by approximately 34-45%. The alphaB crystallin peptides, (113FISREFHR(120 and (156ERTIPITRE(164, inhibited microtubule disassembly by approximately 26-36%, and the peptides (113FISREFHR(120 and (131LTITSSLSSDGV(142 decreased the thermal aggregation of tubulin by approximately 42-44%. The (131LTITSSLSSDGV(142 and (156ERTIPITRE(164 peptides were more effective than the widely used anti-cancer drug, Paclitaxel, in modulating tubulinmicrotubule dynamics. Mutagenesis of these interactive sequences in wt human alphaB crystallin confirmed the effects of the alphaB crystallin peptides on microtubule assembly/disassembly and tubulin aggregation. The regulation of microtubule assembly by alphaB crystallin varied over a narrow range of concentrations. The assembly of microtubules was maximal at alphaB crystallin to tubulin molar ratios between 1:4 and 2:1, while molar ratios >2:1 inhibited microtubule assembly.Interactive sequences on the surface of human alphaB crystallin collectively modulate microtubule assembly through a dynamic subunit exchange mechanism that depends on the concentration and ratio of alphaB crystallin to tubulin. These are the first

  6. Sex, Scavengers, and Chaperones: Transcriptome Secrets of Divergent Symbiodinium Thermal Tolerances.

    Science.gov (United States)

    Levin, Rachel A; Beltran, Victor H; Hill, Ross; Kjelleberg, Staffan; McDougald, Diane; Steinberg, Peter D; van Oppen, Madeleine J H

    2016-09-01

    Corals rely on photosynthesis by their endosymbiotic dinoflagellates (Symbiodinium spp.) to form the basis of tropical coral reefs. High sea surface temperatures driven by climate change can trigger the loss of Symbiodinium from corals (coral bleaching), leading to declines in coral health. Different putative species (genetically distinct types) as well as conspecific populations of Symbiodinium can confer differing levels of thermal tolerance to their coral host, but the genes that govern dinoflagellate thermal tolerance are unknown. Here we show physiological and transcriptional responses to heat stress by a thermo-sensitive (physiologically susceptible at 32 °C) type C1 Symbiodinium population and a thermo-tolerant (physiologically healthy at 32 °C) type C1 Symbiodinium population. After nine days at 32 °C, neither population exhibited physiological stress, but both displayed up-regulation of meiosis genes by ≥ 4-fold and enrichment of meiosis functional gene groups, which promote adaptation. After 13 days at 32 °C, the thermo-sensitive population suffered a significant decrease in photosynthetic efficiency and increase in reactive oxygen species (ROS) leakage from its cells, whereas the thermo-tolerant population showed no signs of physiological stress. Correspondingly, only the thermo-tolerant population demonstrated up-regulation of a range of ROS scavenging and molecular chaperone genes by ≥ 4-fold and enrichment of ROS scavenging and protein-folding functional gene groups. The physiological and transcriptional responses of the Symbiodinium populations to heat stress directly correlate with the bleaching susceptibilities of corals that harbored these same Symbiodinium populations. Thus, our study provides novel, foundational insights into the molecular basis of dinoflagellate thermal tolerance and coral bleaching. PMID:27301593

  7. AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication.

    Science.gov (United States)

    Booth, Laurence; Roberts, Jane L; Ecroyd, Heath; Tritsch, Sarah R; Bavari, Sina; Reid, St Patrick; Proniuk, Stefan; Zukiwski, Alexander; Jacob, Abraham; Sepúlveda, Claudia S; Giovannoni, Federico; García, Cybele C; Damonte, Elsa; González-Gallego, Javier; Tuñón, María J; Dent, Paul

    2016-10-01

    We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286-2302, 2016. © 2016 Wiley Periodicals, Inc. PMID:27187154

  8. Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology

    DEFF Research Database (Denmark)

    Bæk, Kristoffer Torbjørn; Vegge, Christina Skovgaard; Skórko-Glonek, Joanna;

    2011-01-01

    The microaerophilic bacterium Campylobacter jejuni is the most common cause of bacterial food-borne infections in the developed world. Tolerance to environmental stress relies on proteases and chaperones in the cell envelope such as HtrA and SurA. HtrA displays both chaperone and protease activity......, but little is known about how each of these activities contributes to stress tolerance in bacteria. In vitro experiments showed temperature dependent protease and chaperone activities of C. jejuni HtrA. A C. jejuni mutant lacking only the protease activity of HtrA was used to show that the HtrA chaperone...

  9. Mechanisms of Hsp90 regulation

    Science.gov (United States)

    Prodromou, Chrisostomos

    2016-01-01

    Heat shock protein 90 (Hsp90) is a molecular chaperone that is involved in the activation of disparate client proteins. This implicates Hsp90 in diverse biological processes that require a variety of co-ordinated regulatory mechanisms to control its activity. Perhaps the most important regulator is heat shock factor 1 (HSF1), which is primarily responsible for upregulating Hsp90 by binding heat shock elements (HSEs) within Hsp90 promoters. HSF1 is itself subject to a variety of regulatory processes and can directly respond to stress. HSF1 also interacts with a variety of transcriptional factors that help integrate biological signals, which in turn regulate Hsp90 appropriately. Because of the diverse clientele of Hsp90 a whole variety of co-chaperones also regulate its activity and some are directly responsible for delivery of client protein. Consequently, co-chaperones themselves, like Hsp90, are also subject to regulatory mechanisms such as post translational modification. This review, looks at the many different levels by which Hsp90 activity is ultimately regulated. PMID:27515256

  10. Yeast prions are useful for studying protein chaperones and protein quality control.

    Science.gov (United States)

    Masison, Daniel C; Reidy, Michael

    2015-01-01

    Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell's protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions.

  11. Molecular Chaperones of Leishmania: Central Players in Many Stress-Related and -Unrelated Physiological Processes

    Directory of Open Access Journals (Sweden)

    Jose M. Requena

    2015-01-01

    Full Text Available Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges for drug discovery and improving of current treatments against leishmaniasis.

  12. Engineering and evolution of molecular chaperones and protein disaggregases with enhanced activity

    Directory of Open Access Journals (Sweden)

    Korrie eMack

    2016-03-01

    Full Text Available Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson’s disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector.

  13. The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism and the symbiotic interaction with alfalfa

    Directory of Open Access Journals (Sweden)

    Jiménez-Zurdo José I

    2010-03-01

    Full Text Available Abstract Background The bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs, and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. Sinorhizobium meliloti is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in S. meliloti has remained unexplored. Results Two independent S. meliloti mutants, 2011-3.4 and 1021Δhfq, were obtained by disruption and deletion of the hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Δhfq revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64% elicited by the 1021Δhfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of nifA and fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixK is only aerobiosis dependent. Finally, we found that some of the recently

  14. Structural basis for proteasome formation controlled by an assembly chaperone nas2.

    Science.gov (United States)

    Satoh, Tadashi; Saeki, Yasushi; Hiromoto, Takeshi; Wang, Ying-Hui; Uekusa, Yoshinori; Yagi, Hirokazu; Yoshihara, Hidehito; Yagi-Utsumi, Maho; Mizushima, Tsunehiro; Tanaka, Keiji; Kato, Koichi

    2014-05-01

    Proteasome formation does not occur due to spontaneous self-organization but results from a highly ordered process assisted by several assembly chaperones. The assembly of the proteasome ATPase subunits is assisted by four client-specific chaperones, of which three have been structurally resolved. Here, we provide the structural basis for the working mechanisms of the last, hereto structurally uncharacterized assembly chaperone, Nas2. We revealed that Nas2 binds to the Rpt5 subunit in a bivalent mode: the N-terminal helical domain of Nas2 masks the Rpt1-interacting surface of Rpt5, whereas its C-terminal PDZ domain caps the C-terminal proteasome-activating motif. Thus, Nas2 operates as a proteasome activation blocker, offering a checkpoint during the formation of the 19S ATPase prior to its docking onto the proteolytic 20S core particle. PMID:24685148

  15. The fictile coordination chemistry of cuprous-thiolate sites in copper chaperones.

    Science.gov (United States)

    Pushie, M Jake; Zhang, Limei; Pickering, Ingrid J; George, Graham N

    2012-06-01

    Copper plays vital roles in the active sites of cytochrome oxidase and in several other enzymes essential for human health. Copper is also highly toxic when dysregulated; because of this an elaborate array of accessory proteins have evolved which act as intracellular carriers or chaperones for the copper ions. In most cases chaperones transport cuprous copper. This review discusses some of the chemistry of these copper sites, with a view to some of the structural factors in copper coordination which are important in the biological function of these chaperones. The coordination chemistry and accessible geometries of the cuprous oxidation state are remarkably plastic and we discuss how this may relate to biological function. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

  16. ADP-ribosylation factor 6 mediates E-cadherin recovery by chemical chaperones.

    Directory of Open Access Journals (Sweden)

    Joana Figueiredo

    Full Text Available E-cadherin plays a powerful tumor suppressor role. Germline E-cadherin mutations justify 30% of Hereditary Diffuse Gastric Cancer (HDGC and missense mutations are found in 30% of these families. We found possible to restore in vitro mutant E-cadherin associated to HDGC syndrome by using Chemical Chaperones (CCs. Herein, our aim was to disclose the molecular mechanisms underlying the CCs effects in E-cadherin regulation. Using cells stably expressing WT E-cadherin or two HDGC-associated missense mutations, we show that upon DMSO treatment, not only mutant E-cadherin is restored and stabilized at the plasma membrane (PM, but also Arf6 and PIPKIγ expressions are altered. We show that modulation of Arf6 expression partially mimics the effect of CCs, suggesting that the cellular effects observed upon CCs treatment are mediated by Arf6. Further, we show that E-cadherin expression recovery is specifically linked to Arf6 due to its role on endocytosis and recycling pathways. Finally, we demonstrated that, as DMSO, several others CCs are able to modulate the trafficking machinery through an Arf6 dependent mechanism. Interestingly, the more effective compounds in E-cadherin recovery to PM are those that simultaneously inhibit Arf6 and stimulate PIPKIγ expression and binding to E-cadherin. Here, we present the first evidence of a direct influence of CCs in cellular trafficking machinery and we show that this effect is of crucial importance in the context of juxtamembrane E-cadherin missense mutations associated to HDGC. We propose that this influence should be taken into account when exploring the therapeutic potential of this type of chemicals in genetic diseases associated to protein-misfolding.

  17. A protective role of HSP90 chaperone in gamma-irradiated Arabidopsis thaliana seeds

    Science.gov (United States)

    Kozeko, Liudmyla; Talalaiev, Oleksandr; Neimash, Volodymyr; Povarchuk, Vasyl

    2015-07-01

    The heat shock protein 90 (HSP90) is required for the maturation and conformational regulation of many regulatory proteins affecting morphogenetic pathways and stress tolerance. The purpose of this work is to disclose a role of HSP90 in radioresistance of seeds. Arabidopsis thaliana (Ler) seeds were exposed to γ-ray irradiation with doses of 0.1-1 kGy using 60Co source to obtain a viable but polymorphic material. A comet assay of the seeds showed a dose-dependent increase in DNA damage. Phenotypic consequences of irradiation included growth stimulation at doses of 0.1-0.25 kGy and negative growth effects at doses from 0.5 kGy and beyond, along with increasing heterogeneity of seedling growth rate and phenotype. The frequencies of abnormal phenotypes were highly correlated with the degree of DNA damage in seeds. Treatment of seeds with geldanamycin (GDA), an inhibitor of HSP90, stimulated the seedling growth at all radiation doses and, at the same time, enhanced the growth rate and morphological diversity. It was also found that HSP70 induction by γ-rays was increased following GDA treatment (shown at 1 kGy). We suppose that the GDA-induced HSP70 can be involved in elimination of detrimental radiation effects that ultimately results in growth stimulation. On the other hand, the increase in phenotypic variation, when HSP90 function was impaired, confirms the supposition that the chaperone may control the concealment of cryptic genetic alterations and the developmental stability. In general, these results demonstrate that HSP90 may interface the stress response and phenotypic expression of genetic alterations induced by irradiation.

  18. The RNA chaperone Hfq is involved in stress tolerance and virulence in uropathogenic Proteus mirabilis.

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    Min-Cheng Wang

    Full Text Available Hfq is a bacterial RNA chaperone involved in the riboregulation of diverse genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Proteus mirabilis to effectively colonize the bladder and kidneys in a murine urinary tract infection (UTI model and to establish burned wound infection of the rats. In this regard, we found the hfq mutant induced higher IL-8 and MIF levels of uroepithelial cells and displayed reduced intra-macrophage survival. The loss of hfq affected bacterial abilities to handle H2O2 and osmotic pressures and to grow at 50 °C. Relative to wild-type, the hfq mutant had reduced motility, fewer flagella and less hemolysin expression and was less prone to form biofilm and to adhere to and invade uroepithelial cells. The MR/P fimbrial operon was almost switched to the off phase in the hfq mutant. In addition, we found the hfq mutant exhibited an altered outer membrane profile and had higher RpoE expression, which indicates the hfq mutant may encounter increased envelope stress. With the notion of envelope disturbance in the hfq mutant, we found increased membrane permeability and antibiotic susceptibilities in the hfq mutant. Finally, we showed that Hfq positively regulated the RpoS level and tolerance to H2O2 in the stationary phase seemed largely mediated through the Hfq-dependent RpoS expression. Together, our data indicate that Hfq plays a critical role in P. mirabilis to establish UTIs by modulating stress responses, surface structures and virulence factors. This study suggests Hfq may serve as a scaffold molecule for development of novel anti-P. mirabilis drugs and P. mirabilis hfq mutant is a vaccine candidate for preventing UTIs.

  19. The RNA chaperone Hfq impacts growth, metabolism and production of virulence factors in Yersinia enterocolitica.

    Directory of Open Access Journals (Sweden)

    Tamara Kakoschke

    Full Text Available To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin.

  20. Mechanisms of Translocation of ER Chaperones to the Cell Surface and Immunomodulatory Roles in Cancer and Autoimmunity

    NARCIS (Netherlands)

    Wiersma, Valerie; Michalak, Marek; Abdullah, Trefa M; Bremer, Edwin; Eggleton, Paul

    2015-01-01

    Endoplasmic reticulum (ER) chaperones (e.g., calreticulin, heat shock proteins, and isomerases) perform a multitude of functions within the ER. However, many of these chaperones can translocate to the cytosol and eventually the surface of cells, particularly during ER stress induced by e.g., drugs,

  1. Chaperone protein L-isoaspartate (D-aspartyl) O-methyltransferase as a novel predictor of poor prognosis in lung adenocarcinoma.

    Science.gov (United States)

    Saito, Heisuke; Yamashita, Masahiro; Ogasawara, Masahito; Yamada, Noriyuki; Niisato, Miyuki; Tomoyasu, Makoto; Deguchi, Hiroyuki; Tanita, Tatsuo; Ishida, Kazuyuki; Sugai, Tamotsu; Yamauchi, Kohei

    2016-04-01

    Endoplasmic reticulum stress and chaperone dysfunction have recently been associated with poor prognoses in various cancers. The newly discovered chaperone protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) regulates the viability of cancer cells in various cancers, although no clinical information regarding the relationship between lung cancer and PIMT expression has been reported. In this study, we aimed to elucidate the relationship between PIMT expression and the prognosis of lung adenocarcinoma. Paraffin-embedded lung tissues obtained from 208 patients with surgically resected lung adenocarcinoma were subjected to immunohistochemical analyses using primary antibodies against PIMT. Kaplan-Meier curves, log-rank tests, and the Cox proportional hazards model were used to analyze the association between PIMT expression and patient survival. Strong PIMT expression was detected in 106 (50.9%) patients, being particularly observed in patients with advanced stages of lung adenocarcinoma. Strong PIMT expression was associated with that of 78-kDa glucose-regulated protein, a marker of endoplasmic reticulum stress. Patients with strong PIMT expression had a shorter survival time (Kaplan-Meier analysis, Plung adenocarcinoma, including those with stage I disease (hazard ratios, 6.45 and 6.81, respectively; 95% confidence intervals, 2.46-16.9 and 1.79-25.8, respectively; Plung adenocarcinoma, and this finding might help clinicians determine the need for postoperative adjuvant chemotherapy in patients with stage I lung adenocarcinoma.

  2. The Molecular Chaperone GRP78 Contributes to Toll-like Receptor 3-mediated Innate Immune Response to Hepatitis C Virus in Hepatocytes.

    Science.gov (United States)

    Wei, Dahai; Li, Nan L; Zeng, Yanli; Liu, Baoming; Kumthip, Kattareeya; Wang, Tony T; Huo, Dezheng; Ingels, Jesse F; Lu, Lu; Shang, Jia; Li, Kui

    2016-06-01

    Toll-like receptor-3 (TLR3) senses double-stranded RNA intermediates produced during hepatitis C virus (HCV) replication, leading to activation of interferon regulatory factor-3 (IRF3) and NF-κB and subsequent antiviral and proinflammatory responses. Yet, how this TLR3-dependent pathway operates in hepatocytes is unclear. Upon fractionating cultured hepatocytes into various cellular organelles, we observed that TLR3 predominantly resides in endolysosomes of hepatocytes. To determine the critical regulators of TLR3 signaling in response to HCV infection in human hepatocytes, we isolated endolysosome fractions from mock-infected and HCV-infected hepatoma Huh7.5 cells that had been reconstituted for TLR3 expression, separated these fractions on two-dimensional gels, and identified up-regulated/down-regulated proteins by mass spectrometry. Approximately a dozen of cellular proteins were found to be differentially expressed in endolysosome fractions following HCV infection. Of these, expression of several molecular chaperone proteins was elevated. Knockdown of one of these chaperones, glucose-regulated protein 78 kDa (GRP78), compromised TLR3-dependent induction of interferon-stimulated genes and chemokines following HCV infection or poly(I:C) stimulation in cultured hepatocytes. Consistent with this finding, GRP78 depletion impaired TLR3-mediated establishment of an antiviral state. Mechanistically, although TLR3 trafficking to endolysosomes was not affected, phosphorylated IRF3 diminished faster following GRP78 knockdown. Remarkably, GRP78 transcript was significantly up-regulated in liver biopsies of chronic hepatitis C patients as compared with normal liver tissues. Moreover, the GRP78 expression level correlated with that of RANTES (regulated upon activation, normal T-cell expressed and secreted) and CXCL10, two inflammatory chemokines most frequently elevated in HCV-infected liver. Altogether, our data suggest that GRP78 contributes to TLR3-mediated, IRF3

  3. The Yersinia enterocolitica type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1

    Directory of Open Access Journals (Sweden)

    Heesemann Jürgen

    2007-07-01

    Full Text Available Abstract Background Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica share a virulence plasmid encoding a type three secretion system (T3SS. This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins, the specific Yop chaperones (Sycs, and the Ysc (Yop secretion proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study. Results We have established the large-scale production of recombinant SycO in its outright form. We confirm that Y. enterocolitica SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in Y. enterocolitica decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. We demonstrate that in vitro SycO interacts with YscM1, a negative regulator of Yop expression in Y. enterocolitica. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants. Conclusion We present evidence that SycO is integrated into the regulatory network of the Yersinia T3SS. Our picture of the Yersinia T3SS interactome is supplemented by identification of the SycO/YscM1 interaction. Further, our results suggest that at least one additional interaction partner of SycO has to be identified.

  4. Chaperone driven polymer translocation through Nanopore: spatial distribution and binding energy

    CERN Document Server

    Abdolvahab, Rouhollah Haji

    2016-01-01

    Chaperones are binding proteins which work as a driving force to bias the biopolymer translocation by binding to it near the pore and preventing its backsliding. Chaperones may have different spatial distribution. Recently we show the importance of their spatial distribution in translocation and how it effects on sequence dependency of the translocation time. Here we focus on homopolymers and exponential distribution. As a result of the exponential distribution of chaperones, energy dependency of the translocation time will changed and one see a minimum in translocation time versus effective energy curve. The same trend can be seen in scaling exponent of time versus polymer length, $\\beta$ ($T\\sim\\beta$). Interestingly in some special cases e.g. chaperones of size $\\lambda=6$ and with exponential distribution rate of $\\alpha=5$, the minimum reaches even to amount of less than $1$ ($\\beta<1$). We explain the possibility of this rare result and base on a theoretical discussion we show that by taking into acc...

  5. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    Science.gov (United States)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  6. Hsp40 function in yeast prion propagation: Amyloid diversity necessitates chaperone functional complexity.

    Science.gov (United States)

    Sporn, Zachary A; Hines, Justin K

    2015-01-01

    Yeast prions are heritable protein-based elements, most of which are formed of amyloid aggregates that rely on the action of molecular chaperones for transmission to progeny. Prions can form distinct amyloid structures, known as 'strains' in mammalian systems, that dictate both pathological progression and cross-species infection barriers. In yeast these same amyloid structural polymorphisms, called 'variants', dictate the intensity of prion-associated phenotypes and stability in mitosis. We recently reported that [PSI(+)] prion variants differ in the fundamental domain requirements for one chaperone, the Hsp40/J-protein Sis1, which are mutually exclusive between 2 different yeast prions, demonstrating a functional plurality for Sis1. Here we extend that analysis to incorporate additional data that collectively support the hypothesis that Sis1 has multiple functional roles that can be accomplished by distinct sets of domains. These functions are differentially required by distinct prions and prion variants. We also present new data regarding Hsp104-mediated prion elimination and show that some Sis1 functions, but not all, are conserved in the human homolog Hdj1/DNAJB1. Importantly, of the 10 amyloid-based prions indentified to date in Saccharomyces cerevisiae, the chaperone requirements of only 4 are known, leaving a great diversity of amyloid structures, and likely modes of amyloid-chaperone interaction, largely unexplored.

  7. Iminosugars and isoiminosugars as pharmacological chaperones for Krabbe disease - a SAR study

    DEFF Research Database (Denmark)

    Viuff, Agnete

    2015-01-01

    The overall theme of this thesis is the use of iminosugars and isoiminosugars as inhibitors or chaperones for glycosidases. Glycosidases are a group of enzymes with a large variety of biological roles, and the ability to modulate their activity is, therefore, relevant for the development of new...

  8. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Zhang, Lingling; Picking, Wendy L.; Geisbrecht, Brian V. (UMKC); (OKLU)

    2010-10-05

    Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. In this study, we present the 3.3 {angstrom} crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC{sup 1-151}). Specifically, we observe a rotationally-symmetric 'head-to-head' dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC{sup 1-151}. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions: From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may

  9. The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica.

    Science.gov (United States)

    Kakoschke, Tamara Katharina; Kakoschke, Sara Carina; Zeuzem, Catharina; Bouabe, Hicham; Adler, Kristin; Heesemann, Jürgen; Rossier, Ombeline

    2016-07-08

    In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies.

  10. MITRAC7 Acts as a COX1-Specific Chaperone and Reveals a Checkpoint during Cytochrome c Oxidase Assembly

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    Sven Dennerlein

    2015-09-01

    Full Text Available Cytochrome c oxidase, the terminal enzyme of the respiratory chain, is assembled from mitochondria- and nuclear-encoded subunits. The MITRAC complex represents the central assembly intermediate during this process as it receives imported subunits and regulates mitochondrial translation of COX1 mRNA. The molecular processes that promote and regulate the progression of assembly downstream of MITRAC are still unknown. Here, we identify MITRAC7 as a constituent of a late form of MITRAC and as a COX1-specific chaperone. MITRAC7 is required for cytochrome c oxidase biogenesis. Surprisingly, loss of MITRAC7 or an increase in its amount causes selective cytochrome c oxidase deficiency in human cells. We demonstrate that increased MITRAC7 levels stabilize and trap COX1 in MITRAC, blocking progression in the assembly process. In contrast, MITRAC7 deficiency leads to turnover of newly synthesized COX1. Accordingly, MITRAC7 affects the biogenesis pathway by stabilizing newly synthesized COX1 in assembly intermediates, concomitantly preventing turnover.

  11. The assembly and intermolecular properties of the Hsp70-Tomm34-Hsp90 molecular chaperone complex.

    Science.gov (United States)

    Trcka, Filip; Durech, Michal; Man, Petr; Hernychova, Lenka; Muller, Petr; Vojtesek, Borivoj

    2014-04-01

    Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.

  12. Conformational selection underlies recognition of a molybdoenzyme by its dedicated chaperone.

    Directory of Open Access Journals (Sweden)

    Magali Lorenzi

    Full Text Available Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism.

  13. The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x.

    Directory of Open Access Journals (Sweden)

    Michael O Daniyan

    Full Text Available Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1 or a human Hsp70 (HSPA1A, indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.

  14. Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.

    Science.gov (United States)

    Olesen, Sanne H; Ingles, Donna J; Zhu, Jin-Yi; Martin, Mathew P; Betzi, Stephane; Georg, Gunda I; Tash, Joseph S; Schönbrunn, Ernst

    2015-01-19

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  15. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Directory of Open Access Journals (Sweden)

    Sanne H. Olesen

    2015-01-01

    Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  16. Protein arginine methyltransferase Prmt5-Mep50 methylates histones H2A and H4 and the histone chaperone nucleoplasmin in Xenopus laevis eggs.

    Science.gov (United States)

    Wilczek, Carola; Chitta, Raghu; Woo, Eileen; Shabanowitz, Jeffrey; Chait, Brian T; Hunt, Donald F; Shechter, David

    2011-12-01

    Histone proteins carry information contained in post-translational modifications. Eukaryotic cells utilize this histone code to regulate the usage of the underlying DNA. In the maturing oocytes and eggs of the frog Xenopus laevis, histones are synthesized in bulk in preparation for deposition during the rapid early developmental cell cycles. During this key developmental time frame, embryonic pluripotent chromatin is established. In the egg, non-chromatin-bound histones are complexed with storage chaperone proteins, including nucleoplasmin. Here we describe the identification and characterization of a complex of the protein arginine methyltransferase 5 (Prmt5) and the methylosome protein 50 (Mep50) isolated from Xenopus eggs that specifically methylates predeposition histones H2A/H2A.X-F and H4 and the histone chaperone nucleoplasmin on a conserved motif (GRGXK). We demonstrate that nucleoplasmin (Npm), an exceedingly abundant maternally deposited protein, is a potent substrate for Prmt5-Mep50 and is monomethylated and symmetrically dimethylated at Arg-187. Furthermore, Npm modulates Prmt5-Mep50 activity directed toward histones, consistent with a regulatory role for Npm in vivo. We show that H2A and nucleoplasmin methylation appears late in oogenesis and is most abundant in the laid egg. We hypothesize that these very abundant arginine methylations are constrained to pre-mid blastula transition events in the embryo and therefore may be involved in the global transcriptional repression found in this developmental time frame.

  17. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Science.gov (United States)

    Olesen, Sanne H.; Ingles, Donna J.; Zhu, Jin-Yi; Martin, Mathew P.; Betzi, Stephane; Georg, Gunda I.; Tash, Joseph S.; Schönbrunn, Ernst

    2015-01-01

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in-vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands. PMID:25608045

  18. Calreticulin chaperones regulate functional expression of vomeronasal type 2 pheromone receptors

    OpenAIRE

    Dey, Sandeepa; Matsunami, Hiroaki

    2011-01-01

    A variety of social behaviors like intermale aggression, fear, and mating rituals are important for sustenance of a species. In mice, these behaviors have been implicated to be mediated by peptide pheromones that are sensed by a class of G protein-coupled receptors, vomeronasal receptor type 2 (V2Rs), expressed in the pheromone detecting vomeronasal organ. Matching V2Rs with their cognate ligands is required to learn what receptors the biologically relevant pheromones are acting on. However, ...

  19. Drosophila Frataxin: An Iron Chaperone During Cellular Fe-S Cluster Bioassembly

    Energy Technology Data Exchange (ETDEWEB)

    Kondapalli, K.C.; Kok, N.M.; Dancis, A.; Stemmler, T.L.

    2009-05-20

    Frataxin, a mitochondrial protein that is directly involved in regulating cellular iron homeostasis, has been suggested to serve as an iron chaperone during cellular Fe-S cluster biosynthesis. In humans, decreased amounts or impaired function of frataxin causes the autosomal recessive neurodegenerative disorder Friedreich's ataxia. Cellular production of Fe-S clusters is accomplished by the Fe cofactor assembly platform enzymes Isu (eukaryotes) and IscU (prokaryotes). In this report, we have characterized the overall stability and iron binding properties of the Drosophila frataxin homologue (Dfh). Dfh is highly folded with secondary structural elements consistent with the structurally characterized frataxin orthologs. While the melting temperature (T{sub M} {approx} 59 C) and chemical stability ([urea]{sub 50} {approx} 2.4 M) of Drosophila frataxin, measured using circular dichroism (CD) and fluorescence spectroscopy, closely match values determined for the human ortholog, pure Dfh is more stable against autodegradation than both the human and yeast proteins. The ferrous iron binding affinity (K{sub d} {approx} 6.0 {micro}M) and optimal metal to protein stoichiometry (1:1) for Dfh have been measured using isothermal titration calorimetry (ITC). Under anaerobic conditions with salt present, holo-Dfh is a stable iron-loaded protein monomer. Frataxin prevents reactive oxygen species-induced oxidative damage to DNA when presented with both Fe(II) and H{sub 2}O{sub 2}. Ferrous iron bound to Dfh is high-spin and held in a partially symmetric Fe-(O/N){sub 6} coordination environment, as determined by X-ray absorption spectroscopy (XAS). Extended X-ray absorption fine structure (EXAFS) simulations indicate the average Fe-O/N bond length in Dfh is 2.13 {angstrom}, consistent with a ligand geometry constructed by water and carboxylate oxygens most likely supplied in part by surface-exposed conserved acidic residues located on helix 1 and strand 1 in the structurally

  20. 分子伴侣的多重功能%The mutifunction of chaperones

    Institute of Scientific and Technical Information of China (English)

    翟静; 张凤珍; 蒋汉明

    2004-01-01

    分子伴侣是一类能帮助其他蛋白质进行正确折叠、组装、转运、介导错误折叠的蛋白质进行降解的蛋白质.它们还参与染色体的复制、抗原的加工与提呈,并作用于一些信息转导分子以调节生长和发育.分子伴侣发挥功能依赖于ATP的结合与水解.热休克应答是生物界从细菌到植物和动物普遍存在的,它是保护细胞免受像热休克、酒精、能量代谢抑制剂、重金属、抗原加工与提呈、凋亡等有害环境损伤的一个基本的防御机制.这些蛋白质的相对水平是非常重要的,过多或过少的Hsp70或Hsp90可以导致生长异常、发育畸形甚至细胞死亡.%Moleculer Chaperones are proteins that can help target protein to acquire one possible conformation, translocation, refolding of intermediates, chromosome replication, proteases, such as the ubiquitin-dependent proteasome, ensure that damaged and short-lived proteins are degraded efficiently.They can also interact with multiple key components of signaling pathways that regulate growth and development. They function in ATP binding and hydrolysis. Heat shock response is ubiquitous and highly conserved-in all organisms from bacteria to plants and animals-as an essential defense mechanism for protection of cells from a wide range of harmful conditions, including heat shock, alcohols, inhibitors of energy metabolism, heavy metals, oxidative stress, antigen processing and presentation, apoptosis. The relative levels of these proteins may be important, as too little or too much Hsp70 or Hsp90 can result in aberrant growth control, developmental malformations and cell death.

  1. A Rational Design Strategy for the Selective Activity Enhancement of a Molecular Chaperone toward a Target Substrate.

    Science.gov (United States)

    Aprile, Francesco A; Sormanni, Pietro; Vendruscolo, Michele

    2015-08-18

    Molecular chaperones facilitate the folding and assembly of proteins and inhibit their aberrant aggregation. They thus offer several opportunities for biomedical and biotechnological applications, as for example they can often prevent protein aggregation more effectively than other therapeutic molecules, including small molecules and antibodies. Here we present a method of designing molecular chaperones with enhanced activity against specific amyloidogenic substrates while leaving unaltered their functions toward other substrates. The method consists of grafting onto a molecular chaperone a peptide designed to bind specifically an epitope in the target substrate. We illustrate this strategy by describing Hsp70 variants with increased affinities for α-synuclein and Aβ42 but otherwise unaltered affinities for other substrates. These designed variants inhibit protein aggregation and disaggregate preformed fibrils significantly more effectively than wild-type Hsp70 indicating that the strategy presented here provides a possible route for tailoring rationally molecular chaperones for specific purposes.

  2. Unique Residues Involved in Activation of the Multitasking Protease/Chaperone HtrA from Chlamydia trachomatis

    OpenAIRE

    Huston, Wilhelmina M.; Joel D. A. Tyndall; Lott, William B.; Stansfield, Scott H.; Timms, Peter

    2011-01-01

    DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrat...

  3. Thioredoxin Reductase Type C (NTRC) Orchestrates Enhanced Thermotolerance to Arabidopsis by Its Redox-Dependent Holdase Chaperone Function

    Institute of Scientific and Technical Information of China (English)

    Ho Byoung Chae; Jeong Chan Moon; Mi Rim Shin; Yong Hun Chi; Young Jun Jung; Sun Yong Lee; Ganesh M.Nawkar

    2013-01-01

    Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reductase,type C (NTRC) in the light.Here we show overexpression of NTRC in Arabidopsis (NTRCoE) resulting in enhanced tolerance to heat shock,whereas NTRC knockout mutant plants (ntrcl) exhibit a temperature sensitive phenotype.To investigate the underlying mechanism of this phenotype,we analyzed the protein's biochemical properties and protein structure.NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase,a foldase chaperone,and as a holdase chaperone.The multiple functions of NTRC are closely correlated with protein structure.Complexes of higher molecular weight (HMW) showed stronger activity as a holdase chaperone,while low molecular weight (LMW) species exhibited weaker holdase chaperone activity but stronger disulfide reductase and foldase chaperone activities.Heat shock converted LMW proteins into HMW complexes.Mutations of the two active site Cys residues of NTRC into Ser (C217/454S-NTRC) led to a complete inactivation of its disulfide reductase and foldase chaperone functions,but conferred only a slight decrease in its holdase chaperone function.The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRCoE plants.However,after prolonged incubation under heat shock,NTRCoE plants tolerated the stress to a higher degree than C217/454S-NTRCoE plants.The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH.

  4. Immunoglobulin-like PapD chaperone caps and uncaps interactive surfaces of nascently translocated pilus subunits.

    OpenAIRE

    Kuehn, M J; Normark, S; Hultgren, S. J.

    1991-01-01

    Molecular chaperones are found in the cytoplasm of bacteria and in various cellular compartments in eukaryotes to maintain proteins in nonnative conformations that permit their secretion across membranes or assembly into oligomeric structures. Virtually nothing, however, has been reported about a similar requirement for molecular chaperones in the periplasm of Gram-negative bacteria. We used the well-characterized P pilus biogenesis system in Escherichia coli as a model to elucidate the mecha...

  5. Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality.

    Science.gov (United States)

    Bibova, Ilona; Hot, David; Keidel, Kristina; Amman, Fabian; Slupek, Stephanie; Cerny, Ondrej; Gross, Roy; Vecerek, Branislav

    2015-01-01

    Bordetella pertussis, the causative agent of human whooping cough (pertussis) produces a complex array of virulence factors in order to establish efficient infection in the host. The RNA chaperone Hfq and small regulatory RNAs are key players in posttranscriptional regulation in bacteria and have been shown to play an essential role in virulence of a broad spectrum of bacterial pathogens. This study represents the first attempt to characterize the Hfq regulon of the human pathogen B. pertussis under laboratory conditions as well as upon passage in the host and indicates that loss of Hfq has a profound effect on gene expression in B. pertussis. Comparative transcriptional profiling revealed that Hfq is required for expression of several virulence factors in B. pertussis cells including the Type III secretion system (T3SS). In striking contrast to the wt strain, T3SS did not become operational in the hfq mutant passaged either through mice or macrophages thereby proving that Hfq is required for the functionality of the B. pertussis T3SS. Likewise, expression of virulence factors vag8 and tcfA encoding autotransporter and tracheal colonization factor, respectively, was strongly reduced in the hfq mutant. Importantly, for the first time we demonstrate that B. pertussis T3SS can be activated upon contact with macrophage cells in vitro.

  6. Transcriptional coactivator HCF-1 couples the histone chaperone Asf1b to HSV-1 DNA replication components.

    Science.gov (United States)

    Peng, Hua; Nogueira, Mauricio L; Vogel, Jodi L; Kristie, Thomas M

    2010-02-01

    The cellular transcriptional coactivator HCF-1 interacts with numerous transcription factors as well as other coactivators and is a component of multiple chromatin modulation complexes. The protein is essential for the expression of the immediate early genes of both herpes simplex virus (HSV) and varicella zoster virus and functions, in part, by coupling chromatin modification components including the Set1 or MLL1 histone methyltransferases and the histone demethylase LSD1 to promote the installation of positive chromatin marks and the activation of viral immediately early gene transcription. Although studies have investigated the role of HCF-1 in both cellular and viral transcription, little is known about other processes that the protein may be involved in. Here we demonstrate that HCF-1 localizes to sites of HSV replication late in infection. HCF-1 interacts directly and simultaneously with both HSV DNA replication proteins and the cellular histone chaperone Asf1b, a protein that regulates the progression of cellular DNA replication forks via chromatin reorganization. Asf1b localizes with HCF-1 in viral replication foci and depletion of Asf1b results in significantly reduced viral DNA accumulation. The results support a model in which the transcriptional coactivator HCF-1 is a component of the HSV DNA replication assembly and promotes viral DNA replication by coupling Asf1b to DNA replication components. This coupling provides a novel function for HCF-1 and insights into the mechanisms of modulating chromatin during DNA replication.

  7. Molecular Mechanism Underlying Pathogenesis of Lewisite-Induced Cutaneous Blistering and Inflammation: Chemical Chaperones as Potential Novel Antidotes.

    Science.gov (United States)

    Li, Changzhao; Srivastava, Ritesh K; Weng, Zhiping; Croutch, Claire R; Agarwal, Anupam; Elmets, Craig A; Afaq, Farrukh; Athar, Mohammad

    2016-10-01

    Lewisite is a potent arsenic-based chemical warfare agent known to induce painful cutaneous inflammation and blistering. Only a few modestly effective antidotes have so far been described in the literature. However, the discovery of effective antidotes for lewisite was hampered by the paucity of the exact molecular mechanism underlying its cutaneous pathogenesis. We investigated the molecular mechanism underlying lewisite-induced cutaneous blistering and inflammation and describe its novel antidotes. On the basis of our initial screening, we used a highly sensitive murine model that recapitulates the known human pathogenesis of arsenicals-induced cutaneous inflammation and blistering. Topically administered lewisite induced potent acute inflammation and microvesication in the skin of Ptch1(+/-)/SKH-1 mice. Even at a very low dose, lewisite up-regulates unfolded protein response signaling, inflammatory response, and apoptosis. These cutaneous lesions were associated with production of reactive oxygen species and extensive apoptosis of the epidermal keratinocytes. We confirmed that activation of reactive oxygen species-dependent unfolded protein response signaling is the underlying molecular mechanism of skin damage. Similar alterations were noticed in lewisite-treated cultured human skin keratinocytes. We discovered that chemical chaperone 4-phenyl butyric acid and antioxidant N-acetylcysteine, which significantly attenuate lewisite-mediated skin injury, can serve as potent antidotes. These data reveal a novel molecular mechanism underlying the cutaneous pathogenesis of lewisite-induced lesions. We also identified novel potential therapeutic targets for lewisite-mediated cutaneous injury. PMID:27528504

  8. The chemical chaperones tauroursodeoxycholic and 4-phenylbutyric acid accelerate thyroid hormone activation and energy expenditure

    Science.gov (United States)

    da-Silva, Wagner S.; Ribich, Scott; e Drigo, Rafael Arrojo; Castillo, Melany; Patty, Mary-Elizabeth; Bianco, Antonio C.

    2011-01-01

    Exposure of cell lines endogenously expressing the thyroid hormone activating enzyme type 2 deiodinase (D2) to the chemical chaperones tauroursodeoxycholic acid (TUDCA) or 4-phenylbutiric acid (4-PBA) increases D2 expression, activity and T3 production. In brown adipocytes, TUDCA or 4-PBA induced T3-dependent genes and oxygen consumption (~2-fold), an effect partially lost in D2 knockout cells. In wild type, but not in D2 knockout mice, administration of TUDCA lowered the respiratory quotient, doubled brown adipose tissue D2 activity and normalized the glucose intolerance associated with high fat feeding. Thus, D2 plays a critical role in the metabolic effects of chemical chaperones. PMID:21237159

  9. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Chingakham R. [Kansas State University, 338 Ackert Hall, Manhattan, KS 66506 (United States); Lovell, Scott; Mehzabeen, Nurjahan [University of Kansas, Del Shankel Structural Biology Center, Lawrence, KS 66047 (United States); Chowdhury, Wasimul Q.; Geanes, Eric S. [Kansas State University, 338 Ackert Hall, Manhattan, KS 66506 (United States); Battaile, Kevin P. [IMCA-CAT Hauptman–Woodward Medical Research Institute, 9700 South Cass Avenue, Building 435A, Argonne, IL 60439 (United States); Roelofs, Jeroen, E-mail: jroelofs@ksu.edu [Kansas State University, 338 Ackert Hall, Manhattan, KS 66506 (United States)

    2014-03-25

    The proteasome-assembly chaperone Nas2 binds to the proteasome subunit Rpt5 using its PDZ domain. The structure of the Nas2 PDZ domain has been determined. The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Å resolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly.

  10. The Role of System-Specific Molecular Chaperones in the Maturation of Molybdoenzymes in Bacteria

    Directory of Open Access Journals (Sweden)

    Meina Neumann

    2011-01-01

    Full Text Available Biogenesis of prokaryotic molybdoenzymes is a complex process with the final step representing the insertion of a matured molybdenum cofactor (Moco into a folded apoenzyme. Usually, specific chaperones of the XdhC family are required for the maturation of molybdoenzymes of the xanthine oxidase family in bacteria. Enzymes of the xanthine oxidase family are characterized to contain an equatorial sulfur ligand at the molybdenum center of Moco. This sulfur ligand is inserted into Moco while bound to the XdhC-like protein and before its insertion into the target enzyme. In addition, enzymes of the xanthine oxidase family bind either the molybdopterin (Mo-MPT form of Moco or the modified molybdopterin cytosine dinucleotide cofactor (MCD. In both cases, only the matured cofactor is inserted by a proofreading process of XdhC. The roles of these specific XdhC-like chaperones during the biogenesis of enzymes of the xanthine oxidase family in bacteria are described.

  11. Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease.

    Directory of Open Access Journals (Sweden)

    Gonçalo da Costa

    Full Text Available Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.

  12. Structure of the hypothetical Mycoplasma protein, MPN555, suggestsa chaperone function

    Energy Technology Data Exchange (ETDEWEB)

    Schulze-Gahmen, Ursula; Aono, Shelly; Chen, Shengfeng; Yokota,Hisao; Kim, Rosalind; Kim, Sung-Hou

    2005-06-15

    The crystal structure of the hypothetical protein MPN555from Mycoplasma pneumoniae (gi pbar 1673958) has been determined to a resolution of 2.8 Angstrom using anomalous diffraction data at the Sepeak wavelength. Structure determination revealed a mostly alpha-helical protein with a three-lobed shape. The three lobes or fingers delineate a central binding groove and additional grooves between lobes 1 and 3, and between lobes 2 and 3. For one of the molecules in the asymmetric unit,the central binding pocket was filled with a peptide from the uncleaved N-terminal affinity tag. The MPN555 structure has structural homology to two bacterial chaperone proteins, SurA and trigger factor from Escherichia coli. The structural data and the homology to other chaperone for MPN555.

  13. Structure of Glycerol Dehydratase Reactivase: A New Type of Molecular Chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Der-Ing; Reiss, Lisa; Turner, Jr., Ivan; Dotson, Garry (Dupont)

    2010-03-08

    The function of glycerol dehydratase (GDH) reactivase is to remove damaged coenzyme B{sub 12} from GDH that has suffered mechanism-based inactivation. The structure of GDH reactivase from Klebsiella pneumoniae was determined at 2.4 {angstrom} resolution by the single isomorphous replacement with anomalous signal (SIR/AS) method. Each tetramer contains two elongated 63 kDa {alpha} subunits and two globular 14 kDa {beta} subunits. The {alpha} subunit contains structural features resembling both GroEL and Hsp70 groups of chaperones, and it appears chaperone like in its interactions with ATP. The fold of the {beta} subunit resembles that of the {beta} subunit of glycerol dehydratase, except that it lacks some coenzyme B12 binding elements. A hypothesis for the reactivation mechanism of reactivase is proposed based on these structural features.

  14. Quantifying the role of chaperones in protein translocation by computational modelling

    OpenAIRE

    Salvatore eAssenza; Paolo eDe Los Rios; Alessandro eBarducci

    2015-01-01

    The molecular chaperone Hsp70 plays a central role in the import of cytoplasmic proteins into organelles,driving their translocation by binding them from the organellar interior. Starting from the experimentally-determined structure of the E. coli Hsp70, we computed, by means of molecular simulations,the effective free-energy profile for substrate translocation uponchaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import process, whos...

  15. Unique Photobleaching Phenomena of the Twin-Arginine Translocase Respiratory Enzyme Chaperone DmsD

    OpenAIRE

    Rivardo, Fabrizio; Leach, Thorin G.H.; Chan, Catherine S.; Winstone, Tara M.L.; Ladner, Carol L.; Sarfo, Kwabena J.; Turner, Raymond J.

    2014-01-01

    DmsD is a chaperone of the redox enzyme maturation protein family specifically required for biogenesis of DMSO reductase in Escherichia coli. It exists in multiple folding forms, all of which are capable of binding its known substrate, the twin-arginine leader sequence of the DmsA catalytic subunit. It is important for maturation of the reductase and targeting to the cytoplasmic membrane for translocation. Here, we demonstrate that DmsD exhibits an irreversible photobleaching phenomenon upon ...

  16. Chemical chaperone 4-phenylbutyrate prevents endoplasmic reticulum stress induced by T17M rhodopsin

    OpenAIRE

    Jiang, Haibo; Xiong, Siqi; Xia, Xiaobo

    2014-01-01

    Background Rhodopsin mutations are associated with the autosomal dominant form of retinitis pigmentosa. T17M mutation in rhodopsin predisposes cells to endoplasmic reticulum (ER) stress and induces cell death. This study aimed to examine whether chemical chaperone 4-phenylbutyrate prevents ER stress induced by rhodopsin T17M. Results ARPE-19 cells were transfected with myc-tagged wild-type (WT) and T17M rhodopsin constructs. Turnover of WT and T17M rhodopsin was measured by cycloheximide chas...

  17. Lipase Maturation Factor 1: a lipase chaperone involved in lipid metabolism

    OpenAIRE

    Péterfy, Miklós

    2011-01-01

    Mutations in lipase maturation factor 1 (LMF1) are associated with severe hypertriglyceridemia in mice and human subjects. The underlying cause is impaired lipid clearance due to lipase deficiency. LMF1 is a chaperone of the endoplasmic reticulum (ER) and it is critically required for the post-translational activation of three vascular lipases: lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). As LMF1 is only required for the maturation of homodimeric, but not monomer...

  18. Enhanced Transport Capabilities via Nanotechnologies: Impacting Bioefficacy, Controlled Release Strategies, and Novel Chaperones

    Directory of Open Access Journals (Sweden)

    Thomai Panagiotou

    2011-01-01

    side affects and providing improved therapeutic interventions. Innovative nanotechnology applications, such as simultaneous targeting, imaging and delivery to tumors, are now possible through use of novel chaperones. Other examples include nanoparticles attachment to T-cells, release from novel hydrogel implants, and functionalized encapsulants. Difficult tasks such as drug delivery to the brain via the blood brain barrier and/or the cerebrospinal fluid are now easier to accomplish.

  19. Structure of a Chaperone-Usher Pilus reveals the molecular basis of rod uncoiling

    OpenAIRE

    Hospenthal, M. K.; Redzej, A.; Dodson, K.; Ukleja, M.; Frenz, B.; Rodrigues, C; Hultgren, S. J.; DiMaio, F.; Egelman, E. H.; Waksman, G

    2016-01-01

    Summary Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the...

  20. A Clp/Hsp100 Chaperone Functions in Myxococcus xanthus Sporulation and Self-Organization

    OpenAIRE

    Yan, Jinyuan; Garza, Anthony G.; Michael D. Bradley; Welch, Roy D.

    2012-01-01

    The Clp/Hsp100 proteins are chaperones that play a role in protein degradation and reactivation. In bacteria, they exhibit a high degree of pleiotropy, affecting both individual and multicellular phenotypes. In this article, we present the first characterization of a Clp/Hsp100 homolog in Myxococcus xanthus (MXAN_4832 gene locus). Deletion of MXAN_4832 causes defects in both swarming and aggregation related to cell motility and the production of fibrils, which are an important component of th...

  1. Rapid induction of Alternative Lengthening of Telomeres by depletion of the histone chaperone ASF1

    OpenAIRE

    O'Sullivan, Roderick J; Arnoult, Nausica; Daniel H Lackner; Oganesian, Liana; Haggblom, Candy; Corpet, Armelle; Almouzni, Genevieve; Karlseder, Jan

    2014-01-01

    The mechanism of activation of the Alternative Lengthening of Telomeres (ALT) pathway of mammalian chromosome end maintenance has remained an unresolved issue. We have discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced all hallmarks of ALT in both primary and cancer cells. These included the formation of ALT associated PML bodies (APBs), extra-chromosomal telomeric DNA species an elevated frequency of telomeric sister chromatid exchanges (t-SCE) even...

  2. Rescue of vasopressin V2 receptor mutants by chemical chaperones: specificity and mechanism.

    OpenAIRE

    Robben, J.H.; Sze, M.; Knoers, N.V.A.M.; Deen, P. M. T.

    2006-01-01

    Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca2+ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogeni...

  3. Modulation of the chaperone-like activity of bovine α-crystallin

    OpenAIRE

    Clark, John I.; Huang, Qing-ling

    1996-01-01

    The effects of pantethine, glutathione, and selected chemical reagents on the anti-aggregation activity of α-crystallin was evaluated. Protein aggregation was monitored by light scattering of solutions of denatured βL-crystallin or alcohol dehydrogenase (ADH). The ratios of βL-crystallin/α-crystallin and ADH/α-crystallin were adjusted so that partial inhibition of protein aggregation at 60°C or 37°C, respectively, was observed and modulation of the chaperone ac...

  4. Complex Lipid Requirements for SNARE- and SNARE Chaperone-dependent Membrane Fusion*

    OpenAIRE

    Mima, Joji; Wickner, William

    2009-01-01

    Membrane fusion without lysis has been reconstituted with purified yeast vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE chaperones Sec17p/Sec18p and the multifunctional HOPS complex, which includes a subunit of the SNARE-interactive Sec1-Munc18 family, and vacuolar lipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin (CL), ergosterol (ERG), d...

  5. Evidence for a Functional Role of the Molecular Chaperone Clusterin in Amyloidotic Cardiomyopathy

    OpenAIRE

    Michael J Greene; Sam, Flora; Soo Hoo, Pamela T.; Patel, Rupesh S.; Seldin, David C.; Connors, Lawreen H.

    2011-01-01

    Molecular chaperones, including the extracellular protein clusterin (CLU), play a significant role in maintaining proteostasis; they have a unique capacity to bind and stabilize non-native protein conformations, prevent aggregation, and keep proteins in a soluble folding-competent state. In this study, we investigated amyloid-infiltrated cardiac tissue for the presence of CLU and measured serum levels of CLU in patients with and without amyloidotic cardiomyopathy (CMP). Cardiac tissues contai...

  6. Metabolic and chaperone gene loss marks the origin of animals: evidence for Hsp104 and Hsp78 chaperones sharing mitochondrial enzymes as clients.

    Directory of Open Access Journals (Sweden)

    Albert J Erives

    Full Text Available The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i sixteen distinct biosynthetic functions; (ii the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones.

  7. Hsp70/Hsp90 organising protein (hop): beyond interactions with chaperones and prion proteins.

    Science.gov (United States)

    Baindur-Hudson, Swati; Edkins, Adrienne L; Blatch, Gregory L

    2015-01-01

    The Hsp70/Hsp90 organising protein (Hop), also known as stress-inducible protein 1 (STI1), has received considerable attention for diverse cellular functions in both healthy and diseased states. There is extensive evidence that intracellular Hop is a co-chaperone of the major chaperones Hsp70 and Hsp90, playing an important role in the productive folding of Hsp90 client proteins. Consequently, Hop is implicated in a number of key signalling pathways, including aberrant pathways leading to cancer. However, Hop is also secreted and it is now well established that Hop also serves as a receptor for the prion protein, PrP(C). The intracellular and extracellular forms of Hop most likely represent two different isoforms, although the molecular determinants of these divergent functions are yet to be identified. There is also a growing body of research that reports the involvement of Hop in cellular activities that appear independent of either chaperones or PrP(C). While Hop has been shown to have various cellular functions, its biological function remains elusive. However, recent knockout studies in mammals suggest that Hop has an important role in embryonic development. This review provides a critical overview of the latest molecular, cellular and biological research on Hop, critically evaluating its function in healthy systems and how this function is adapted in diseases states.

  8. Chaperone therapy for Krabbe disease: potential for late-onset GALC mutations.

    Science.gov (United States)

    Hossain, Mohammad Arif; Higaki, Katsumi; Saito, Seiji; Ohno, Kazuki; Sakuraba, Hitoshi; Nanba, Eiji; Suzuki, Yoshiyuki; Ozono, Keiichi; Sakai, Norio

    2015-09-01

    Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on mutant GM1 β-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations. PMID:26108143

  9. Chemical chaperones improve protein secretion and rescue mutant factor VIII in mice with hemophilia A.

    Directory of Open Access Journals (Sweden)

    Stefanie D Roth

    Full Text Available Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII, which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P. We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.

  10. A review of acquired thermotolerance, heat shock proteins, and molecular chaperones in archaea

    Energy Technology Data Exchange (ETDEWEB)

    Trent, J.D.

    1996-05-01

    Acquired thermotolerance, the associated synthesis of heat-shock proteins (HSPs) under stress conditions, and the role of HSPs as molecular chaperones under normal growth conditions have been studied extensively in eukaryotes and bacteria, whereas research in these areas in archaea is only beginning. All organisms have evolved a variety of strategies for coping with high-temperature stress, and among these strategies is the increased synthesis of HSPs. The facts that both high temperatures and chemical stresses induce the HSPs and that some of the HSPs recognize and bind to unfolded proteins in vitro have led to the theory that the function of HSPs is to prevent protein aggregation in vivo. The facts that some HSPs are abundant under normal growth conditions and that they assist in protein folding in vitro have led to the theory that they assist protein folding in vivo; in this role, they are referred to as molecular chaperones. The limited research on acquired thermotolerance, HSPs, and molecular chaperones in archaea, particularly the hyperthermophilic archaea, suggests that these extremophiles provide a new perspective in these areas of research, both because they are members of a separate phylogenetic domain and because they have evolved to live under extreme conditions.

  11. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisné, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  12. Conserved C-terminal nascent peptide binding domain of HYPK facilitates its chaperone-like activity

    Indian Academy of Sciences (India)

    Swasti Raychaudhuri; Rachana Banerjee; Subhasish Mukhopadhyay; Nitai P Bhattacharyya

    2014-09-01

    Human HYPK (Huntingtin Yeast-two-hybrid Protein K) is an intrinsically unstructured chaperone-like protein with no sequence homology to known chaperones. HYPK is also known to be a part of ribosome-associated protein complex and present in polysomes. The objective of the present study was to investigate the evolutionary influence on HYPK primary structure and its impact on the protein’s function. Amino acid sequence analysis revealed 105 orthologs of human HYPK from plants, lower invertebrates to mammals. C-terminal part of HYPK was found to be particularly conserved and to contain nascent polypeptide-associated alpha subunit (NPAA) domain. This region experiences highest selection pressure, signifying its importance in the structural and functional evolution. NPAA domain of human HYPK has unique amino acid composition preferring glutamic acid and happens to be more stable from a conformational point of view having higher content of -helices than the rest. Cell biology studies indicate that overexpressed C-terminal human HYPK can interact with nascent proteins, co-localizes with huntingtin, increases cell viability and decreases caspase activities in Huntington’s disease (HD) cell culture model. This domain is found to be required for the chaperone-like activity of HYPK in vivo. Our study suggested that by virtue of its flexibility and nascent peptide binding activity, HYPK may play an important role in assisting protein (re)folding.

  13. Nucleic Acid Chaperone Activity of HIV-1 NC Proteins Investigated by Single Molecule DNA Stretching

    Science.gov (United States)

    Williams, Mark C.; Gorelick, Robert J.; Musier-Forsyth, Karin; Bloomfield, Victor A.

    2002-03-01

    HIV-1 Nucleocapsid Protein (NC) is a nucleic acid chaperone protein that is responsible for facilitating numerous nucleic acid rearrangements throughout the reverse transcription cycle of HIV-1. To understand the mechanism of NC’s chaperone function, we carried out single molecule DNA stretching studies in the presence of NC and mutant forms of NC. Using an optical tweezers instrument, we stretch single DNA molecules from the double-stranded helical state to the single-stranded (coil) state. Based on the observed cooperativity of DNA force-induced melting, we find that the fraction of melted base pairs at room temperature is increased dramatically in the presence of NC. Thus, upon NC binding, increased thermal fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations in order to find the lowest energy state. While NC destabilizes the double-stranded form of DNA, a mutant form of NC that lacks the zinc finger structures does not. DNA stretching experiments carried out in the presence of NC variants containing more subtle changes in the zinc finger structures were conducted to elucidate the contribution of each individual finger to NC’s chaperone activity, and these results will be reported.

  14. The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding*

    Science.gov (United States)

    Baaklini, Imad; Wong, Michael J. H.; Hantouche, Christine; Patel, Yogita; Shrier, Alvin; Young, Jason C.

    2012-01-01

    DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70. PMID:23091061

  15. The DNAJA2 substrate release mechanism is essential for chaperone-mediated folding.

    Science.gov (United States)

    Baaklini, Imad; Wong, Michael J H; Hantouche, Christine; Patel, Yogita; Shrier, Alvin; Young, Jason C

    2012-12-01

    DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70. PMID:23091061

  16. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A. (McMaster U.); (Melbourne); (Toronto); (Deakin); (HWMRI)

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  17. The yeast histone chaperone hif1p functions with RNA in nucleosome assembly.

    Directory of Open Access Journals (Sweden)

    Amy R Knapp

    Full Text Available Hif1p is an H3/H4-specific histone chaperone that associates with the nuclear form of the Hat1p/Hat2p complex (NuB4 complex in the yeast Saccharomyces cerevisiae. While not capable of depositing histones onto DNA on its own, Hif1p can act in conjunction with a yeast cytosolic extract to assemble nucleosomes onto a relaxed circular plasmid.To identify the factor(s that function with Hif1p to carry out chromatin assembly, multiple steps of column chromatography were carried out to fractionate the yeast cytosolic extract. Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein. Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA. In addition, the RNA-mediated chromatin assembly activity was blocked by mutations in the human homolog of Hif1p, sNASP, that prevent the association of this histone chaperone with histone H3 and H4 without altering its electrostatic properties.These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.

  18. TRP and Rhodopsin Transport Depends on Dual XPORT ER Chaperones Encoded by an Operon

    Directory of Open Access Journals (Sweden)

    Zijing Chen

    2015-10-01

    Full Text Available TRP channels and G protein-coupled receptors (GPCRs play critical roles in sensory reception. However, the identities of the chaperones that assist GPCRs in translocating from the endoplasmic reticulum (ER are limited, and TRP ER chaperones are virtually unknown. The one exception for TRPs is Drosophila XPORT. Here, we show that the xport locus is bicistronic and encodes unrelated transmembrane proteins, which enable the signaling proteins that initiate and culminate phototransduction, rhodopsin 1 (Rh1 and TRP, to traffic to the plasma membrane. XPORT-A and XPORT-B are ER proteins, and loss of either has a profound impact on TRP and Rh1 targeting to the light-sensing compartment of photoreceptor cells. XPORT-B complexed in vivo with the Drosophila homolog of the mammalian HSP70 protein, GRP78/BiP, which, in turn, associated with Rh1. Our work highlights a coordinated network of chaperones required for the biosynthesis of the TRP channel and rhodopsin in Drosophila photoreceptor cells.

  19. TRP and Rhodopsin Transport Depends on Dual XPORT ER Chaperones Encoded by an Operon.

    Science.gov (United States)

    Chen, Zijing; Chen, Hsiang-Chin; Montell, Craig

    2015-10-20

    TRP channels and G protein-coupled receptors (GPCRs) play critical roles in sensory reception. However, the identities of the chaperones that assist GPCRs in translocating from the endoplasmic reticulum (ER) are limited, and TRP ER chaperones are virtually unknown. The one exception for TRPs is Drosophila XPORT. Here, we show that the xport locus is bicistronic and encodes unrelated transmembrane proteins, which enable the signaling proteins that initiate and culminate phototransduction, rhodopsin 1 (Rh1) and TRP, to traffic to the plasma membrane. XPORT-A and XPORT-B are ER proteins, and loss of either has a profound impact on TRP and Rh1 targeting to the light-sensing compartment of photoreceptor cells. XPORT-B complexed in vivo with the Drosophila homolog of the mammalian HSP70 protein, GRP78/BiP, which, in turn, associated with Rh1. Our work highlights a coordinated network of chaperones required for the biosynthesis of the TRP channel and rhodopsin in Drosophila photoreceptor cells. PMID:26456832

  20. Discovery of a novel target for the dysglycemic chromogranin A fragment pancreastatin: interaction with the chaperone GRP78 to influence metabolism.

    Directory of Open Access Journals (Sweden)

    Nilima Biswas

    Full Text Available RATIONALE: The chromogranin A-derived peptide pancreastatin (PST is a dysglycemic, counter-regulatory peptide for insulin action, especially in liver. Although previous evidence for a PST binding protein has been reported, such a receptor has not been identified or sequenced. METHODS AND RESULTS: We used ligand affinity to purify the PST target, with biotinylated human PST (hCHGA273-301-amide as "bait" and mouse liver homogenate as "prey", and identified GRP78 (a.k.a. "78 kDa Glucose Regulated Protein", HSPA5, BIP as a major interacting partner of PST. GRP78 belongs to the family of heat shock proteins (chaperones, involved in several cellular processes including protein folding and glucose metabolism. We analyzed expression of GRP78 in the absence of PST in a mouse knockout model lacking its precursor CHGA: hepatic transcriptome data revealed global over-expression of not only GRP78 but also other heat shock transcripts (of the "adaptive UPR" in CHGA(-/- mice compared to wild-type (+/+. By contrast, we found a global decline in expression of hepatic pro-apoptotic transcripts in CHGA(-/- mice. GRP78's ATPase enzymatic activity was dose-dependently inhibited by PST (IC50∼5.2 µM. PST also inhibited the up-regulation of GRP78 expression during UPR activation (by tunicamycin in hepatocytes. PST inhibited insulin-stimulated glucose uptake in adipocytes, and increased hepatic expression of G6Pase (the final step in gluconeogenesis/glycogenolysis. In hepatocytes not only PST but also other GRP78-ATPase inhibitors (VER-155008 or ADP increased G6Pase expression. GRP78 over-expression inhibited G6Pase expression in hepatocytes, with partial restoration by GRP78-ATPase inhibitors PST, VER-155008, or ADP. CONCLUSIONS: Our results indicate that an unexpected major hepatic target of PST is the adaptive UPR chaperone GRP78. PST not only binds to GRP78 (in pH-dependent fashion, but also inhibits GRP78's ATPase enzymatic activity, and impairs its biosynthetic

  1. Oxidative modification of the molecular chaperone family in a PC12 cell model of Parkinson's disease induced by Z-lle-Glu(OtBu)-Ala-Leucinal

    Institute of Scientific and Technical Information of China (English)

    Ying Zhang; Yimin Yang; Jing Bai; Ming Chang; Linsen Hu

    2011-01-01

    Previous studies have demonstrated that ubiquitin-proteasome system function is significantly decreased in the substantia nigra of Parkinson's disease patients.In the present study, proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leucinal (PSI) was used to inhibit the function of the ubiquitin-proteasome system in PC12 cells to simulate Parkinson's disease.Oxidatively modified proteins were identified to determine pathogenesis of Parkinson's disease.Results demonstrated that 24 hours of 10 μmol/L PSI-treatment in PC12 cells simulated pathological characteristics of Parkinson's disease: neuronal degeneration and eosinophilic inclusion formation in neurons.In PSI-treated PC12 cells, three oxidative proteins and a molecular chaperone family member were detected: chaperonin containing t-complex polypeptide 1 subunit 3, glucose-regulated protein 58,and heat shock protein 70.This is the first study to demonstrate oxidative modification of a molecule family in a cell model of Parkinson's disease induced with PSI.

  2. Water and molecular chaperones act as weak links of protein folding networks: energy landscape and punctuated equilibrium changes point towards a game theory of proteins

    OpenAIRE

    Kovacs, Istvan A.; Szalay, Mate S.; Csermely, Peter

    2004-01-01

    Water molecules and molecular chaperones efficiently help the protein folding process. Here we describe their action in the context of the energy and topological networks of proteins. In energy terms water and chaperones were suggested to decrease the activation energy between various local energy minima smoothing the energy landscape, rescuing misfolded proteins from conformational traps and stabilizing their native structure. In kinetic terms water and chaperones may make the punctuated equ...

  3. A role for the RNA chaperone Hfq in controlling adherent-invasive Escherichia coli colonization and virulence.

    Directory of Open Access Journals (Sweden)

    Karina T Simonsen

    Full Text Available Adherent-invasive Escherichia coli (AIEC has been linked with the onset and perpetuation of inflammatory bowel diseases. The AIEC strain LF82 was originally isolated from an ileal biopsy from a patient with Crohn's disease. The pathogenesis of LF82 results from its abnormal adherence to and subsequent invasion of the intestinal epithelium coupled with its ability to survive phagocytosis by macrophages once it has crossed the intestinal barrier. To gain further insight into AIEC pathogenesis we employed the nematode Caenorhabditis elegans as an in vivo infection model. We demonstrate that AIEC strain LF82 forms a persistent infection in C. elegans, thereby reducing the host lifespan significantly. This host killing phenotype was associated with massive bacterial colonization of the nematode intestine and damage to the intestinal epithelial surface. C. elegans killing was independent of known LF82 virulence determinants but was abolished by deletion of the LF82 hfq gene, which encodes an RNA chaperone involved in mediating posttranscriptional gene regulation by small non-coding RNAs. This finding reveals that important aspects of LF82 pathogenesis are controlled at the posttranscriptional level by riboregulation. The role of Hfq in LF82 virulence was independent of its function in regulating RpoS and RpoE activity. Further, LF82Δhfq mutants were non-motile, impaired in cell invasion and highly sensitive to various chemical stress conditions, reinforcing the multifaceted function of Hfq in mediating bacterial adaptation. This study highlights the usefulness of simple non-mammalian infection systems for the identification and analysis of bacterial virulence factors.

  4. In silico identification of potential chaperone genes that belong to type III and type IV secretion systems in Xanthomonas axonopodis pv citri

    Directory of Open Access Journals (Sweden)

    Letícia Khater

    2005-01-01

    Full Text Available The secretion of bacterial virulence factors and flagellar components requires the assistance of specific type III and flagellar chaperones. Standard computational annotation of the genome of Xanthomonas axonopodis pv citri, a plant pathogen that causes citrus canker, initially did not identify any genes belonging to these chaperone categories since the primary sequence homology between them was very low. However, in a search for hypothetical proteins with characteristics similar to these chaperones, we have now identified 30 chromosomal and 10 plasmidial potential genes encoding chaperones belonging to types III/IV, and flagellar secretion systems in this organism. The significance of these findings is discussed.

  5. Parkinson disease-linked GBA mutation effects reversed by molecular chaperones in human cell and fly models.

    Science.gov (United States)

    Sanchez-Martinez, Alvaro; Beavan, Michelle; Gegg, Matthew E; Chau, Kai-Yin; Whitworth, Alexander J; Schapira, Anthony H V

    2016-01-01

    GBA gene mutations are the greatest cause of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase) but the mechanisms by which loss of GCase contributes to PD remain unclear. Inhibition of autophagy and the generation of endoplasmic reticulum (ER) stress are both implicated. Mutant GCase can unfold in the ER and be degraded via the unfolded protein response, activating ER stress and reducing lysosomal GCase. Small molecule chaperones that cross the blood brain barrier help mutant GCase refold and traffic correctly to lysosomes are putative treatments for PD. We treated fibroblast cells from PD patients with heterozygous GBA mutations and Drosophila expressing human wild-type, N370S and L444P GBA with the molecular chaperones ambroxol and isofagomine. Both chaperones increased GCase levels and activity, but also GBA mRNA, in control and mutant GBA fibroblasts. Expression of mutated GBA in Drosophila resulted in dopaminergic neuronal loss, a progressive locomotor defect, abnormal aggregates in the ER and increased levels of the ER stress reporter Xbp1-EGFP. Treatment with both chaperones lowered ER stress and prevented the loss of motor function, providing proof of principle that small molecule chaperones can reverse mutant GBA-mediated ER stress in vivo and might prove effective for treating PD. PMID:27539639

  6. Reversible Interactions of Proteins with Mixed Shell Polymeric Micelles: Tuning the Surface Hydrophobic/Hydrophilic Balance toward Efficient Artificial Chaperones.

    Science.gov (United States)

    Wang, Jianzu; Song, Yiqing; Sun, Pingchuan; An, Yingli; Zhang, Zhenkun; Shi, Linqi

    2016-03-22

    Molecular chaperones can elegantly fine-tune its hydrophobic/hydrophilic balance to assist a broad spectrum of nascent polypeptide chains to fold properly. Such precious property is difficult to be achieved by chaperone mimicking materials due to limited control of their surface characteristics that dictate interactions with unfolded protein intermediates. Mixed shell polymeric micelles (MSPMs), which consist of two kinds of dissimilar polymeric chains in the micellar shell, offer a convenient way to fine-tune surface properties of polymeric nanoparticles. In the current work, we have fabricated ca. 30 kinds of MSPMs with finely tunable hydrophilic/hydrophobic surface properties. We investigated the respective roles of thermosensitive and hydrophilic polymeric chains in the thermodenaturation protection of proteins down to the molecular structure. Although the three kinds of thermosensitive polymers investigated herein can form collapsed hydrophobic domains on the micellar surface, we found distinct capability to capture and release unfolded protein intermediates, due to their respective affinity for proteins. Meanwhile, in terms of the hydrophilic polymeric chains in the micellar shell, poly(ethylene glycol) (PEG) excels in assisting unfolded protein intermediates to refold properly via interacting with the refolding intermediates, resulting in enhanced chaperone efficiency. However, another hydrophilic polymer-poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) severely deteriorates the chaperone efficiency of MSPMs, due to its protein-resistant properties. Judicious combination of thermosensitive and hydrophilic chains in the micellar shell lead to MSPM-based artificial chaperones with optimal efficacy.

  7. New therapeutic approaches for Krabbe disease: The potential of pharmacological chaperones.

    Science.gov (United States)

    Spratley, Samantha J; Deane, Janet E

    2016-11-01

    Missense mutations in the lysosomal hydrolase β-galactocerebrosidase (GALC) account for at least 40% of known cases of Krabbe disease (KD). Most of these missense mutations are predicted to disrupt the fold of the enzyme, preventing GALC in sufficient amounts from reaching its site of action in the lysosome. The predominant central nervous system (CNS) pathology and the absence of accumulated primary substrate within the lysosome mean that strategies used to treat other lysosomal storage disorders (LSDs) are insufficient in KD, highlighting the still unmet clinical requirement for successful KD therapeutics. Pharmacological chaperone therapy (PCT) is one strategy being explored to overcome defects in GALC caused by missense mutations. In recent studies, several small-molecule inhibitors have been identified as promising chaperone candidates for GALC. This Review discusses new insights gained from these studies and highlights the importance of characterizing both the chaperone interaction and the underlying mutation to define properly a responsive population and to improve the translation of existing lead molecules into successful KD therapeutics. We also highlight the importance of using multiple complementary methods to monitor PCT effectiveness. Finally, we explore the exciting potential of using combination therapy to ameliorate disease through the use of PCT with existing therapies or with more generalized therapeutics, such as proteasomal inhibition, that have been shown to have synergistic effects in other LSDs. This, alongside advances in CNS delivery of recombinant enzyme and targeted rational drug design, provides a promising outlook for the development of KD therapeutics. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc. PMID:27638604

  8. OSU-03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood-Brain Barrier: Implications for Anti-Cancer Therapies.

    Science.gov (United States)

    Booth, Laurence; Roberts, Jane L; Tavallai, Mehrad; Nourbakhsh, Aida; Chuckalovcak, John; Carter, Jori; Poklepovic, Andrew; Dent, Paul

    2015-08-01

    We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality. PMID:25736380

  9. OSU‐03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood–Brain Barrier: Implications for Anti‐Cancer Therapies

    Science.gov (United States)

    Booth, Laurence; Roberts, Jane L.; Tavallai, Mehrad; Nourbakhsh, Aida; Chuckalovcak, John; Carter, Jori; Poklepovic, Andrew

    2015-01-01

    We examined the interaction between OSU‐03012 (also called AR‐12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose‐regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU‐03012 to kill stem‐like GBM cells. Treatment of cells with OSU‐03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK‐eIF2α‐ATF4‐CHOP signaling and was blocked by GRP78 over‐expression. In vivo OSU‐03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU‐03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over‐expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU‐03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU‐03012/sildenafil lethality. J. Cell. Physiol. 230: 1982–1998, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:25736380

  10. Theoretical and experimental investigation of chaperone effects on soluble recombinant proteins in Escherichia coli: effect of free DnaK level on temperature-induced recombinant streptokinase production.

    Science.gov (United States)

    Balagurunathan, Balaji; Jayaraman, Guhan

    2008-06-01

    Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK-DnaJ-GrpE and GroEL-GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a sigma(32) mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in

  11. Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models

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    Kung PJ

    2014-10-01

    Full Text Available Pin-Jui Kung,1,* Yu-Chen Tao,1,* Ho-Chiang Hsu,1 Wan-Ling Chen,1 Te-Hsien Lin,1 Donala Janreddy,2 Ching-Fa Yao,2 Kuo-Hsuan Chang,3 Jung-Yaw Lin,1 Ming-Tsan Su,1 Chung-Hsin Wu,1 Guey-Jen Lee-Chen,1 Hsiu-Mei Hsieh-Li1 1Department of Life Science, 2Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan; 3Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan *These authors contributed equally to this work Abstract: In spinocerebellar ataxia type 17 (SCA17, the expansion of a translated CAG repeat in the TATA box binding protein (TBP gene results in a long polyglutamine (polyQ tract in the TBP protein, leading to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On SH-SY5Y cells with inducible SCA17 TBP/Q79-green fluorescent protein (GFP expression to test indole and synthetic derivative NC001-8 for neuroprotection. We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells. The effects on promoting neurite outgrowth and on reduction of aggregation on Purkinje cells were also confirmed with cerebellar primary and slice cultures of SCA17 transgenic mice. Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment. Keywords: spinocerebellar ataxia type 17, TATA box binding protein, polyQ aggregation, indole and derivative, therapeutics

  12. Identification of two p23 co-chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities.

    Science.gov (United States)

    Batista, Fernanda A H; Almeida, Glessler S; Seraphim, Thiago V; Silva, Kelly P; Murta, Silvane M F; Barbosa, Leandro R S; Borges, Júlio C

    2015-01-01

    The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90. PMID:25369258

  13. Effects of pH and Iminosugar Pharmacological Chaperones on Lysosomal Glycosidase Structure and Stability

    Energy Technology Data Exchange (ETDEWEB)

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.; (Harvard-Med); (Brandeis)

    2009-06-05

    Human lysosomal enzymes acid-{beta}-glucosidase (GCase) and acid-{alpha}-galactosidase ({alpha}-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and {alpha}-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using {alpha}-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of {alpha}-Gal A with DGJ. Both GCase and {alpha}-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in {alpha}-Gal A are not seen. Thermodynamic parameters obtained from {alpha}-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and {alpha}-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological

  14. Fabry_CEP: a tool to identify Fabry mutations responsive to pharmacological chaperones.

    Science.gov (United States)

    Cammisa, Marco; Correra, Antonella; Andreotti, Giuseppina; Cubellis, Maria Vittoria

    2013-01-01

    Fabry_CEP is a user-friendly web-application designed to help clinicians Choose Eligible Patients for the therapy with pharmacological chaperones. It provides a database and a predictive tool to evaluate the responsiveness of lysosomal alpha-galactosidase mutants to a small molecule drug, namely 1-Deoxy-galactonojirimycin. The user can introduce any missense/nonsense mutation in the coding sequence, learn whether it is has been tested and gain access to appropriate reference literature. In the absence of experimental data structural, functional and evolutionary analysis provides a prediction and the probability that a given mutation is responsive to the drug. PMID:23883437

  15. The wonderous chaperones: A highlight on therapeutics of cancer and potentially malignant disorders

    Directory of Open Access Journals (Sweden)

    Nutan Tyagi

    2015-01-01

    Full Text Available Diverse environmental and physiological factors are known to induce the transcription of a set of genes encoding special protective molecules known as "molecular chaperones" within our cells. Literature abounds in evidence regarding the varied roles; these "guides" can effectively perform in our system. Highly conserved through evolution, from the prokaryotes to the eukaryotes, these make perfect study tools for verifying their role in both the pathogenesis as well as the therapeutics of varied neurodegenerative, autoimmune and potentially malignant disorders and varied cancer states. We present a concise review of this ever dynamic molecule, highlighting the probable role in a potentially malignant disorder, oral lichen planus.

  16. Outer-membrane PapC molecular usher discriminately recognizes periplasmic chaperone-pilus subunit complexes.

    OpenAIRE

    Dodson, K W; Jacob-Dubuisson, F; Striker, R T; Hultgren, S. J.

    1993-01-01

    P pili are highly ordered composite structures consisting of thin fibrillar tips joined end-to-end to rigid helical rods. The production of these virulence-associated structures requires a periplasmic chaperone (PapD) and an outer membrane protein (PapC) that is the prototype member of a newly recognized class of proteins that we have named "molecular ushers." Two in vitro assays showed that the preassembly complexes that PapD forms with the three most distal tip fibrillar proteins (PapG, Pap...

  17. Quantifying the role of chaperones in protein translocation by computational modelling

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    Salvatore eAssenza

    2015-03-01

    Full Text Available The molecular chaperone Hsp70 plays a central role in the import of cytoplasmic proteins into organelles,driving their translocation by binding them from the organellar interior. Starting from the experimentally-determined structure of the E. coli Hsp70, we computed, by means of molecular simulations,the effective free-energy profile for substrate translocation uponchaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import process, whose comparison with unassisted translocation highlights the essential role played by Hsp70 in importing cytoplasmic proteins.

  18. PpiD is a player in the network of periplasmic chaperones in Escherichia coli

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    Behrens-Kneip Susanne

    2010-09-01

    Full Text Available Abstract Background The inner membrane-anchored periplasmic folding factor PpiD is described as a parvulin-like peptidyl prolyl isomerase (PPIase that assists in the maturation of the major beta-barrel outer membrane proteins (OMPs of Escherichia coli. More recent work however, calls these findings into question. Here, we re-examined the role of PpiD in the E. coli periplasm by analyzing its functional interplay with other folding factors that influence OMP maturation as well as general protein folding in the periplasmic compartment of the cell, such as SurA, Skp, and DegP. Results The analysis of the effects of both deletion and overexpression of ppiD on cell envelope phenotypes revealed that PpiD in contrast to prior observations plays only a minor role, if any, in the maturation of OMPs and cannot compensate for the lack of SurA in the periplasm. On the other hand, our results show that overproduction of PpiD rescues a surA skp double mutant from lethality. In the presence of increased PpiD levels surA skp cells show reduced activities of both the SigmaE-dependent and the Cpx envelope stress responses, and contain increased amounts of folded species of the major OMP OmpA. These effects require the anchoring of PpiD in the inner membrane but are independent of its parvulin-like PPIase domain. Moreover, a PpiD protein lacking the PPIase domain also complements the growth defects of an fkpA ppiD surA triple PPIase mutant and exhibits chaperone activity in vitro. In addition, PpiD appears to collaborate with DegP, as deletion of ppiD confers a temperature-dependent conditional synthetic phenotype in a degP mutant. Conclusions This study provides first direct evidence that PpiD functions as a chaperone and contributes to the network of periplasmic chaperone activities without being specifically involved in OMP maturation. Consistent with previous work, our data support a model in which the chaperone function of PpiD is used to aid in the early

  19. Ebb-and-flow of macroautophagy and chaperone-mediated autophagy in Raji cells induced by starvation and arsenic trioxide.

    Science.gov (United States)

    Li, Cai-Li; Wei, Hu-Lai; Chen, Jing; Wang, Bei; Xie, Bei; Fan, Lin-Lan; Li, Lin-Jing

    2014-01-01

    Autophagy is crucial in the maintenance of homeostasis and regenerated energy of mammalian cells. Macroautophagy and chaperone-mediated autophagy(CMA) are the two best-identified pathways. Recent research has found that in normal cells, decline of macroautophagy is appropriately parallel with activation of CMA. However, whether it is also true in cancer cells has been poorly studied. Here we focused on cross-talk and conversion between macroautophagy and CMA in cultured Burkitt lymphoma Raji cells when facing serum deprivation and exposure to a toxic compound, arsenic trioxide. The results showed that both macroautophagy and CMA were activated sequentially instead of simultaneously in starvation-induced Raji cells, and macroautophagy was quickly activated and peaked during the first hours of nutrition deprivation, and then gradually decreased to near baseline. With nutrient deprivation persisted, CMA progressively increased along with the decline of macroautophagy. On the other hand, in arsenic trioxide-treated Raji cells, macroautophagy activity was also significantly increased, but CMA activity was not rapidly enhanced until macroautophagy was inhibited by 3-methyladenine, an inhibitor. Together, we conclude that cancer cells exhibit differential responses to diverse stressor-induced damage by autophagy. The sequential switch of the first-aider macroautophagy to the homeostasis-stabilizer CMA, whether active or passive, might be conducive to the adaption of cancer cells to miscellaneous intracellular or extracellular stressors. These findings must be helpful to understand the characteristics, compensatory mechanisms and answer modes of different autophagic pathways in cancer cells, which might be very important and promising to the development of potential targeting interventions for cancer therapies via regulation of autophagic pathways. PMID:25081691

  20. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases.

    Science.gov (United States)

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies.

  1. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    Science.gov (United States)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  2. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

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    Hongjie Xia

    2015-07-01

    Full Text Available RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71, which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16, another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings

  3. Water and molecular chaperones act as weak links of protein folding networks: energy landscape and punctuated equilibrium changes point towards a game theory of proteins.

    Science.gov (United States)

    Kovács, István A; Szalay, Máté S; Csermely, Peter

    2005-04-25

    Water molecules and molecular chaperones efficiently help the protein folding process. Here we describe their action in the context of the energy and topological networks of proteins. In energy terms water and chaperones were suggested to decrease the activation energy between various local energy minima smoothing the energy landscape, rescuing misfolded proteins from conformational traps and stabilizing their native structure. In kinetic terms water and chaperones may make the punctuated equilibrium of conformational changes less punctuated and help protein relaxation. Finally, water and chaperones may help the convergence of multiple energy landscapes during protein-macromolecule interactions. We also discuss the possibility of the introduction of protein games to narrow the multitude of the energy landscapes when a protein binds to another macromolecule. Both water and chaperones provide a diffuse set of rapidly fluctuating weak links (low affinity and low probability interactions), which allow the generalization of all these statements to a multitude of networks. PMID:15848154

  4. A Salmonella type three secretion effector/chaperone complex adopts a hexameric ring-like structure.

    Science.gov (United States)

    Roblin, Pierre; Dewitte, Frédérique; Villeret, Vincent; Biondi, Emanuele G; Bompard, Coralie

    2015-02-15

    Many bacterial pathogens use type three secretion systems (T3SS) to inject virulence factors, named effectors, directly into the cytoplasm of target eukaryotic cells. Most of the T3SS components are conserved among plant and animal pathogens, suggesting a common mechanism of recognition and secretion of effectors. However, no common motif has yet been identified for effectors allowing T3SS recognition. In this work, we performed a biochemical and structural characterization of the Salmonella SopB/SigE chaperone/effector complex by small-angle X-ray scattering (SAXS). Our results showed that the SopB/SigE complex is assembled in dynamic homohexameric-ring-shaped structures with an internal tunnel. In this ring, the chaperone maintains a disordered N-terminal end of SopB molecules, in a good position to be reached and processed by the T3SS. This ring dimensionally fits the ring-organized molecules of the injectisome, including ATPase hexameric rings; this organization suggests that this structural feature is important for ATPase recognition by T3SS. Our work constitutes the first evidence of the oligomerization of an effector, analogous to the organization of the secretion machinery, obtained in solution. As effectors share neither sequence nor structural identity, the quaternary oligomeric structure could constitute a strategy evolved to promote the specificity and efficiency of T3SS recognition.

  5. A mathematical model of the dynamics of prion aggregates with chaperone-mediated fragmentation.

    Science.gov (United States)

    Davis, Jason K; Sindi, Suzanne S

    2016-05-01

    Prions are proteins most commonly associated with fatal neurodegenerative diseases in mammals but are also responsible for a number of harmless heritable phenotypes in yeast. These states arise when a misfolded form of a protein appears and, rather than be removed by cellular quality control mechanisms, persists. The misfolded prion protein forms aggregates and is capable of converting normally folded protein to the misfolded state through direct interaction between the two forms. The dominant mathematical model for prion aggregate dynamics has been the nucleated polymerization model (NPM) which considers the dynamics of only the normal protein and the aggregates. However, for yeast prions the molecular chaperone Hsp104 is essential for prion propagation. Further, although mammals do not express Hsp104, experimental assays have shown Hsp104 also interacts with mammalian prion aggregates. In this study, we generalize the NPM to account for molecular chaperones and develop what we call the enzyme-limited nucleated polymerization model (ELNPM). We discuss existence, uniqueness and stability of solutions to our model and demonstrate that the NPM represents a quasi-steady-state reduction of our model. We validate the ELNPM by demonstrating agreement with experimental results on the yeast prion [Formula: see text] PSI [Formula: see text] that could not be supported by the NPM. Finally, we demonstrate that, in contrast to the NPM, the ELNPM permits the coexistence of multiple prion strains. PMID:26297259

  6. Heterologous gln/asn-rich proteins impede the propagation of yeast prions by altering chaperone availability.

    Directory of Open Access Journals (Sweden)

    Zi Yang

    Full Text Available Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q/asparagine (N-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller "seeds." We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI(+] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI(+] or [URE3] prions. We explore in detail the events leading to the loss (curing of [PSI(+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI(+].

  7. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein.

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M; Sim, Valerie L; Woodside, Michael T

    2016-01-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. PMID:27346148

  8. Hsp40s specify functions of Hsp104 and Hsp90 protein chaperone machines.

    Directory of Open Access Journals (Sweden)

    Michael Reidy

    2014-10-01

    Full Text Available Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.

  9. Chaperone potential of Pulicaria undulata extract in preventing aggregation of stressed proteins.

    Science.gov (United States)

    Ghahghaei, Arezou; Valizadeh, Jafar; Nazari, Shahrzad; Ravandeh, Mehdi

    2014-06-01

    This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease.

  10. Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?

    Science.gov (United States)

    Nakanishi, Kotaro

    2016-09-01

    RNA silencing is a eukaryote-specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA-induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference-based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone-assisted duplex loading, and the slicer-dependent and slicer-independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637-660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. PMID:27184117

  11. The Hsp110 molecular chaperone stabilizes apolipoprotein B from endoplasmic reticulum-associated degradation (ERAD).

    Science.gov (United States)

    Hrizo, Stacy L; Gusarova, Viktoria; Habiel, David M; Goeckeler, Jennifer L; Fisher, Edward A; Brodsky, Jeffrey L

    2007-11-01

    Apolipoprotein B (apoB) is the most abundant protein in low density lipoproteins and plays key roles in cholesterol homeostasis. The co-translational degradation of apoB is controlled by fatty acid levels in the endoplasmic reticulum (ER) and is mediated by the proteasome. To define the mechanism of apoB degradation, we employed a cell-free system in which proteasome-dependent degradation is recapitulated with yeast cytosol, and we developed an apoB yeast expression system. We discovered that a yeast Hsp110, Sse1p, associates with and stabilizes apoB, which contrasts with data indicating that select Hsp70s and Hsp90s facilitate apoB degradation. However, the Ssb Hsp70 chaperones have no effect on apoB turnover. To determine whether our results are relevant in mammalian cells, Hsp110 was overexpressed in hepatocytes, and enhanced apoB secretion was observed. This study indicates that chaperones within distinct complexes can play unique roles during ER-associated degradation (ERAD), establishes a role for Sse1/Hsp110 in ERAD, and identifies Hsp110 as a target to lower cholesterol. PMID:17823116

  12. Glutathione selectively modulates the binding of platinum drugs to human copper chaperone Cox17.

    Science.gov (United States)

    Zhao, Linhong; Wang, Zhen; Wu, Han; Xi, Zhaoyong; Liu, Yangzhong

    2015-12-01

    The copper chaperone Cox17 (cytochrome c oxidase copper chaperone) has been shown to facilitate the delivery of cisplatin to mitochondria, which contributes to the overall cytotoxicity of the drug [Zhao et al. (2014) Chem. Commun. 50: , 2667-2669]. Kinetic data indicate that Cox17 has reactivity similar to glutathione (GSH), the most abundant thiol-rich molecule in the cytoplasm. In the present study, we found that GSH significantly modulates the reaction of platinum complexes with Cox17. GSH enhances the reactivity of three anti-cancer drugs (cisplatin, carboplatin and oxaliplatin) to Cox17, but suppresses the reaction of transplatin. Surprisingly, the pre-formed cisplatin-GSH adducts are highly reactive to Cox17; over 90% platinum transfers from GSH to Cox17. On the other hand, transplatin-GSH adducts are inert to Cox17. These different effects are consistent with the drug activity of these platinum complexes. In addition, GSH attenuates the protein aggregation of Cox17 induced by platination. These results indicate that the platinum-protein interactions could be substantially influenced by the cellular environment.

  13. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-06-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

  14. Modulation of the chaperone-like activity of bovine alpha-crystallin.

    Science.gov (United States)

    Clark, J I; Huang, Q L

    1996-12-24

    The effects of pantethine, glutathione, and selected chemical reagents on the anti-aggregation activity of alpha-crystallin was evaluated. Protein aggregation was monitored by light scattering of solutions of denatured beta L-crystallin or alcohol dehydrogenase (ADH). The ratios of beta L-crystallin/alpha-crystallin and ADH/alpha-crystallin were adjusted so that partial inhibition of protein aggregation at 60 degrees C or 37 degrees C, respectively, was observed and modulation of the chaperone action of alpha-crystallin could be evaluated easily with selected endogenous metabolites. Enhancement of the anti-aggregation activity in the beta L-crystallin assay was strongest with pantethine, which appeared to interact with alpha-crystallin. Enhancement of the anti-aggregation activity in the ADH assay was strongest with glutathione which appeared to interact with ADH. The results indicated that the products of common metabolic pathways can modulate the chaperone-like effects of alpha-crystallin on protein aggregation. PMID:8986785

  15. Chaperones are necessary for the expression of catalytically active potato apyrases in prokaryotic cells.

    Science.gov (United States)

    Porowińska, Dorota; Czarnecka, Joanna; Komoszyński, Michał

    2014-07-01

    NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

  16. The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase

    Directory of Open Access Journals (Sweden)

    Maria Ranieri-Raggi

    2014-05-01

    Full Text Available Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD and the metal binding protein histidine-proline-rich glycoprotein (HPRG acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua(μ-carboxylatodizinc(II core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated.

  17. Contributions of chaperone and glycosyltransferase activities of O-fucosyltransferase 1 to Notch signaling

    Directory of Open Access Journals (Sweden)

    Irvine Kenneth D

    2008-01-01

    Full Text Available Abstract Background O-fucosyltransferase1 (OFUT1 is a conserved ER protein essential for Notch signaling. OFUT1 glycosylates EGF domains, which can then be further modified by the N-acetylglucosaminyltransferase Fringe. OFUT1 also possesses a chaperone activity that promotes the folding and secretion of Notch. Here, we investigate the respective contributions of these activities to Notch signaling in Drosophila. Results We show that expression of an isoform lacking fucosyltransferase activity, Ofut1R245A, rescues the requirement for Ofut1 in embryonic neurogenesis. Lack of requirement for O-fucosylation is further supported by the absence of embryonic phenotypes in Gmd mutants, which lack all forms of fucosylation. Requirements for O-fucose during imaginal development were evaluated by characterizing clones of cells expressing only Ofut1R245A. These clones phenocopy fringe mutant clones, indicating that the absence of O-fucose is functionally equivalent to the absence of elongated O-fucose. Conclusion Our results establish that Notch does not need to be O-fucosylated for fringe-independent Notch signaling in Drosophila; the chaperone activity of OFUT1 is sufficient for the generation of functional Notch.

  18. The archaic chaperone-usher pathways may depend on donor strand exchange for intersubunit interactions.

    Science.gov (United States)

    Wu, Miaomiao; Xu, Shihui; Zhu, Wei; Mao, Xiaohua

    2014-10-01

    Subunit-subunit interactions of the classical and alternate chaperone-usher (CU) systems have been shown to proceed through a donor strand exchange (DSE) mechanism. However, it is not known whether DSE is required for intersubunit interactions in the archaic CU system. We have previously shown that the Myxococcus xanthus Mcu system, a member of the archaic CU family that functions in spore coat formation, is likely to use the principle of donor strand complementation to medicate chaperone-subunit interactions analogous to the classical CU pathway. Here we describe the results of studies on Mcu subunit-subunit interactions. We constructed a series of N-terminal-deleted, single amino acid-mutated and donor strand-complemented Mcu subunits, and characterized their abilities to participate in subunit-subunit interactions. It appears that certain residues in both the N and C termini of McuA, a subunit of the Mcu system, play a critical role in intersubunit interactions and these interactions may involve the general principle of DSE of the classical and alternate CU systems. In addition, the specificity of the M. xanthus CU system for Mcu subunits over other spore coat proteins is demonstrated.

  19. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-01-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. PMID:27346148

  20. Structure, Function and Regulation of the Hsp90 Machinery

    Directory of Open Access Journals (Sweden)

    Jing Li

    2013-06-01

    Full Text Available Heat shock protein 90 (Hsp90 is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

  1. Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis.

    Directory of Open Access Journals (Sweden)

    Wilhelmina M Huston

    Full Text Available DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog. We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.

  2. Chaperone-Assisted Formation of Cucurbit[8]uril-Based Molecular Porous Materials with One-Dimensional Channel Structure.

    Science.gov (United States)

    Zhu, Wei; Wang, Chen; Lan, Yue; Li, Jian; Wang, Hui; Gao, Ning; Ji, Jingwei; Li, Guangtao

    2016-09-01

    Exploiting "chaperone molecule" to navigate the successful assembly energy landscapes has been extensively used in biological systems, whereas in artifical supramolecular systems the "chaperone-assisted" assembly strategy to be used for the synthesis of materials with novel structures or the structures to be hardly prepared by "conventional" methods are still far from realizing the potential functions. In this work, we present a new example of small organic molecule acting as "chaperone molecule" in the facile formation of organic molecular porous materials. This porous material is composed of pure cucurbit[8]uril (CB[8]) macrocycle and possesses a honeycomb-like structure with an isolated and relatively large one-dimensional (1D) nanochannel. Moreover, it has good chemical and thermal stability, and shows a good adsorption capability for large molecule loading. Importantly, with the assistance of chaperone molecules, pure CB[8] could also be recycled even from a complex aqueous solution, demonstrating a powerful purification method of CB[8] from complex systems. PMID:27539793

  3. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks

    DEFF Research Database (Denmark)

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia;

    2015-01-01

    , chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required...

  4. Structure of Human J-type Co-chaperone HscB Reveals a Tetracysteine Metal-binding Domain

    Energy Technology Data Exchange (ETDEWEB)

    Bitto, Eduard; Bingman, Craig A.; Bittova, Lenka; Kondrashov, Dmitry A.; Bannen, Ryan M.; Fox, Brian G.; Markley, John L.; Phillips, Jr., George N. (UW); (UC)

    2008-11-24

    Iron-sulfur proteins play indispensable roles in a broad range of biochemical processes. The biogenesis of iron-sulfur proteins is a complex process that has become a subject of extensive research. The final step of iron-sulfur protein assembly involves transfer of an iron-sulfur cluster from a cluster-donor to a cluster-acceptor protein. This process is facilitated by a specialized chaperone system, which consists of a molecular chaperone from the Hsc70 family and a co-chaperone of the J-domain family. The 3.0 A crystal structure of a human mitochondrial J-type co-chaperone HscB revealed an L-shaped protein that resembles Escherichia coli HscB. The important difference between the two homologs is the presence of an auxiliary metal-binding domain at the N terminus of human HscB that coordinates a metal via the tetracysteine consensus motif CWXCX(9-13)FCXXCXXXQ. The domain is found in HscB homologs from animals and plants as well as in magnetotactic bacteria. The metal-binding site of the domain is structurally similar to that of rubredoxin and several zinc finger proteins containing rubredoxin-like knuckles. The normal mode analysis of HscB revealed that this L-shaped protein preferentially undergoes a scissors-like motion that correlates well with the conformational changes of human HscB observed in the crystals.

  5. Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis.

    Science.gov (United States)

    Valenzuela, Nicolas; Fan, Qiying; Fa'ak, Faisal; Soibam, Benjamin; Nagandla, Harika; Liu, Yu; Schwartz, Robert J; McConnell, Bradley K; Stewart, M David

    2016-03-01

    HIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis. PMID:26935106

  6. A chaperone function of NO CATALASE ACTIVITY1 is required to maintain catalase activity and for multiple stress responses in Arabidopsis.

    Science.gov (United States)

    Li, Jing; Liu, Juntao; Wang, Guoqiang; Cha, Joon-Yung; Li, Guannan; Chen, She; Li, Zhen; Guo, Jinghua; Zhang, Caiguo; Yang, Yongqing; Kim, Woe-Yeon; Yun, Dae-Jin; Schumaker, Karen S; Chen, Zhongzhou; Guo, Yan

    2015-03-01

    Catalases are key regulators of reactive oxygen species homeostasis in plant cells. However, the regulation of catalase activity is not well understood. In this study, we isolated an Arabidopsis thaliana mutant, no catalase activity1-3 (nca1-3) that is hypersensitive to many abiotic stress treatments. The mutated gene was identified by map-based cloning as NCA1, which encodes a protein containing an N-terminal RING-finger domain and a C-terminal tetratricopeptide repeat-like helical domain. NCA1 interacts with and increases catalase activity maximally in a 240-kD complex in planta. In vitro, NCA1 interacts with CATALASE2 (CAT2) in a 1:1 molar ratio, and the NCA1 C terminus is essential for this interaction. CAT2 activity increased 10-fold in the presence of NCA1, and zinc ion binding of the NCA1 N terminus is required for this increase. NCA1 has chaperone protein activity that may maintain the folding of catalase in a functional state. NCA1 is a cytosol-located protein. Expression of NCA1 in the mitochondrion of the nca1-3 mutant does not rescue the abiotic stress phenotypes of the mutant, while expression in the cytosol or peroxisome does. Our results suggest that NCA1 is essential for catalase activity.

  7. High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK

    Directory of Open Access Journals (Sweden)

    Schloot Nanette C

    2010-11-01

    Full Text Available Abstract Background Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (preproinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK. Results Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further region in each proinsulin domain (A- and B-chain, C-peptide. Among the latter, peptides covering most of the B-chain region B11-23 exhibited strongest binding, which was in the range of known high-affinity DnaK ligands, dissociation equilibrium constant (K'd of 2.2 ± 0.4 μM. The B-chain region B11-23 is located at the interface between two insulin molecules and not accessible in insulin oligomers. Indeed, native insulin oligomers showed very low DnaK affinity (K'd 67.8 ± 20.8 μM whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity (K'd 11.3 ± 7.8 μM. Conclusions Intact insulin only weakly interacts with the hsp70 chaperone DnaK whereas monomeric proinsulin and peptides from 3 distinct proinsulin regions show substantial chaperone binding. Strongest binding was seen for the B-chain peptide B 11-23. Interestingly, peptide B11-23 represents a dominant autoantigen in type 1 diabetes.

  8. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B).

    Science.gov (United States)

    Haigney, Allison; Ricketts, M Daniel; Marmorstein, Ronen

    2015-12-18

    The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

  9. The Role of Bacterial Chaperones in the Circulative Transmission of Plant Viruses by Insect Vectors

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    Murad Ghanim

    2013-06-01

    Full Text Available Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of Potato leafroll virus (PLRV by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed.

  10. The role of bacterial chaperones in the circulative transmission of plant viruses by insect vectors.

    Science.gov (United States)

    Kliot, Adi; Ghanim, Murad

    2013-06-01

    Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of Potato leafroll virus (PLRV) by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV) by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed. PMID:23783810

  11. Mechanism of thermal aggregation of yeast alcohol dehydrogenase I: role of intramolecular chaperone.

    Science.gov (United States)

    Markossian, Kira A; Golub, Nikolay V; Khanova, Helen A; Levitsky, Dmitrii I; Poliansky, Nikolay B; Muranov, Konstantin O; Kurganov, Boris I

    2008-09-01

    Kinetics of thermal aggregation of yeast alcohol dehydrogenase I (yADH) have been studied using dynamic light scattering at a fixed temperature (56 degrees C) and under the conditions where the temperature was elevated at a constant rate (1 K/min). The initial parts of the dependences of the hydrodynamic radius on time (or temperature) follow the exponential law. At rather high values of time splitting of the population of aggregates into two components occurs. It is assumed that such peculiarities of the kinetics of thermal aggregation of yADH are due to the presence of a sequence -YSGVCHTDLHAWHGDWPLPVK- in the polypeptide chain possessing chaperone-like activity. Thermodynamic parameters for thermal denaturation of yADH have been calculated from the differential scanning calorimetry data.

  12. Heat shock protein 70 chaperoned alpha-fetoprotein in human hepatocellular carcinoma cell line BEL-7402

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ping Wang; Qiao-Xia Wang; Hai-Yan Li; Rui-Fen Chen

    2005-01-01

    AIM: To investigate the interaction between heat shock protein 70 (HSP70) and α-fetoprotein (AFP) in human hepatocellular carcinoma (HCC) cell line BEL7402.METHODS: The expression and localization of HSP70 and AFP in human HCC cell line BEL-7402 were determined by immunocytochemistry and indirect immunofluorescence cytochemical staining. The interaction between HSP70 and AFP in HCC cells was analyzed by immunoprecipitation and Western blot.RESULTS: Immunocytochemical staining detection showed that HCC cell BEL-7402 expressed a high level of HSP70 and AFP synchronously. Both were stained in cell plasma.AFP existed in the immunoprecipitate of anti-HSP70 mAb,while there was HSP70 in the immunoprecipitate of antiAFP mAb.CONCLUSION: HSP70 chaperones AFP in human HCCcell BEL-7402. The interaction between HSP70 and AFP in human HCC cell can be a new route to study the pathogenesis and immunotherapy of HCC.

  13. The extracellular chaperone clusterin sequesters oligomeric forms of the Aβ1–40 peptide

    Science.gov (United States)

    Narayan, Priyanka; Orte, Angel; Clarke, Richard W.; Bolognesi, Benedetta; Hook, Sharon; Ganzinger, Kristina A.; Meehan, Sarah; Wilson, Mark R.; Dobson, Christopher M.; Klenerman, David

    2016-01-01

    In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a novel risk factor in Alzheimer’s disease (AD). We have examined the interactions between clusterin and the AD-associated amyloid-β1–40 peptide (Aβ1–40) which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single molecule fluorescence methods, we have found that Aβ1–40 forms a heterogeneous distribution of small oligomers (from dimers to 50mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence strongly both the aggregation and disaggregation of Aβ1–40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and AD. PMID:22179788

  14. A mutant chaperone converts a wild-type protein into a tumor-specific antigen.

    Science.gov (United States)

    Schietinger, Andrea; Philip, Mary; Yoshida, Barbara A; Azadi, Parastoo; Liu, Hui; Meredith, Stephen C; Schreiber, Hans

    2006-10-13

    Monoclonal antibodies have become important therapeutic agents against certain cancers. Many tumor-specific antigens are mutant proteins that are predominantly intracellular and thus not readily accessible to monoclonal antibodies. We found that a wild-type transmembrane protein could be transformed into a tumor-specific antigen. A somatic mutation in the chaperone gene Cosmc abolished function of a glycosyltransferase, disrupting O-glycan Core 1 synthesis and creating a tumor-specific glycopeptidic neo-epitope consisting of a monosaccharide and a specific wild-type protein sequence. This epitope induced a high-affinity, highly specific, syngeneic monoclonal antibody with antitumor activity. Such tumor-specific glycopeptidic neo-epitopes represent potential targets for monoclonal antibody therapy.

  15. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2

    Science.gov (United States)

    2016-01-01

    One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation. PMID:27454736

  16. Single-step Purification of Molecular Chaperone GroEL by Expanded Bed Chromatography

    Institute of Scientific and Technical Information of China (English)

    佟晓冬; 杨征; 董晓燕; 孙彦

    2003-01-01

    Expanded bed adsorption (EBA) is an integrative downstream processing technique for the purification of biological substances directly from unclarified feedstock. In this study, molecular chaperone GroEL, an important protein folding helper both in vivo and in vitro, was purified by the single-step EBA technique from the unclarified homogenate of recombinant E. coli cells. Compared with packed bed adsorption, the EBA technique provided a single-step approach to yield an electrophoretic purity of GroEL. After the homogenate loading and column washing in the expanded bed mode, the GroEL protein was recovered by stepwise salt-gradient elution in packed-bed or expanded-bed modes, respectively. The expanded-bed elution mode was found as efficient as the packed-bed mode in the purification of GroEL from cell disruptate.

  17. Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

    Science.gov (United States)

    Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

    2015-05-15

    The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.

  18. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2.

    Directory of Open Access Journals (Sweden)

    Angela Lavado-Roldán

    2016-07-01

    Full Text Available One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2 acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation.

  19. Molecular cloning, organellar targeting and developmental expression of mitochondrial chaperone HSP60 in Toxoplasma gondii.

    Science.gov (United States)

    Toursel, C; Dzierszinski, F; Bernigaud, A; Mortuaire, M; Tomavo, S

    2000-12-01

    The obligate intracellular protozoan parasite Toxoplasma gondii has a single tubular mitochondrion. During infection, it recruits the host cell's mitochondria abutting to the intracellular vacuole, that contains the parasites. The respective contribution of host and parasitic mitochondria in the intracellular growth of T. gondii remains unknown. Heat shock protein, HSP60 has been reported in all eukaryotes examined, as an essential chaperone required for the folding and multimeric complex assembly of mitochondrial proteins. Here, we report the isolation and molecular characterization of two cDNAs corresponding to a single T. gondii gene coding for HSP60. Using a model fusion protein, preHSP60-chloramphenicol acetyl transferase (CAT), we demonstrate that the classical 22 amino acid mitochondrial presequence and the adjacent 32 amino acids of the mature protein are both required for the in vivo import into T. gondii mitochondria. The T. gondii HSP60 gene composed of five introns and six exons is transcribed into two related but differently spliced transcripts. Whereas the two transcripts can be detected in both developmental stages within the intermediate host, their levels are significantly increased in bradyzoites when compared to tachyzoites. By immunoblot analysis, the predicted 60-kDa protien corresponding to HSP60 was detected in both tachyzoite and bradyzoite forms. Using immunofluorescence assays. the polyclonal antibodies specific to T. gondii HSP60 recognized the mitochondrion in tachyzoites, as expected. In contrast, these antibodies reacted against two unknown vesicular bodies which are distinct from the classical mitochondrial pattern in bradyzoites. Taken together. these expression patterns of mitochondrial chaperone HSP60 suggests stage-specific induction of the respiratory pathway in the protozoan parasite T. gondii.

  20. Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Qualley, Dominic F., E-mail: dqualley@berry.edu; Sokolove, Victoria L.; Ross, James L.

    2015-03-13

    Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC.

  1. The mitochondrial chaperone protein TRAP1 mitigates α-Synuclein toxicity.

    Directory of Open Access Journals (Sweden)

    Erin K Butler

    2012-02-01

    Full Text Available Overexpression or mutation of α-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human [A53T]α-Synuclein-induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1 was found to enhance age-dependent loss of fly head dopamine (DA and DA neuron number resulting from [A53T]α-Synuclein expression. In addition, decreased TRAP1 expression in [A53T]α-Synuclein-expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued [A53T]α-Synuclein-induced sensitivity to rotenone treatment. In human (nonneuronal cell lines, small interfering RNA directed against TRAP1 enhanced [A53T]α-Synuclein-induced sensitivity to oxidative stress treatment. [A53T]α-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by [A53T]α-Synuclein overexpression in human SH-SY5Y cells. These results indicate that [A53T]α-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to α-Synuclein.

  2. Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

    International Nuclear Information System (INIS)

    Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC

  3. Cloning, expression and crystallisation of SGT1 co-chaperone protein from Glaciozyma antarctica

    Science.gov (United States)

    Yusof, Nur Athirah; Bakar, Farah Diba Abu; Beddoe, Travis; Murad, Abdul Munir Abdul

    2013-11-01

    Studies on psycrophiles are now in the limelight of today's post genomic era as they fascinate the research and development industries. The discovery from Glaciozyma antarctica, an extreme cold adapted yeast from Antarctica shows promising future to provide cost effective natural sustainable energy and create wider understanding of the property that permits this organisms to adapt to extreme temperature downshift. In plants and yeast, studies show the interaction between SGT1 and HSP90 are essential for disease resistance and heat stress by activating a number of resistance proteins. Here we report for the first time cloning, expression and crystallization of the recombinant SGT1 protein of G. antarctica (rGa_SGT1), a highly conserved eukaryotic protein that interacts with the molecular chaperones HSP90 (heat shock protein 90) apparently associated in a role of co-chaperone that may play important role in cold adaptation. The sequence analysis of rGa_SGT1 revealed the presence of all the characteristic features of SGT1 protein. In this study, we present the outlines and results of protein structural study of G. antarctica SGT1 protein. We validate this approach by starting with cloning the target insert into Ligation Independent Cloning system proceeded with expression using E. coli system, and crystallisation of the target rGA_SGT1 protein. The work is still on going with the target subunit of the complex proteins yielded crystals. These results, still ongoing, open a platform for better understanding of the uniqueness of this crucial molecular machine function in cold adaptation.

  4. Evaluation of molecular chaperons Hsp72 and neuropeptide Y as characteristic markers of adaptogenic activity of plant extracts.

    Science.gov (United States)

    Asea, Alexzander; Kaur, Punit; Panossian, Alexander; Wikman, Karl Georg

    2013-11-15

    We have previously demonstrated that ADAPT-232, a fixed combination of adaptogenic substances derived from Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract stimulated the expression and release of neuropeptide Y (NPY) and molecular chaperone Hsp72 from isolated human neurolgia cells. Both of these mediators of stress response are known to play an important role in regulation of neuroendocrine system and immune response. We further demonstrated that ADAPT-232 induced release of Hsp70 is mediated by NPY, suggesting an existence of NPY-mediated pathway of activation of Hsp72 release into the blood circulation system. The objective of this study was to determine whether this pathway is common for adaptogens and whether NPY and/or Hsp72 can be considered as necessary specific biomarkers for adaptogenic activity. The release of NPY and Hsp72 from neuroglia cells in response to treatment with various plant extracts (n=23) including selected validated adaptogens, partly validated adaptogens, claimed but negligibly validated adaptogens and some other plant extracts affecting neuroendocrine and immune systems but never considered as adaptogens was measured using high throughput ELISA techniques. We demonstrated that adaptogens, e.g. R. rosea, S. chinensis and E. senticosus stimulate both NPY and Hsp70 release from neuroblastoma cells, while tonics and stimulants have no significant effect on NPY in this in vitro test. In the groups of partly validated adaptogens the effect of Panax ginseng and Withania somnifera was not statistically significant both on NPY and Hsp70 release, while the activating effect of Bryonia alba and Rhaponticum cartamoides was significant only on Hsp70. In contrast, all tested non-adaptogens, such as antiinflammatoty plant extracts Matricaria recutita, Pelargonium sidoides, Hedera helix and Vitis vinifera significantly inhibit Hsp70 release and have no influence on NPY release from neuroblastoma

  5. Drug Development in Conformational Diseases: A Novel Family of Chemical Chaperones that Bind and Stabilise Several Polymorphic Amyloid Structures.

    Directory of Open Access Journals (Sweden)

    Marquiza Sablón-Carrazana

    Full Text Available The increasing prevalence of conformational diseases, including Alzheimer's disease, type 2 Diabetes Mellitus and Cancer, poses a global challenge at many different levels. It has devastating effects on the sufferers as well as a tremendous economic impact on families and the health system. In this work, we apply a cross-functional approach that combines ideas, concepts and technologies from several disciplines in order to study, in silico and in vitro, the role of a novel chemical chaperones family (NCHCHF in processes of protein aggregation in conformational diseases. Given that Serum Albumin (SA is the most abundant protein in the blood of mammals, and Bovine Serum Albumin (BSA is an off-the-shelf protein available in most labs around the world, we compared the ligandability of BSA:NCHCHF with the interaction sites in the Human Islet Amyloid Polypeptide (hIAPP:NCHCHF, and in the amyloid pharmacophore fragments (Aβ17-42 and Aβ16-21:NCHCHF. We posit that the merging of this interaction sites is a meta-structure of pharmacophore which allows the development of chaperones that can prevent protein aggregation at various states from: stabilizing the native state to destabilizing oligomeric state and protofilament. Furthermore to stabilize fibrillar structures, thus decreasing the amount of toxic oligomers in solution, as is the case with the NCHCHF. The paper demonstrates how a set of NCHCHF can be used for studying and potentially treating the various physiopathological stages of a conformational disease. For instance, when dealing with an acute phase of cytotoxicity, what is needed is the recruitment of cytotoxic oligomers, thus chaperone F, which accelerates fiber formation, would be very useful; whereas in a chronic stage it is better to have chaperones A, B, C, and D, which stabilize the native and fibril structures halting self-catalysis and the creation of cytotoxic oligomers as a consequence of fiber formation. Furthermore, all the

  6. The modulation of endoplasmic reticulum stress by chemical chaperone upregulates immune negative cytokine IL-35 in apolipoprotein E-deficient mice.

    Science.gov (United States)

    Wang, Bo; Dai, Shen; Dong, Zhaojing; Sun, Yue; Song, Xingguo; Guo, Chun; Zhu, Faliang; Wang, Qun; Zhang, Lining

    2014-01-01

    Interleukin (IL)-35 is a newly identified immune negative molecule which is secreted by CD4(+)Foxp3(+) T regulatory cells (Tregs) and contributes to their suppressive capacity. Early data have shown that IL-35 inhibits development of several autoimmune diseases. However, the role of IL-35 in atherosclerosis, a lipid-driven chronic inflammatory disease in arterial wall, remains to be investigated. Here, we found that IL-35 was involved in atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. ApoE(-/-) mice with established atherosclerotic lesion displayed a lower level of IL-35 compared to age-matched wild type C57BL/6 mice without plaque. However, IL-35 expression increased significantly in ApoE(-/-) mice with attenuated plaque. More importantly, we found that modulation of ER stress treated by chemical chaperone, 4-Phenyl butyric acid (PBA) in vivo, mainly upregulated immune negative regulating molecule IL-35, as well as IL-10 and Foxp3, accompanied by increased Tregs. However, no obvious impact on pro-inflammatory molecules such as TNF-α, IFN-γ, IL-17 and IL-23 was observed, which provides new insight into the benefit of ER stress recovery from attenuated plaque. Our results suggest that IL-35 might have a potential value for atherosclerotic therapy.

  7. Discovery of novel interacting partners of PSMD9, a proteasomal chaperone: Role of an Atypical and versatile PDZ-domain motif interaction and identification of putative functional modules

    Science.gov (United States)

    Sangith, Nikhil; Srinivasaraghavan, Kannan; Sahu, Indrajit; Desai, Ankita; Medipally, Spandana; Somavarappu, Arun Kumar; Verma, Chandra; Venkatraman, Prasanna

    2014-01-01

    PSMD9 (Proteasome Macropain non-ATPase subunit 9), a proteasomal assembly chaperone, harbors an uncharacterized PDZ-like domain. Here we report the identification of five novel interacting partners of PSMD9 and provide the first glimpse at the structure of the PDZ-domain, including the molecular details of the interaction. We based our strategy on two propositions: (a) proteins with conserved C-termini may share common functions and (b) PDZ domains interact with C-terminal residues of proteins. Screening of C-terminal peptides followed by interactions using full-length recombinant proteins, we discovered hnRNPA1 (an RNA binding protein), S14 (a ribosomal protein), CSH1 (a growth hormone), E12 (a transcription factor) and IL6 receptor as novel PSMD9-interacting partners. Through multiple techniques and structural insights, we clearly demonstrate for the first time that human PDZ domain interacts with the predicted Short Linear Sequence Motif (SLIM) at the C-termini of the client proteins. These interactions are also recapitulated in mammalian cells. Together, these results are suggestive of the role of PSMD9 in transcriptional regulation, mRNA processing and editing, hormone and receptor activity and protein translation. Our proof-of-principle experiments endorse a novel and quick method for the identification of putative interacting partners of similar PDZ-domain proteins from the proteome and for discovering novel functions. PMID:25009770

  8. Discovery of novel interacting partners of PSMD9, a proteasomal chaperone: Role of an Atypical and versatile PDZ-domain motif interaction and identification of putative functional modules

    Directory of Open Access Journals (Sweden)

    Nikhil Sangith

    2014-01-01

    Full Text Available PSMD9 (Proteasome Macropain non-ATPase subunit 9, a proteasomal assembly chaperone, harbors an uncharacterized PDZ-like domain. Here we report the identification of five novel interacting partners of PSMD9 and provide the first glimpse at the structure of the PDZ-domain, including the molecular details of the interaction. We based our strategy on two propositions: (a proteins with conserved C-termini may share common functions and (b PDZ domains interact with C-terminal residues of proteins. Screening of C-terminal peptides followed by interactions using full-length recombinant proteins, we discovered hnRNPA1 (an RNA binding protein, S14 (a ribosomal protein, CSH1 (a growth hormone, E12 (a transcription factor and IL6 receptor as novel PSMD9-interacting partners. Through multiple techniques and structural insights, we clearly demonstrate for the first time that human PDZ domain interacts with the predicted Short Linear Sequence Motif (SLIM at the C-termini of the client proteins. These interactions are also recapitulated in mammalian cells. Together, these results are suggestive of the role of PSMD9 in transcriptional regulation, mRNA processing and editing, hormone and receptor activity and protein translation. Our proof-of-principle experiments endorse a novel and quick method for the identification of putative interacting partners of similar PDZ-domain proteins from the proteome and for discovering novel functions.

  9. In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E. coli

    Directory of Open Access Journals (Sweden)

    Wu Xiangping

    2012-09-01

    Full Text Available Abstract Background Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA and its chaperone (LipB from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E. coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. Results In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp and lipase specific foldase gene lipB (1023 bp. One single expression plasmid system E. coli BL21/pET28a-lipAB and two dual expression plasmid systems E. coli BL21/pETDuet-lipA-lipB and E. coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E. coli BL21/pET28a-lipAB and E. coli BL21/pETDuet-lipA-lipB were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by E. coli BL21/pACYCDuet-lipA-lipB was up to 8500 U/L. The lipase LipA had an optimal temperature of 30°C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol

  10. Interplay between Molecular Chaperones and the Ubiquitin-Proteasome System in Targeting of Misfolded Proteins for Degradation

    DEFF Research Database (Denmark)

    Poulsen, Esben Guldahl

    The focus of this PhD study has been the ubiquitin-proteasome system (UPS), with a particular emphasis on the recognition and degradation of misfolded proteins. Given the large number of proteins potentially involved in any given degradation pathway, several high-throughput screening techniques....... This study was based on the implementation of high-throughput siRNA screening, using a library against more than 600 UPS components. The second study was focused on studying proteins associated with the 26S proteasome in the fission yeast S. pombe. This study includes a proteomics analysis of proteins...... involved in the specific degradation pathway were identified. This work had its origin in epistasis mapping of two Hsp70 co-chaperone proteins, which also formed the basis for the fourth study. In this final study, the Hsp70 co-chaperones were overexpressed in S. pombe. This led to a cell growth defect...

  11. Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro.

    Science.gov (United States)

    Kuryan, Benjamin G; Kim, Jessica; Tran, Nancy Nga H; Lombardo, Sarah R; Venkatesh, Swaminathan; Workman, Jerry L; Carey, Michael

    2012-02-01

    ATPases and histone chaperones facilitate RNA polymerase II (pol II) elongation on chromatin. In vivo, the coordinated action of these enzymes is necessary to permit pol II passage through a nucleosome while restoring histone density afterward. We have developed a biochemical system recapitulating this basic process. Transcription through a nucleosome in vitro requires the ATPase remodels structure of chromatin (RSC) and the histone chaperone nucleosome assembly protein 1 (NAP1). In the presence of NAP1, RSC generates a hexasome. Despite the propensity of RSC to evict histones, NAP1 reprograms the reaction such that the hexasome is retained on the template during multiple rounds of transcription. This work has implications toward understanding the mechanism of pol II elongation on chromatin.

  12. An interaction network predicted from public data as a discovery tool: application to the Hsp90 molecular chaperone machine.

    Directory of Open Access Journals (Sweden)

    Pablo C Echeverría

    Full Text Available Understanding the functions of proteins requires information about their protein-protein interactions (PPI. The collective effort of the scientific community generates far more data on any given protein than individual experimental approaches. The latter are often too limited to reveal an interactome comprehensively. We developed a workflow for parallel mining of all major PPI databases, containing data from several model organisms, and to integrate data from the literature for a protein of interest. We applied this novel approach to build the PPI network of the human Hsp90 molecular chaperone machine (Hsp90Int for which previous efforts have yielded limited and poorly overlapping sets of interactors. We demonstrate the power of the Hsp90Int database as a discovery tool by validating the prediction that the Hsp90 co-chaperone Aha1 is involved in nucleocytoplasmic transport. Thus, we both describe how to build a custom database and introduce a powerful new resource for the scientific community.

  13. Increasing the catalytic activity of Bilirubin oxidase from Bacillus pumilus: Importance of host strain and chaperones proteins.

    Science.gov (United States)

    Gounel, Sébastien; Rouhana, Jad; Stines-Chaumeil, Claire; Cadet, Marine; Mano, Nicolas

    2016-07-20

    Aggregation of recombinant proteins into inclusion bodies (IBs) is the main problem of the expression of multicopper oxidase in Escherichia coli. It is usually attributed to inefficient folding of proteins due to the lack of copper and/or unavailability of chaperone proteins. The general strategies reported to overcome this issue have been focused on increasing the intracellular copper concentration. Here we report a complementary method to optimize the expression in E. coli of a promising Bilirubin oxidase (BOD) isolated from Bacillus pumilus. First, as this BOD has a disulfide bridge, we switched E.coli strain from BL21 (DE3) to Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm. In a second step, we investigate the effect of co-expression of chaperone proteins on the protein production and specific activity. Our strategy allowed increasing the final amount of enzyme by 858% and its catalytic rate constant by 83%.

  14. Heterologous expression, chaperone mediated solubilization and purification of parasitic nematode-specific growth factor-like protein of Setaria digitata

    Institute of Scientific and Technical Information of China (English)

    W WP Rodrigo; R S Dassanayake; E H Karunanayake; YIN Silva Gunawardene; O VDS J Weerasena

    2014-01-01

    Objective:To clone, express and purify a putative parasitic nematode specific protein of Setaria digitata (S. digitata), filarial nematode that infects livestock and cause significant economic losses inFarEast andAsia to be used for structural and functional analyses. Methods:To characterize uncharacterized gene ofS. digitata(SDUG), the herterologous expression ofSDUG was carried out in the pET [cloned into pET45b(+)] expression system initially and co-expression ofSDUG using chaperone plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2 and pTf16 containing chaperone proteins of dnaK-dnaJ-grpE-groES-gro-E, groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, and tig respectively, was carried out subsequently.Results:Expression ofSDUG was seen whenEscherichia coli strainBL21(DE3) is used, while concentrating protein largely into the insoluble fraction.The co-expression ofSDUG using chaperone plasmid mediated system indicated a significant increase of the protein in the soluble fraction.Of the chaperon plasmid sets, the highest amount of recombinantSDUP in the soluble fraction was seen when pGro7 was used in the presence of 2 mg/mLL-arabinose and0.6MIPTG concentration in the culture medium and for3 h of incubation at the temperature of28 ℃.RecombinantSDUG was purified both from soluble and insoluble fractions usingNi affinity chromatography.SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of ~24 kDa.Conclusions:SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.

  15. Chaperons expressions and search for new gravity-related genes in the embryos of crabs and amphibians

    Science.gov (United States)

    Gusev, O.; Kashiwagi, A.; Saigusa, M.

    Molecular mechanism of influence of gravity on living system is a subject of controversy for many years. Influence of gravity directly or indirectly affects to wide variety of biological processes, including biological clocks and general patterns in development of vertebrates and invertebrates. cDNA subtraction method was used for detection of the genes related to the hatching of the embryos semi-terrestrial crab Chiromantes haematocheir. Timing of the hatching of the embryos is highly synchronized with Moon phase and tides. While no new genes were found, we found that expression of chaperon hsp-90 increase in the embryos within two days before hatching, while expression of other stress proteins doesn't show any significant difference. Another model we used -- is a development of amphibian embryos. In order to clarify the effect of high gravity environment on development of Xenopus laevis, embryos on several developmental stages were subjected to the short-time high-gravity pulses (3G, 5G, and 9G). Analysis of stress-protein expression level and cDNA subtraction among high-gravity stressed embryos and control group revealed some changes in level of RNA expression of stress-proteins in experimental group. At the same time, we found two new genes expressed exclusively in the embryos under high gravity stress. The expression of the genes dramatically increased within several hours after the gravity stress, while the expression of the typical chaperons showed just slight difference. The genes expression pattern and its comparison with previously reported chaperons let us assume the presence physiological mechanism of specific gravity-stress response using previously unreported, special type of chaperons.

  16. Placental endoplasmic reticulum stress in gestational diabetes: the potential for therapeutic intervention with chemical chaperones and antioxidants

    OpenAIRE

    Yung, H. W.; Alaes-Katjavivi, P.; Jones, C.J.P.; El-Bacha, T.; Golic, M.; A.C. Staff; Burton, G J

    2016-01-01

    AIMS/HYPOTHESIS: The aim of this work was to determine whether placental endoplasmic reticulum (ER) stress may contribute to the pathophysiology of gestational diabetes mellitus (GDM) and to test the efficacy of chemical chaperones and antioxidant vitamins in ameliorating that stress in a trophoblast-like cell line in vitro. METHODS: Placental samples were obtained from women suffering from GDM and from normoglycaemic controls and were frozen immediately. Women with GDM had 2 h serum glucose ...

  17. GABA Acts as a Ligand Chaperone in the Early Secretory Pathway to Promote Cell Surface Expression of GABAA Receptors

    OpenAIRE

    Eshaq, Randa S.; Stahl, Letha D.; Stone, Randolph; Smith, Sheryl S.; Robinson, Lucy C.; Leidenheimer, Nancy J.

    2010-01-01

    GABA (γ-aminobutyric acid) is the primary inhibitory neurotransmitter in brain. The fast inhibitory effect of GABA is mediated through the GABAA receptor, a postsynaptic ligand-gated chloride channel. We propose that GABA can act as a ligand chaperone in the early secretory pathway to facilitate GABAA receptor cell surface expression. Forty-two hrs of GABA treatment increased the surface expression of recombinant receptors expressed in HEK 293 cells, an effect accompanied by an increase in GA...

  18. Modeling signal propagation mechanisms and ligand-based conformational dynamics of the Hsp90 molecular chaperone full-length dimer.

    Directory of Open Access Journals (Sweden)

    Giulia Morra

    2009-03-01

    Full Text Available Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine "hot spots" involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a "conformational selection model" of the Hsp90 mechanism, whereby the protein may

  19. Induction and adaptation of chaperone-assisted selective autophagy CASA in response to resistance exercise in human skeletal muscle

    OpenAIRE

    Ulbricht, Anna; Gehlert, Sebastian; Leciejewski, Barbara; Schiffer, Thorsten; Bloch, Wilhelm; Höhfeld, Jörg

    2015-01-01

    Chaperone-assisted selective autophagy (CASA) is a tension-induced degradation pathway essential for muscle maintenance. Impairment of CASA causes childhood muscle dystrophy and cardiomyopathy. However, the importance of CASA for muscle function in healthy individuals has remained elusive so far. Here we describe the impact of strength training on CASA in a group of healthy and moderately trained men. We show that strenuous resistance exercise causes an acute induction of CASA in affected mus...

  20. Serum antibodies to Porphyromonas gingivalis chaperone HtpG predict health in periodontitis susceptible patients.

    Directory of Open Access Journals (Sweden)

    Charles E Shelburne

    Full Text Available BACKGROUND: Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG might be associated with protection/resistance against destructive periodontitis. METHODOLOGY/PRINCIPAL FINDINGS: ELISA assays using cloned HtpG and peptide antigens confirmed gingivitis subjects colonized with P. gingivalis had higher serum levels of anti-HtpG and, concomitantly, lower levels of attachment loss. Additionally, serum antibody levels to P. gingivalis HtpG protein were higher in healthy subjects compared to patients with either chronic or aggressive periodontitis. We found a negative association between tooth attachment loss and anti-P. gingivalis HtpG (p = 0.043 but not anti-Fusobacterium nucleatum (an oral opportunistic commensal HtpG levels. Furthermore, response to periodontal therapy was more successful in subjects having higher levels of anti-P. gingivalis HtpG before treatment (p = 0.018. There was no similar relationship to anti-F. nucleatum HtpG levels. Similar results were obtained when these experiments were repeated with a synthetic peptide of a region of P. gingivalis HtpG. CONCLUSIONS/SIGNIFICANCE: OUR RESULTS SUGGEST: 1 anti-P. gingivalis HtpG antibodies are protective and therefore predict health periodontitis-susceptable patients; 2 may augment the host defence to periodontitis and 3 a

  1. Identification of peptides in human Hsp20 and Hsp27 that possess molecular chaperone and anti-apoptotic activities

    Science.gov (United States)

    Nahomi, Rooban B.; DiMauro, Michael A.; Wang, Benlian; Nagaraj, Ram H.

    2015-01-01

    Previous studies have identified peptides in the ‘crystallin-domain’ of the small heat-shock protein (sHSP) α-crystallin with chaperone and anti-apoptotic activities. We found that peptides in heat-shock protein Hsp20 (G71HFSVLLDVKHFSPEEIAVK91) and Hsp27 (D93RWRVSLDVNHFAPDELTVK113) with sequence homology to α-crystallin also have robust chaperone and anti-apoptotic activities. Both peptides inhibited hyperthermic and chemically induced aggregation of client proteins. The scrambled peptides of Hsp20 and Hsp27 showed no such effects. The chaperone activities of the peptides were better than those from αA- and αB-crystallin. HeLa cells took up the FITC-conjugated Hsp20 peptide and, when the cells were thermally stressed, the peptide was translocated from the cytoplasm to the nucleus. The two peptides inhibited apoptosis in HeLa cells by blocking cytochrome c release from the mitochondria and caspase-3 activation. We found that scrambling the last four amino acids in the two peptides (KAIV in Hsp20 and KTLV in Hsp27) made them unable to enter cells and ineffective against stress-induced apoptosis. Intraperitoneal injection of the peptides prevented sodium-selenite-induced cataract formation in rats by inhibiting protein aggregation and oxidative stress. Our study has identified peptides from Hsp20 and Hsp27 that may have therapeutic benefit in diseases where protein aggregation and apoptosis are contributing factors. PMID:25332102

  2. Heat shock protein 90 (Hsp90) chaperone complex. A molecular target for enhancement of thermosensitivity and radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Akimoto, Tetsuo; Nonaka, Tetsuo; Kitamoto, Yoshizumi; Sakurai, Hideyuki [Gunma Univ., Maebashi (Japan). School of Medicine; Mitsuhashi, Norio [Tokyo Women' s Medical Coll. (Japan)

    2002-09-01

    Heat shock protein 90 (Hsp90) is a highly conserved heat shock protein in animal and plants, and exists abundantly in the cytoplasm in unstressed condition, accounting for 1-2% in cytoplasmic proteins. Main difference of Hsp90 from other Hsps are its substrate that Hsp90 binds to. These substrates include various signal transduction proteins, kinase, steroid receptors and transcription factors, therefore, Hsp90 plays a key role in maintaining cellular signal transduction networks. Many chaperoned proteins (client proteins) of Hsp90 are associated with cellular proliferation or malignant transformation, thus Hsp90 chaperone complex has been focused as targets for cancer therapy. Among the client proteins, there are several molecules that have been defined as targets or factors for determination or enhancement of radiosensitivity or thermosensitivity. Thus, it is easily speculated that Hsp90 chaperone complex inhibitors that disrupt association of Hsp90 and client protein in combination with radiation or/and heat has potential effect on enhancement of radiosensitivity or thermosensitivity. In this paper, possible mechanisms in enhancing radiosensitivity or thermosensitivity according to the client proteins will be summarized. (author)

  3. Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease.

    Science.gov (United States)

    Zheng, Wei; Padia, Janak; Urban, Daniel J; Jadhav, Ajit; Goker-Alpan, Ozlem; Simeonov, Anton; Goldin, Ehud; Auld, Douglas; LaMarca, Mary E; Inglese, James; Austin, Christopher P; Sidransky, Ellen

    2007-08-01

    Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene. Missense mutations result in reduced enzyme activity that may be due to misfolding, raising the possibility of small-molecule chaperone correction of the defect. Screening large compound libraries by quantitative high-throughput screening (qHTS) provides comprehensive information on the potency, efficacy, and structure-activity relationships (SAR) of active compounds directly from the primary screen, facilitating identification of leads for medicinal chemistry optimization. We used qHTS to rapidly identify three structural series of potent, selective, nonsugar glucocerebrosidase inhibitors. The three structural classes had excellent potencies and efficacies and, importantly, high selectivity against closely related hydrolases. Preliminary SAR data were used to select compounds with high activity in both enzyme and cell-based assays. Compounds from two of these structural series increased N370S mutant glucocerebrosidase activity by 40-90% in patient cell lines and enhanced lysosomal colocalization, indicating chaperone activity. These small molecules have potential as leads for chaperone therapy for Gaucher disease, and this paradigm promises to accelerate the development of leads for other rare genetic disorders.

  4. Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR.

    Science.gov (United States)

    Tao, Jiahui; Petrova, Kseniya; Ron, David; Sha, Bingdong

    2010-04-16

    P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 A resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting with misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).

  5. Chaperone-like activity of alpha-cyclodextrin via hydrophobic nanocavity to protect native structure of ADH.

    Science.gov (United States)

    Barzegar, Abolfazl; Moosavi-Movahedi, Ali A; Mahnam, Karim; Ashtiani, Saman Hosseini

    2010-01-26

    The chaperone action of alpha-cyclodextrin (alpha-CyD), based on providing beneficial microenvironment of hydrophobic nanocavity to form molecular complex with alcohol dehydrogenase (ADH) was examined by experimental and computational techniques. The results of UV-vis and dynamic light scattering (DLS) indicated that the chaperone-like activity of alpha-CyD depends on molecular complex formation between alpha-CyD and ADH, which caused to decrease the amount and size of polymerized molecules. Computational calculations of molecular dynamic (MD) simulations and blind docking (BD) demonstrated that alpha-CyD acts as an artificial chaperone because of its high affinity to the region of ADH's two chains interface. The hydrophobic nanocavity of alpha-CyD has the ability to form inclusion complex due to the presence of phenyl ring of aromatic phenylalanine (Phe) residue in the dimeric intersection area. Delocalization of ADH subunits, which causes the exposure of Phe110, takes part in the enzyme polymerization and has proven to be beneficial for aggregation inhibition and solubility enhancement within the host alpha-CyD-nanocavity.

  6. Enhancement of soluble expression of codon-optimized Thermomicrobium roseum sarcosine oxidase in Escherichia coli via chaperone co-expression.

    Science.gov (United States)

    Tong, Yanjun; Feng, Shoushuai; Xin, Yu; Yang, Hailin; Zhang, Ling; Wang, Wu; Chen, Wei

    2016-01-20

    The codon-optimized sarcosine oxidase from Thermomicrobium roseum (TrSOX) was successfully expressed in Escherichia coli and its soluble expression was significantly enhanced via the co-expression of chaperones. With the assistance of whole-genome analysis of T. roseum DSM 5159, the sox gene was predicated and its sequence was optimized based on the codon bias of E. coli. The TrSOX gene was successfully constructed in the pET28a plasmid. After induction with IPTG for 8h, SDS-PAGE analysis of crude enzyme solutions showed a significant 43 kDa protein band, indicating SOX was successfully expressed in E. coli. However, the dark band corresponding to the intracellular insoluble fraction indicated that most of TrSOX enzyme existed in the inactive form in "inclusion bodies" owing to the "hot spots" of TrSOX. Furthermore, the co-expression of five different combinations of chaperones indicated that the soluble expression of TrSOX was greatly improved by the co-expression of molecular chaperones GroES-GroEL and DnaK-DnaJ-GrpE-GroES-GroEL. Additionally, the analysis of intramolecular forces indicated that the hydrophobic amino acids, hydrogen bonds, and ionic bonds were favorable for enhancing the interaction and stability of TrSOX secondary structure. This study provides a novel strategy for enhancing the soluble expression of TrSOX in E. coli. PMID:26626227

  7. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  8. Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins.

    Directory of Open Access Journals (Sweden)

    Yiwen Xiang

    Full Text Available BACKGROUND: Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP. METHODOLOGY/PRINCIPAL FINDINGS: Lactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y, whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes. CONCLUSIONS/SIGNIFICANCE: These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.

  9. The Cell Wall Polymer Lipoteichoic Acid Becomes Nonessential in Staphylococcus aureus Cells Lacking the ClpX Chaperone

    Science.gov (United States)

    Bowman, Lisa; Millership, Charlotte; Dupont Søgaard, Mia; Kaever, Volkhard; Siljamäki, Pia; Savijoki, Kirsi; Varmanen, Pekka; Nyman, Tuula A.

    2016-01-01

    ABSTRACT Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and a promising target for the development of vaccines and antimicrobial compounds against Staphylococcus aureus. Here we demonstrate that mutations in the conditionally essential ltaS (LTA synthase) gene arise spontaneously in an S. aureus mutant lacking the ClpX chaperone. A wide variety of ltaS mutations were selected, and among these, a substantial portion resulted in premature stop codons and other changes predicted to abolish LtaS synthesis. Consistent with this assumption, the clpX ltaS double mutants did not produce LTA, and genetic analyses confirmed that LTA becomes nonessential in the absence of the ClpX chaperone. In fact, inactivation of ltaS alleviated the severe growth defect conferred by the clpX deletion. Microscopic analyses showed that the absence of ClpX partly alleviates the septum placement defects of an LTA-depleted strain, while other phenotypes typical of LTA-negative S. aureus mutants, including increased cell size and decreased autolytic activity, are retained. In conclusion, our results indicate that LTA has an essential role in septum placement that can be bypassed by inactivating the ClpX chaperone. PMID:27507828

  10. Chaperone-like effect of the linker on the isolated C-terminal domain of rabbit muscle creatine kinase.

    Science.gov (United States)

    Chen, Zhe; Chen, Xiang-Jun; Xia, Mengdie; He, Hua-Wei; Wang, Sha; Liu, Huihui; Gong, Haipeng; Yan, Yong-Bin

    2012-08-01

    Intramolecular chaperones (IMCs), which are specific domains/segments encoded in the primary structure of proteins, exhibit chaperone-like activity against the aggregation of the other domains in the same molecule. In this research, we found that the truncation of the linker greatly promoted the thermal aggregation of the isolated C-terminal domain (CTD) of rabbit muscle creatine kinase (RMCK). Either the existence of the linker covalently linked to CTD or the supply of the synthetic linker peptide additionally could successfully protect the CTD of RMCK against aggregation in a concentration-dependent manner. Truncated fragments of the linker also behaved as a chaperone-like effect with lower efficiency, revealing the importance of its C-terminal half in the IMC function of the linker. The aggregation sites in the CTD of RMCK were identified by molecular dynamics simulations. Mutational analysis of the three key hydrophobic residues resulted in opposing effects on the thermal aggregation between the CTD with intact or partial linker, confirming the role of linker as a lid to protect the hydrophobic residues against exposure to solvent. These observations suggested that the linkers in multidomain proteins could act as IMCs to facilitate the correct folding of the aggregation-prone domains. Furthermore, the intactness of the IMC linker after proteolysis modulates the production of off-pathway aggregates, which may be important to the onset of some diseases caused by the toxic effects of aggregated proteolytic fragments.

  11. Unique Photobleaching Phenomena of the Twin-Arginine Translocase Respiratory Enzyme Chaperone DmsD

    Science.gov (United States)

    Rivardo, Fabrizio; Leach, Thorin G.H.; Chan, Catherine S.; Winstone, Tara M.L.; Ladner, Carol L.; Sarfo, Kwabena J.; Turner, Raymond J.

    2014-01-01

    DmsD is a chaperone of the redox enzyme maturation protein family specifically required for biogenesis of DMSO reductase in Escherichia coli. It exists in multiple folding forms, all of which are capable of binding its known substrate, the twin-arginine leader sequence of the DmsA catalytic subunit. It is important for maturation of the reductase and targeting to the cytoplasmic membrane for translocation. Here, we demonstrate that DmsD exhibits an irreversible photobleaching phenomenon upon 280 nm excitation irradiation. The phenomenon is due to quenching of the tryptophan residues in DmsD and is dependent on its folding and conformation. We also show that a tryptophan residue involved in DmsA signal peptide binding (W87) is important for photobleaching of DmsD. Mutation of W87, or binding of the DmsA twin-arginine signal peptide to DmsD in the pocket that includes W72, W80, and W91 significantly affects the degree of photobleaching. This study highlights the advantage of a photobleaching phenomenon to study protein folding and conformation changes within a protein that was once considered unusable in fluorescence spectroscopy. PMID:24497893

  12. A Testis-Specific Chaperone and the Chromatin Remodeler ISWI Mediate Repackaging of the Paternal Genome

    Directory of Open Access Journals (Sweden)

    Cécile M. Doyen

    2015-11-01

    Full Text Available During spermatogenesis, the paternal genome is repackaged into a non-nucleosomal, highly compacted chromatin structure. Bioinformatic analysis revealed that Drosophila sperm chromatin proteins are characterized by a motif related to the high-mobility group (HMG box, which we termed male-specific transcript (MST-HMG box. MST77F is a MST-HMG-box protein that forms an essential component of sperm chromatin. The deposition of MST77F onto the paternal genome requires the chaperone function of tNAP, a testis-specific NAP protein. MST77F, in turn, enables the stable incorporation of MST35Ba and MST35Bb into sperm chromatin. Following MST-HMG-box protein deposition, the ATP-dependent chromatin remodeler ISWI mediates the appropriate organization of sperm chromatin. Conversely, at fertilization, maternal ISWI targets the paternal genome and drives its repackaging into de-condensed nucleosomal chromatin. Failure of this transition in ISWI mutant embryos is followed by mitotic defects, aneuploidy, and haploid embryonic divisions. Thus, ISWI enables bi-directional transitions between two fundamentally different forms of chromatin.

  13. Antimyeloma Effects of the Heat Shock Protein 70 Molecular Chaperone Inhibitor MAL3-101

    Directory of Open Access Journals (Sweden)

    Marc J. Braunstein

    2011-01-01

    Full Text Available Multiple myeloma (MM is the second most common hematologic malignancy and remains incurable, primarily due to the treatment-refractory/resistant nature of the disease. A rational approach to this compelling challenge is to develop new drugs that act synergistically with existing effective agents. This approach will reduce drug concentrations, avoid treatment resistance, and also improve treatment effectiveness by targeting new and nonredundant pathways in MM. Toward this goal, we examined the antimyeloma effects of MAL3-101, a member of a new class of non-ATP-site inhibitors of the heat shock protein (Hsp 70 molecular chaperone. We discovered that MAL3-101 exhibited antimyeloma effects on MM cell lines in vitro and in vivo in a xenograft plasmacytoma model, as well as on primary tumor cells and bone marrow endothelial cells from myeloma patients. In combination with a proteasome inhibitor, MAL3-101 significantly potentiated the in vitro and in vivo antimyeloma effects. These data support a preclinical rationale for small molecule inhibition of Hsp70 function, either alone or in combination with other agents, as an effective therapeutic strategy for MM.

  14. Chaperone ligand-discrimination by the TPR-domain protein Tah1.

    Science.gov (United States)

    Millson, Stefan H; Vaughan, Cara K; Zhai, Chao; Ali, Maruf M U; Panaretou, Barry; Piper, Peter W; Pearl, Laurence H; Prodromou, Chrisostomos

    2008-07-15

    Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

  15. Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments.

    Science.gov (United States)

    Perng, Ming-Der; Huang, Yu-Shan; Quinlan, Roy A

    2016-01-01

    Intermediate filament (IF) scaffolds facilitate small heat shock protein (sHSP) function, while IF function is sHSP dependent. sHSPs interact with IFs and the importance of this interaction is to maintain the individuality of the IFs and to modulate interfilament interactions both in networks and in assembly intermediates. Mutations in both sHSPs and their interacting IF proteins phenocopy each other in the human diseases they cause. This establishes a key functional relationship between these two very distinct protein families, and it also evidences the role of this cytoskeleton-chaperone complex in the cellular stress response. In this chapter, we describe the detailed experimental protocols for the preparation of purified IF proteins and sHSPs to facilitate the study in vitro of their functional interactions. In addition, we describe the detailed biochemical procedures to assess the effect of sHSP on the assembly of IFs, the binding to IFs, and the prevention of noncovalent filament-filament interactions using in vitro cosedimentation, electron microscopy, and viscosity assays. These assays are valuable research tools to study and manipulate sHSP-IF complexes in vitro and therefore to determine the structure-function detail of this complex, and how it contributes to cellular, tissue, and organismal homeostasis and the in vivo stress response.

  16. An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis

    Science.gov (United States)

    Yu, Xiang; Kong, Yi; Dore, Louis C.; Abdulmalik, Osheiza; Katein, Anne M.; Zhou, Suiping; Choi, John K.; Gell, David; Mackay, Joel P.; Gow, Andrew J.; Weiss, Mitchell J.

    2007-01-01

    Erythrocyte precursors produce abundant α- and β-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. αHb-stabilizing protein (AHSP) binds free α subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free α-globin caused by β-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free α-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 α-globin genes (genotype Ahsp–/–α-globin*α/αα) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated α-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free α-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess α-globin, AHSP also acts as a molecular chaperone to stabilize nascent α-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances. PMID:17607360

  17. Impaired Chaperone Activity of Human Heat Shock Protein Hsp27 Site-Specifically Modified with Argpyrimidine.

    Science.gov (United States)

    Matveenko, Maria; Cichero, Elena; Fossa, Paola; Becker, Christian F W

    2016-09-12

    Non-enzymatic posttranslational modifications (nPTMs) affect at least ∼30 % of human proteins, but our understanding of their impact on protein structure and function is limited. Studies of nPTMs are difficult because many modifications are not included in common chemical libraries or protein expression systems and should be introduced site-specifically. Herein, we probed the effect of the nPTM argpyrimidine on the structure and function of human protein Hsp27, which acquires argpyrimidine at residue 188 in vivo. We developed a synthetic approach to an argpyrimidine building block, which we then incorporated at position 188 of Hsp27 through protein semisynthesis. This modification did not affect the protein secondary structure, but perturbed the oligomeric assembly and impaired chaperone activity. Our work demonstrates that protein function can be altered by a single nPTM and opens up a new area of investigation only accessible by methods that allow site-selective protein modification. PMID:27440458

  18. The impact of the HIRA histone chaperone upon global nucleosome architecture.

    Science.gov (United States)

    Gal, Csenge; Moore, Karen M; Paszkiewicz, Konrad; Kent, Nicholas A; Whitehall, Simon K

    2015-01-01

    HIRA is an evolutionarily conserved histone chaperone that mediates replication-independent nucleosome assembly and is important for a variety of processes such as cell cycle progression, development, and senescence. Here we have used a chromatin sequencing approach to determine the genome-wide contribution of HIRA to nucleosome organization in Schizosaccharomyces pombe. Cells lacking HIRA experience a global reduction in nucleosome occupancy at gene sequences, consistent with the proposed role for HIRA in chromatin reassembly behind elongating RNA polymerase II. In addition, we find that at its target promoters, HIRA commonly maintains the full occupancy of the -1 nucleosome. HIRA does not affect global chromatin structure at replication origins or in rDNA repeats but is required for nucleosome occupancy in silent regions of the genome. Nucleosome organization associated with the heterochromatic (dg-dh) repeats located at the centromere is perturbed by loss of HIRA function and furthermore HIRA is required for normal nucleosome occupancy at Tf2 LTR retrotransposons. Overall, our data indicate that HIRA plays an important role in maintaining nucleosome architecture at both euchromatic and heterochromatic loci.

  19. Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment.

    Science.gov (United States)

    Borodavka, Alexander; Ault, James; Stockley, Peter G; Tuma, Roman

    2015-08-18

    Reoviruses are important human, animal and plant pathogens having 10-12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA-protein and RNA-RNA interactions between viral genomic segment precursors have been implicated in the process. While non-structural viral RNA-binding proteins, such as avian reovirus σNS, are essential for virus replication, the mechanism by which they assist packaging is unclear. Here we demonstrate that σNS assembles into stable elongated hexamers in vitro, which bind single-stranded nucleic acids with high affinity, but little sequence specificity. Using ensemble and single molecule fluorescence spectroscopy, we show that σNS also binds to a partially double-stranded RNA, resulting in gradual helix unwinding. The hexamer can bind multiple RNA molecules and exhibits strand-annealing activity, thus mediating conversion of metastable, intramolecular stem-loops into more stable heteroduplexes. We demonstrate that the ARV σNS acts as an RNA chaperone facilitating specific RNA-RNA interactions between genomic precursors during segment assortment and packaging.

  20. The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms.

    Science.gov (United States)

    Wang, Huanyu; Ge, Zhongqi; Walsh, Scott T R; Parthun, Mark R

    2012-01-01

    Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.

  1. The myosin chaperone UNC45B is involved in lens development and autosomal dominant juvenile cataract

    DEFF Research Database (Denmark)

    Hansen, Lars; Comyn, Sophie; Mang, Yuan;

    2014-01-01

    -type embryos resulted in development of a phenotype similar to the steif mutant. The p.Arg805Trp alteration in the mammalian UNC45B gene suggests that developmental cataract may be caused by a defect in non-muscle myosin assembly during maturation of the lens fiber cells.European Journal of Human Genetics...... in this region, in ACACA and UNC45B. As alterations of the UNC45B protein have been shown to affect eye development in model organisms, effort was focused on the heterozygous UNC45B missense mutation. UNC45B encodes a myosin-specific chaperone that, together with the general heat shock protein HSP90, is involved...... in myosin assembly. The mutation changes p.Arg805 to Trp in the UCS domain, an amino acid that is highly conserved from yeast to human. UNC45B is strongly expressed in the heart and skeletal muscle tissue, but here we show expression in human embryo eye and zebrafish lens. The zebrafish mutant steif...

  2. In vitro thermodynamic dissection of human copper transfer from chaperone to target protein.

    Directory of Open Access Journals (Sweden)

    Moritz S Niemiec

    Full Text Available Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4. Atox1 and WD4 have the same fold (ferredoxin-like fold and Cu-binding site (two surface exposed cysteine residues and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4. We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4 of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

  3. The pvc Operon Regulates the Expression of the Pseudomonas aeruginosa Fimbrial Chaperone/Usher Pathway (Cup) Genes

    OpenAIRE

    Uzma Qaisar; Liming Luo; Haley, Cecily L.; Brady, Sean F.; Carty, Nancy L.; Colmer-Hamood, Jane A.; Hamood, Abdul N.

    2013-01-01

    The Pseudomonas aeruginosa fimbrial structures encoded by the cup gene clusters (cupB and cupC) contribute to its attachment to abiotic surfaces and biofilm formation. The P. aeruginosa pvcABCD gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the pvc operon and the cup gene clus...

  4. Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4.

    Science.gov (United States)

    Natsume, Ryo; Eitoku, Masamitsu; Akai, Yusuke; Sano, Norihiko; Horikoshi, Masami; Senda, Toshiya

    2007-03-15

    CIA (CCG1-interacting factor A)/ASF1, which is the most conserved histone chaperone among the eukaryotes, was genetically identified as a factor for an anti-silencing function (Asf1) by yeast genetic screening. Shortly after that, the CIA-histone-H3-H4 complex was isolated from Drosophila as a histone chaperone CAF-1 stimulator. Human CIA-I/II (ASF1a/b) was identified as a histone chaperone that interacts with the bromodomain-an acetylated-histone-recognizing domain-of CCG1, in the general transcription initiation factor TFIID. Intensive studies have revealed that CIA/ASF1 mediates nucleosome assembly by forming a complex with another histone chaperone in human cells and yeast, and is involved in DNA replication, transcription, DNA repair and silencing/anti-silencing in yeast. CIA/ASF1 was shown as a major storage chaperone for soluble histones in proliferating human cells. Despite all these biochemical and biological functional analyses, the structure-function relationship of the nucleosome assembly/disassembly activity of CIA/ASF1 has remained elusive. Here we report the crystal structure, at 2.7 A resolution, of CIA-I in complex with histones H3 and H4. The structure shows the histone H3-H4 dimer's mutually exclusive interactions with another histone H3-H4 dimer and CIA-I. The carboxy-terminal beta-strand of histone H4 changes its partner from the beta-strand in histone H2A to that of CIA-I through large conformational change. In vitro functional analysis demonstrated that CIA-I has a histone H3-H4 tetramer-disrupting activity. Mutants with weak histone H3-H4 dimer binding activity showed critical functional effects on cellular processes related to transcription. The histone H3-H4 tetramer-disrupting activity of CIA/ASF1 and the crystal structure of the CIA/ASF1-histone-H3-H4 dimer complex should give insights into mechanisms of both nucleosome assembly/disassembly and nucleosome semi-conservative replication.

  5. The roles of co-chaperone CCRP/DNAJC7 in Cyp2b10 gene activation and steatosis development in mouse livers.

    Directory of Open Access Journals (Sweden)

    Marumi Ohno

    Full Text Available Cytoplasmic constitutive active/androstane receptor (CAR retention protein (CCRP and also known as DNAJC7 is a co-chaperone previously characterized to retain nuclear receptor CAR in the cytoplasm of HepG2 cells. Here we have produced CCRP knockout (KO mice and demonstrated that CCRP regulates CAR at multiple steps in activation of the cytochrome (Cyp 2b10 gene in liver: nuclear accumulation, RNA polymerase II recruitment and epigenetic modifications. Phenobarbital treatment greatly increased nuclear CAR accumulation in the livers of KO males as compared to those of wild type (WT males. Despite this accumulation, phenobarbital-induced activation of the Cyp2b10 gene was significantly attenuated. In ChIP assays, a CAR/retinoid X receptor-α (RXRα heterodimer binding to the Cyp2b10 promoter was already increased before phenobarbital treatment and further pronounced after treatment. However, RNA polymerase II was barely recruited to the promoter even after phenobarbital treatment. Histone H3K27 on the Cyp2b10 promoter was de-methylated only after phenobarbital treatment in WT but was fully de-methylated before treatment in KO males. Thus, CCRP confers phenobarbital-induced de-methylation capability to the promoter as well as the phenobarbital responsiveness of recruiting RNA polymerase II, but is not responsible for the binding between CAR and its cognate sequence, phenobarbital responsive element module. In addition, KO males developed steatotic livers and increased serum levels of total cholesterol and high density lipoprotein in response to fasting. CCRP appears to be involved in various hepatic regulations far beyond CAR-mediated drug metabolism.

  6. Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain.

    Science.gov (United States)

    Yousefi, Reza; Shchutskaya, Yulia Y; Zimny, Jaroslaw; Gaudin, Jean-Charles; Moosavi-Movahedi, Ali A; Muronetz, Vladimir I; Zuev, Yuriy F; Chobert, Jean-Marc; Haertlé, Thomas

    2009-08-01

    As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com. PMID:19322774

  7. Expression levels of hsc70 and hsp60 are developmentally regulated during B-cell maturation and not associated to childhood c-ALL at presentation or relapse

    DEFF Research Database (Denmark)

    Wehner, Peder Skov; Nielsen, Bendt; Hokland, Marianne

    2003-01-01

    Heat shock proteins are potent regulators of apoptosis, and so they may also be involved in normal cellular differentiation and cancerogenesis. We used quantitative two-dimensional gel electrophoresis for determining whether either the constitutive chaperonic heat shock cognate protein 70 (hsc70)...... hsc70 and hsp60 expression during B-cell ontogenesis....

  8. Mechanosensitive liposomes as artificial chaperones for shear-driven acceleration of enzyme-catalyzed reaction.

    Science.gov (United States)

    Natsume, Tomotaka; Yoshimoto, Makoto

    2014-03-12

    Mechanosensitive liposomes were prepared and applied to continuously accelerate the glucose oxidase (GO) reaction in shear flow. The liposome membrane was composed of a ternary lipid mixture containing 20 mol % negatively charged lipid and 30 mol % cholesterol. The liposomes encapsulating GO and catalase were passed through microtubes with inner diameter of 190 or 380 μm at 25 °C to induce the catalytic oxidation of 10 mM glucose with simultaneous decomposition of H2O2 produced. The liposomal GO showed significantly low reactivity in the static liquid system because of the permeation resistance of lipid membranes to glucose. On the other hand, the enzyme activity of liposomal GO observed at the average shear rate of 7.8 × 10(3) s(-1) was significantly larger than its intrinsic activity free of mass transfer effect in the static liquid system. The structure of liposomes was highly shear-sensitive as elucidated on the basis of shear rate-dependent physical stability of liposomes and membrane permeability to 5(6)-carboxyfluorescein as well as to GO. Thus, the above shear-driven acceleration of GO reaction was indicated to be caused by the free GO molecules released from the structurally altered liposomes at high shear rates. Moreover, the shear-induced denaturation of free GO was completely depressed by the interaction with the sheared liposomes with the chaperone-like function. The shear-sensitive liposomal GO system can be a unique catalyst that continuously accelerates and also decelerates the oxidation reaction depending on the applied shear rate.

  9. RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

    International Nuclear Information System (INIS)

    Highlights: ► hPrx1 has RNA-binding properties. ► hPrx1 exhibits helix-destabilizing activity. ► Cold stress increases hPrx1 level in the nuclear fraction. ► hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwound nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem–loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.

  10. RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-Hee; Lee, Jeong-Mi; Lee, Hae Na; Kim, Eun-Kyung; Ha, Bin [Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Korea, Republic of); Ahn, Sung-Min, E-mail: smahn@gachon.ac.kr [Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Korea, Republic of); Department of Translational Medicine, Gachon University Gil Hospital, Incheon (Korea, Republic of); Jang, Ho Hee, E-mail: hhjang@gachon.ac.kr [Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Korea, Republic of); Lee, Sang Yeol [Division of Applied Life Sciences (Brain Korea 21 program), Gyeongsang National University, Jinju 660-701 (Korea, Republic of)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer hPrx1 has RNA-binding properties. Black-Right-Pointing-Pointer hPrx1 exhibits helix-destabilizing activity. Black-Right-Pointing-Pointer Cold stress increases hPrx1 level in the nuclear fraction. Black-Right-Pointing-Pointer hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwound nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem-loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.

  11. DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography.

    Science.gov (United States)

    Batyuk, Alexander; Wu, Yufan; Honegger, Annemarie; Heberling, Matthew M; Plückthun, Andreas

    2016-04-24

    DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 β-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept.

  12. Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

    Directory of Open Access Journals (Sweden)

    Colin A Cooper

    2010-02-01

    Full Text Available Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2 is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2 and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

  13. Tyrosine 601 of Bacillus subtilis DnaK Undergoes Phosphorylation and Is Crucial for Chaperone Activity and Heat Shock Survival.

    Science.gov (United States)

    Shi, Lei; Ravikumar, Vaishnavi; Derouiche, Abderahmane; Macek, Boris; Mijakovic, Ivan

    2016-01-01

    In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple Stable Isotope Labeling by Amino acids in Cell culture-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type (WT), were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the WT dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis. PMID:27148221

  14. Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase.

    Science.gov (United States)

    DuPrez, Kevin T; Scranton, Melissa A; Walling, Linda L; Fan, Li

    2016-05-01

    Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Å resolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A. PMID:27139632

  15. Tyrosine 601 of Bacillus subtilis DnaK undergoes phosphorylation and is crucial for chaperone activity and heat shock survival

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2016-04-01

    Full Text Available In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple SILAC-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type, were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the wild type dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

  16. Chaperone mediated autophagy to the rescue: A new-fangled target for the treatment of neurodegenerative diseases.

    Science.gov (United States)

    Xilouri, Maria; Stefanis, Leonidas

    2015-05-01

    One of the main pathways of lysosomal proteolysis is chaperone-mediated autophagy (CMA), which represents a selective mechanism for the degradation of specific soluble proteins within lysosomes. Along with the other two lysosomal pathways, macro- and micro-autophagy, CMA contributes to cellular quality control through the removal of damaged or malfunctioning proteins. The two intrinsic characteristics of CMA are the selective targeting and the direct translocation of substrate proteins into the lysosomal lumen, in a fine-tuned manner through the orchestrated action of a chaperone/co-chaperone complex localized both at the cytosol and the lysosomes. Even though CMA was originally identified as a stress-induced pathway, basal CMA activity is detectable in most cell types analyzed so far, including neurons. Additionally, CMA activity declines with age and this may become a major aggravating factor contributing to neurodegeneration. More specifically, it has been suggested that CMA impairment may underlie the accumulation of misfolded/aggregated proteins, such as alpha-synuclein or LRRK2, whose levels or conformations are critical to Parkinson's disease pathogenesis. On the other hand, CMA induction might accelerate clearance of pathogenic proteins and promote cell survival, suggesting that CMA represents a viable therapeutic target for the treatment of various proteinopathies. In the current review, we provide an overview of the current state of knowledge regarding the role of CMA under physiological and pathological conditions of the nervous system and discuss the implications of these findings for therapeutic interventions for Parkinson's disease and other neurodegenerative disorders. This article is part of Special Issue entitled "Neuronal Protein". PMID:25724482

  17. Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB.

    Science.gov (United States)

    Garnett, James A; Diallo, Mamou; Matthews, Steve J

    2015-06-01

    Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone-usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.

  18. The hydrophobic region of the DmsA twin-arginine leader peptide determines specificity with chaperone DmsD.

    Science.gov (United States)

    Winstone, Tara M L; Tran, Vy A; Turner, Raymond J

    2013-10-29

    The system specific chaperone DmsD plays a role in the maturation of the catalytic subunit of dimethyl sulfoxide (DMSO) reductase, DmsA. Pre-DmsA contains a 45-amino acid twin-arginine leader peptide that is important for targeting and translocation of folded and cofactor-loaded DmsA by the twin-arginine translocase. DmsD has previously been shown to interact with the complete twin-arginine leader peptide of DmsA. In this study, isothermal titration calorimetry was used to investigate the thermodynamics of binding between synthetic peptides composed of different portions of the DmsA leader peptide and DmsD. Only those peptides that included the complete and contiguous hydrophobic region of the DmsA leader sequence were able to bind DmsD with a 1:1 stoichiometry. Each of the peptides that were able to bind DmsD also showed some α-helical structure as indicated by circular dichroism spectroscopy. Differential scanning calorimetry revealed that DmsD gained very little thermal stability upon binding any of the DmsA leader peptides tested. Together, these results suggest that a portion of the hydrophobic region of the DmsA leader peptide determines the specificity of binding and may produce helical properties upon binding to DmsD. Overall, this study demonstrates that the recognition of the DmsA twin-arginine leader sequence by the DmsD chaperone shows unexpected rules and confirms further that the biochemistry of the interaction of the chaperone with their leaders demonstrates differences in their molecular interactions.

  19. Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins.

    Science.gov (United States)

    Lu, Xinyao; He, Shuying; Zong, Hong; Song, Jian; Chen, Wen; Zhuge, Bin

    2016-09-01

    1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK-dnaJ-grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool. PMID:27430516

  20. Hsp31, a member of the DJ-1 superfamily, is a multitasking stress responder with chaperone activity.

    Science.gov (United States)

    Aslam, Kiran; Hazbun, Tony R

    2016-03-01

    Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson's disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including α-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson's disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31's chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purified from yeast is more active compared to expression and purification from E. coli suggesting that posttranslational modifications could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses. PMID:27097320

  1. Folding of the td pre-RNA with the help of the RNA chaperone StpA.

    Science.gov (United States)

    Mayer, O; Waldsich, C; Grossberger, R; Schroeder, R

    2002-11-01

    The td group I intron is inserted in the reading frame of the thymidylate synthase gene, which is mainly devoid of structural elements. In vivo, translation of the pre-mRNA is required for efficient folding of the intron into its splicing-competent structure. The ribosome probably resolves exon-intron interactions that interfere with splicing. Uncoupling splicing from translation, by introducing a non-sense codon into the upstream exon, reduces the splicing efficiency of the mutant pre-mRNA. Alternatively to the ribosome, co-expression of genes that encode proteins with RNA chaperone activity promote folding of the td pre-mRNA in vivo. These proteins also efficiently accelerate folding of the td pre-mRNA in vitro. In order to understand the mechanism of action of RNA chaperones, we probed the impact of the RNA chaperone StpA on the structure of the td intron in vivo and compared it with that of the well characterized Cyt-18 protein, which is a group-I-intron-specific splicing factor. We found that the two proteins have opposite effects on the structure of the td intron. While StpA loosens the three-dimensional structure, Cyt-18 tightens it up. Furthermore, mutations that destabilize the intron structure render the mutants sensitive to StpA, whereas splicing of these mutants is rescued by Cyt-18. Our results provide direct evidence for protein-induced conformational changes within a catalytic RNA in vivo. Whereas StpA resolves tertiary contacts enabling the RNA to refold, Cyt-18 contributes to the stabilization of the native three-dimensional structure. PMID:12440999

  2. Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB

    Energy Technology Data Exchange (ETDEWEB)

    Garnett, James A.; Diallo, Mamou; Matthews, Steve J., E-mail: s.j.matthews@imperial.ac.uk [Imperial College London, South Kensington, London SW7 2AZ (United Kingdom)

    2015-05-20

    In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher pathway that plays a major role in both early biofilm formation and host-cell adhesion. Initial attempts at crystallizing the chaperone EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. This is the first time that this refolding strategy has been used to purify CU chaperones. Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it

  3. Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

    Science.gov (United States)

    den-Haan, Helena; Pérez-Sánchez, Horacio; Del Prete, Rosita; Liguori, Ludovica; Cimmaruta, Chiara; Lukas, Jan; Andreotti, Giuseppina

    2016-01-01

    Personalized therapies are required for Fabry disease due to its large phenotypic spectrum and numerous different genotypes. In principle, missense mutations that do not affect the active site could be rescued with pharmacological chaperones. At present pharmacological chaperones for Fabry disease bind the active site and couple a stabilizing effect, which is required, to an inhibitory effect, which is deleterious. By in silico docking we identified an allosteric hot-spot for ligand binding where a drug-like compound, 2,6-dithiopurine, binds preferentially. 2,6-dithiopurine stabilizes lysosomal alpha-galactosidase in vitro and rescues a mutant that is not responsive to a mono-therapy with previously described pharmacological chaperones, 1-deoxygalactonojirimycin and galactose in a cell based assay. PMID:27788225

  4. Heat Treatment of Small Heat Shock Proteins α-Crystallin and Hsp16.3: Structural Changes vs. Chaperone-like Activity

    Institute of Scientific and Technical Information of China (English)

    毛启龙; 柯丹霞; 昌增益

    2001-01-01

    Both α-crystallin from bovine eye lens and Hsp16.3 from Mycobacterium tuberculosis are members of the small heat shock protein family, They were preincubated at 100 C for 15 min and then cooled on ice immediately. The chaperone-like activities of preheated proteins were measured at 37 C using DTT-treated insulin B chains as substrates. Both preheated proteins exhibited greatly enhanced chaperone-like activities, accompanied with almost unchanged secondary structures and surface hydrophobicity but with a minor change in tertiary structures. The dramatically enhanced chaperone-like activities of preheated α-crystallln and Hsp16.3 may have resulted from the irreversible change in the tertiary structure as detected by near-UV CD spectra.

  5. A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life

    Directory of Open Access Journals (Sweden)

    Merchant Sabeeha S

    2009-10-01

    Full Text Available Abstract Background COG0523 proteins are, like the nickel chaperones of the UreG family, part of the G3E family of GTPases linking them to metallocenter biosynthesis. Even though the first COG0523-encoding gene, cobW, was identified almost 20 years ago, little is known concerning the function of other members belonging to this ubiquitous family. Results Based on a combination of comparative genomics, literature and phylogenetic analyses and experimental validations, the COG0523 family can be separated into at least fifteen subgroups. The CobW subgroup involved in cobalamin synthesis represents only one small sub-fraction of the family. Another, larger subgroup, is suggested to play a predominant role in the response to zinc limitation based on the presence of the corresponding COG0523-encoding genes downstream from putative Zur binding sites in many bacterial genomes. Zur binding sites in these genomes are also associated with candidate zinc-independent paralogs of zinc-dependent enzymes. Finally, the potential role of COG0523 in zinc homeostasis is not limited to Bacteria. We have predicted a link between COG0523 and regulation by zinc in Archaea and show that two COG0523 genes are induced upon zinc depletion in a eukaryotic reference organism, Chlamydomonas reinhardtii. Conclusion This work lays the foundation for the pursuit by experimental methods of the specific role of COG0523 members in metal trafficking. Based on phylogeny and comparative genomics, both the metal specificity and the protein target(s might vary from one COG0523 subgroup to another. Additionally, Zur-dependent expression of COG0523 and putative paralogs of zinc-dependent proteins may represent a mechanism for hierarchal zinc distribution and zinc sparing in the face of inadequate zinc nutrition.

  6. Expression and Regulation of the Fkbp5 Gene in the Adult Mouse Brain

    OpenAIRE

    Scharf, Sebastian H.; Claudia Liebl; Binder, Elisabeth B.; Schmidt, Mathias V.; Müller, Marianne B.

    2011-01-01

    BACKGROUND: Chronic stress has been found to be a major risk factor for various human pathologies. Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, which is tightly regulated via, among others, the glucocorticoid receptor (GR). The activity of the GR is modulated by a variety of proteins, including the co-chaperone FK506 binding protein 51 (FKBP5). Although FKBP5 has been associated with risk for affective disorders and has been implicated in GR sensitivity, previous studies fo...

  7. Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.

    Science.gov (United States)

    Belviso, Benny D; Galliani, Angela; Lasorsa, Alessia; Mirabelli, Valentina; Caliandro, Rocco; Arnesano, Fabio; Natile, Giovanni

    2016-07-01

    Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the

  8. GroEL to DnaK chaperone network behind the stability modulation of σ(32) at physiological temperature in Escherichia coli.

    Science.gov (United States)

    Patra, Monobesh; Roy, Sourav Singha; Dasgupta, Rakhi; Basu, Tarakdas

    2015-12-21

    The stability of heat-shock transcription factor σ(32) in Escherichia coli has long been known to be modulated only by its own transcribed chaperone DnaK. Very few reports suggest a role for another heat-shock chaperone, GroEL, for maintenance of cellular σ(32) level. The present study demonstrates in vivo physical association between GroEL and σ(32) in E. coli at physiological temperature. This study further reveals that neither DnaK nor GroEL singly can modulate σ(32) stability in vivo; there is an ordered network between them, where GroEL acts upstream of DnaK.

  9. Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation.

    Science.gov (United States)

    Chai, Ye Jin; Sierecki, Emma; Tomatis, Vanesa M; Gormal, Rachel S; Giles, Nichole; Morrow, Isabel C; Xia, Di; Götz, Jürgen; Parton, Robert G; Collins, Brett M; Gambin, Yann; Meunier, Frédéric A

    2016-09-12

    Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body-like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-Syn(WT) and that of the Parkinson's disease-causing α-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the α-Syn(A30P) mutant with Munc18-1 reduced the number of α-Syn(A30P) aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration. PMID:27597756

  10. HSF1-controlled and age-associated chaperone capacity in neurons and muscle cells of C. elegans.

    Directory of Open Access Journals (Sweden)

    Andreas Kern

    Full Text Available Protein stability under changing conditions is of vital importance for the cell and under the control of a fine-tuned network of molecular chaperones. Aging and age-related neurodegenerative diseases are directly associated with enhanced protein instability. Employing C. elegans expressing GFP-tagged luciferase as a reporter for evaluation of protein stability we show that the chaperoning strategy of body wall muscle cells and neurons is significantly different and that both are differently affected by aging. Muscle cells of young worms are largely resistant to heat stress, which is directly mediated by the stress response controlled through Heat Shock Transcription Factor 1. During recovery following heat stress the ability to refold misfolded proteins is missing. Young neurons are highly susceptible to chronic heat stress, but show a high potency to refold or disaggregate proteins during subsequent recovery. The particular proteome instability in neurons results from a delayed induction of the heat shock response. In aged neurons protein stability is increased during heat stress, whereas muscle cells show enhanced protein instability due to a deteriorated heat shock response. An efficient refolding activity is absent in both aged tissues. These results provide molecular insights into the differential protein stabilization capacity in different tissues and during aging.

  11. Effect of heterologous expression of molecular chaperone DnaK from Tetragenococcus halophilus on salinity adaptation of Escherichia coli.

    Science.gov (United States)

    Sugimoto, Shinya; Nakayama, Jiro; Fukuda, Daisuke; Sonezaki, Shino; Watanabe, Maki; Tosukhowong, Amonlaya; Sonomoto, Kenji

    2003-01-01

    Molecular chaperone DnaK of halophilic Tetragenococcus halophilus JCM5888 was characterized under salinity conditions both in vitro and in vivo. The dnaK gene was cloned into an expression vector and transformed into Escherichia coli. The DnaK protein obtained from the recombinant E. coli showed a significantly higher refolding activity of denatured lactate dehydrogenase than that from non-halophilic Lactococcus lactis under NaCl concentrations higher than 1 M. E. coli without the overexpression of DnaK exhibited a growth profile with a prolonged lag phase and suppressed maximum cell density in Luria-Bertani medium containing 5% (0.86 M) NaCl. On the contrary, the overexpression of T. halophilus DnaK greatly shortened this prolonged lag phase with no effect on maximum growth, while that of L. lactis DnaK decreased maximum growth. The amount of protein aggregates was increased by salt stress in the E. coli cells, while this aggregation was greatly suppressed by the overexpression of T, halophilus DnaK. These results suggest that heterologous overexpression of T. halophilus DnaK, via its chaperone activity, promotes salinity adaptation of E. coli. PMID:16233497

  12. Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation.

    Science.gov (United States)

    Chai, Ye Jin; Sierecki, Emma; Tomatis, Vanesa M; Gormal, Rachel S; Giles, Nichole; Morrow, Isabel C; Xia, Di; Götz, Jürgen; Parton, Robert G; Collins, Brett M; Gambin, Yann; Meunier, Frédéric A

    2016-09-12

    Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body-like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-Syn(WT) and that of the Parkinson's disease-causing α-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the α-Syn(A30P) mutant with Munc18-1 reduced the number of α-Syn(A30P) aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.

  13. Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action

    Energy Technology Data Exchange (ETDEWEB)

    Baram, David; Pyetan, Erez; Sittner, Assa; Auerbach-Nevo, Tamar; Bashan, Anat; Yonath, Ada (WIS-I)

    2010-07-13

    Trigger factor (TF), the first chaperone in eubacteria to encounter the emerging nascent chain, binds to the large ribosomal subunit in the vicinity of the protein exit tunnel opening and forms a sheltered folding space. Here, we present the 3.5-{angstrom} crystal structure of the physiological complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with the N-terminal domain of TF (TFa) from the same organism. For anchoring, TFa exploits a small ribosomal surface area in the vicinity of proteins L23 and L29, by using its 'signature motif' as well as additional structural elements. The molecular details of TFa interactions reveal that L23 is essential for the association of TF with the ribosome and may serve as a channel of communication with the nascent chain progressing in the tunnel. L29 appears to induce a conformational change in TFa, which results in the exposure of TFa hydrophobic patches to the opening of the ribosomal exit tunnel, thus increasing its affinity for hydrophobic segments of the emerging nascent polypeptide. This observation implies that, in addition to creating a protected folding space for the emerging nascent chain, TF association with the ribosome prevents aggregation by providing a competing hydrophobic environment and may be critical for attaining the functional conformation necessary for chaperone activity.

  14. BtcA, A class IA type III chaperone, interacts with the BteA N-terminal domain through a globular/non-globular mechanism.

    Directory of Open Access Journals (Sweden)

    Chen Guttman

    Full Text Available Bordetella pertussis, the etiological agent of "whooping cough" disease, utilizes the type III secretion system (T3SS to deliver a 69 kDa cytotoxic effector protein, BteA, directly into the host cells. As with other T3SS effectors, prior to its secretion BteA binds BtcA, a 13.9 kDa protein predicted to act as a T3SS class IA chaperone. While this interaction had been characterized for such effector-chaperone pairs in other pathogens, it has yet to be fully investigated in Bordetella. Here we provide the first biochemical proof that BtcA is indeed a class IA chaperone, responsible for the binding of BteA's N-terminal domain. We bring forth extensive evidence that BtcA binds its substrate effector through a dual-interface binding mechanism comprising of non-globular and bi-globular interactions at a moderate micromolar level binding affinity. We demonstrate that the non-globular interactions involve the first 31 N-terminal residues of BteA287 and their removal leads to destabilization of the effector-chaperone complex and lower binding affinities to BtcA. These findings represent an important first step towards a molecular understanding of BteA secretion and cell entry.

  15. Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Pseudoalteromonas sp. SM9913 is a phychrotrophic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL-PCR (GenBank accession Nos DQ640312, DQ504163). The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

  16. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

    NARCIS (Netherlands)

    Punt, P.J.; Gemeren, I.A. van; Drint-Kuijvenhoven, J.; Hessing, J.G.M.; Muijlwijk van - Harteveld, G.M.; Beijersbergen, A.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    1998-01-01

    The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bip

  17. Isolation of a gene encoding a copper chaperone for the Cu/Zn superoxide dismutase and characterization of its promoter in Solanum tuberosum L.

    NARCIS (Netherlands)

    Trindade, L.M.; Horvath, B.M.; Bergervoet-van Deelen, J.E.M.; Visser, R.G.F.

    2003-01-01

    Gene expression during the potato (Solanum tuberosum) tuber lifecycle was monitored by cDNA-amplified fragment-length polymorphism, and several differentially expressed transcript-derived fragments were isolated. One fragment, named TDFL431, showed high homology to a copper (Cu) chaperone for Cu/zin

  18. Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress.

    Science.gov (United States)

    Sugimoto, Shinya; Yoshida, Hiroyuki; Mizunoe, Yoshimitsu; Tsuruno, Keigo; Nakayama, Jiro; Sonomoto, Kenji

    2006-12-01

    In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions. PMID:16997952

  19. Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress▿

    Science.gov (United States)

    Sugimoto, Shinya; Yoshida, Hiroyuki; Mizunoe, Yoshimitsu; Tsuruno, Keigo; Nakayama, Jiro; Sonomoto, Kenji

    2006-01-01

    In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpBTha) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpBTha forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45°C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpBTha reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJETha) and ATP. Interestingly, the mixture of dimer and monomer ClpBTha, which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpBTha forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJETha and ATP under poststress conditions. PMID:16997952

  20. Lack of the RNA chaperone Hfq attenuates pathogenicity of several Escherichia coli pathotypes towards Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Bojer, Martin Saxtorph; Jakobsen, Henrik; Struve, Carsten;

    2012-01-01

    as a model for virulence characterization and screening for novel antimicrobial entities. Several E. coli human pathotypes are also pathogenic towards C. elegans, and we show here that lack of the RNA chaperone Hfq significantly reduces pathogenicity of VTEC, EAEC, and UPEC in the nematode model. Thus, Hfq...

  1. Isolation of a Latimeria menadoensis heat shock protein 70 (Lmhsp70) that has all the features of an inducible gene and encodes a functional molecular chaperone.

    Science.gov (United States)

    Modisakeng, Keoagile W; Jiwaji, Meesbah; Pesce, Eva-Rachele; Robert, Jacques; Amemiya, Chris T; Dorrington, Rosemary A; Blatch, Gregory L

    2009-08-01

    Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system.

  2. Structure of Human J-type Co-chaperone HscB Reveals a Tetracysteine Metal-binding Domain*S⃞

    Science.gov (United States)

    Bitto, Eduard; Bingman, Craig A.; Bittova, Lenka; Kondrashov, Dmitry A.; Bannen, Ryan M.; Fox, Brian G.; Markley, John L.; Phillips, George N.

    2008-01-01

    Iron-sulfur proteins play indispensable roles in a broad range of biochemical processes. The biogenesis of iron-sulfur proteins is a complex process that has become a subject of extensive research. The final step of iron-sulfur protein assembly involves transfer of an iron-sulfur cluster from a cluster-donor to a cluster-acceptor protein. This process is facilitated by a specialized chaperone system, which consists of a molecular chaperone from the Hsc70 family and a co-chaperone of the J-domain family. The 3.0Å crystal structure of a human mitochondrial J-type co-chaperone HscB revealed an L-shaped protein that resembles Escherichia coli HscB. The important difference between the two homologs is the presence of an auxiliary metal-binding domain at the N terminus of human HscB that coordinates a metal via the tetracysteine consensus motif CWXCX9–13FCXXCXXXQ. The domain is found in HscB homologs from animals and plants as well as in magnetotactic bacteria. The metal-binding site of the domain is structurally similar to that of rubredoxin and several zinc finger proteins containing rubredoxin-like knuckles. The normal mode analysis of HscB revealed that this L-shaped protein preferentially undergoes a scissors-like motion that correlates well with the conformational changes of human HscB observed in the crystals. PMID:18713742

  3. A stress-responsive late embryogenesis abundant protein 7 (CsLEA7) of tea [Camellia sinensis (L.) O. Kuntze] encodes for a chaperone that imparts tolerance to Escherichia coli against stresses.

    Science.gov (United States)

    Paul, Asosii; Singh, Sewa; Sharma, Shweta; Kumar, Sanjay

    2014-11-01

    The present study characterized CsLEA7, a group 7 late embryogenesis abundant (LEA) gene, from tea [Camellia sinensis (L.) O. Kuntze]. The gene had an open reading frame of 462 base pairs encoding 153 amino acids with calculated molecular weight of 16.63 kDa and an isoelectric point (pI) of 4.93. Analysis revealed CsLEA7 to be an intrinsically ordered protein consisting of nine β-strands and two α-helices. CsLEA7 expressed ubiquitously in all the tissues analyzed with highest level of transcripts in mature leaf as compared to in flower bud, younger leaves, stem and fruit. Expression was the least in root tissue. CsLEA7 exhibited up-regulation in response to low temperature, polyethylene glycol-8000, sodium chloride and hydrogen peroxide in tea. Analysis of the promoter of CsLEA7 revealed a core promoter element and distinct cis-acting regulatory elements regulating gene expression under abiotic stresses. CsLEA7 exhibited chaperonic activity as evinced by protection to malate dehydrogenase against heat denaturation assay. Recombinant Escherichia coli cells producing CsLEA7 exhibited improved tolerance against diverse cues: polyethylene glycol-8000, sodium chloride, hydrogen peroxide and low temperature signifying its role in imparting stress tolerance. PMID:25052187

  4. Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics.

    Directory of Open Access Journals (Sweden)

    Ram H Nagaraj

    Full Text Available Methylglyoxal (MGO is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12 is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2-10 µM, R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.

  5. Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion.

    Science.gov (United States)

    Zininga, Tawanda; Achilonu, Ikechukwu; Hoppe, Heinrich; Prinsloo, Earl; Dirr, Heini W; Shonhai, Addmore

    2016-05-01

    The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival. PMID:26894764

  6. Exploring geometric properties of gold nanoparticles using TEM images to explain their chaperone like activity for citrate synthase

    Science.gov (United States)

    Kaushik, Vikas; Lahiri, Tapobrata; Singha, Shantiswaroop; Dasgupta, Anjan Kumar; Mishra, Hrishikesh; Kumar, Upendra; Kumar, Rajeev

    2011-01-01

    Study on geometric properties of nanoparticles and their relation with biomolecular activities, especially protein is quite a new field to explore. This work was carried out towards this direction where images of gold nanoparticles obtained from transmission electron microscopy were processed to extract their size and area profile at different experimental conditions including and excluding a protein, citrate synthase. Since the images were ill-posed, texture of a context-window for each pixel was used as input to a back-propagation network architecture to obtain decision on its membership as nanoparticle. The segmented images were further analysed by k-means clustering to derive geometric properties of individual nanoparticles even from their assembled form. The extracted geometric information was found to be crucial to give a model featuring porous cage like configuration of nanoparticle assembly using which the chaperone like activity of gold nanoparticles can be explained. PMID:22355230

  7. Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture.

    Directory of Open Access Journals (Sweden)

    Ramazan Kurt

    Full Text Available Hepatic steatosis is a risk factor for both liver disease progression and an impaired response to interferon alpha (IFN-α-based combination therapy in chronic hepatitis C virus (HCV infection. Previously, we reported that free fatty acid (FFA-treated HCV cell culture induces hepatocellular steatosis and impairs the expression of interferon alpha receptor-1 (IFNAR1, which is why the antiviral activity of IFN-α against HCV is impaired.To investigate the molecular mechanism by which IFNAR1 expression is impaired in HCV cell culture with or without free fatty acid-treatment.HCV-infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate and unsaturated (oleate long-chain free fatty acids (FFA. Intracytoplasmic fat accumulation in HCV-infected culture was visualized by oil red staining. Clearance of HCV in FFA cell culture treated with type I IFN (IFN-α and Type III IFN (IFN-λ was determined by Renilla luciferase activity, and the expression of HCV core was determined by immunostaining. Activation of Jak-Stat signaling in the FFA-treated HCV culture by IFN-α alone and IFN-λ alone was examined by Western blot analysis and confocal microscopy. Lysosomal degradation of IFNAR1 by chaperone-mediated autophagy (CMA in the FFA-treated HCV cell culture model was investigated.FFA treatment induced dose-dependent hepatocellular steatosis and lipid droplet accumulation in HCV-infected Huh-7.5 cells. FFA treatment of infected culture increased HCV replication in a concentration-dependent manner. Intracellular lipid accumulation led to reduced Stat phosphorylation and nuclear translocation, causing an impaired IFN-α antiviral response and HCV clearance. Type III IFN (IFN-λ, which binds to a separate receptor, induces Stat phosphorylation, and nuclear translocation as well as antiviral clearance in FFA-treated HCV cell culture. We show here that the HCV-induced autophagy response is increased in FFA-treated cell culture

  8. Induction and adaptation of chaperone-assisted selective autophagy CASA in response to resistance exercise in human skeletal muscle.

    Science.gov (United States)

    Ulbricht, Anna; Gehlert, Sebastian; Leciejewski, Barbara; Schiffer, Thorsten; Bloch, Wilhelm; Höhfeld, Jörg

    2015-01-01

    Chaperone-assisted selective autophagy (CASA) is a tension-induced degradation pathway essential for muscle maintenance. Impairment of CASA causes childhood muscle dystrophy and cardiomyopathy. However, the importance of CASA for muscle function in healthy individuals has remained elusive so far. Here we describe the impact of strength training on CASA in a group of healthy and moderately trained men. We show that strenuous resistance exercise causes an acute induction of CASA in affected muscles to degrade mechanically damaged cytoskeleton proteins. Moreover, repeated resistance exercise during 4 wk of training led to an increased expression of CASA components. In human skeletal muscle, CASA apparently acts as a central adaptation mechanism that responds to acute physical exercise and to repeated mechanical stimulation.

  9. Chaperone-Like Activity of ß-Casein and Its Effect on Residual in Vitro Activity of Food Enzymes

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria

    . The negative effect of BSA on enzyme was not observed before. The residual activity of horseradish peroxidase was also improved by the reconstituted skim milk: addition of reconstituted skim milk prior to heat treatment resulted in higher residual activity of HRP compared to no addition (58±3% and 30......±1%, respectively) The findings of this study show that β-casein can influence the response of food enzymes to heat treatment. β-Casein is not a universal chaperone and its effect on different targets needs to be evaluated on a case-by-case basis. This study also shows that proteins as e.g. BSA may affect......ABSTRACT Activity of endogenous enzymes may cause browning of fruits and vegetables. These enzymes can be inactivated, for example by heat treatment, but the response of enzymes to heat treatment depends on many factors. Foods are very complex systems and the stability of enzymes...

  10. Structural insights into yeast histone chaperone Hif1: a scaffold protein recruiting protein complexes to core histones.

    Science.gov (United States)

    Liu, Hejun; Zhang, Mengying; He, Wei; Zhu, Zhongliang; Teng, Maikun; Gao, Yongxiang; Niu, Liwen

    2014-09-15

    Yeast Hif1 [Hat1 (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hif1 consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.

  11. SUMO: regulating the regulator

    Directory of Open Access Journals (Sweden)

    Bossis Guillaume

    2006-06-01

    Full Text Available Abstract Post-translational modifiers of the SUMO (Small Ubiquitin-related Modifier family have emerged as key regulators of protein function and fate. While the past few years have seen an enormous increase in knowledge on SUMO enzymes, substrates, and consequences of modification, regulation of SUMO conjugation is far from being understood. This brief review will provide an overview on recent advances concerning (i the interplay between sumoylation and other post-translational modifications at the level of individual targets and (ii global regulation of SUMO conjugation and deconjugation.

  12. Virus-Induced Chaperone-Enriched (VICE domains function as nuclear protein quality control centers during HSV-1 infection.

    Directory of Open Access Journals (Sweden)

    Christine M Livingston

    2009-10-01

    Full Text Available Virus-Induced Chaperone-Enriched (VICE domains form adjacent to nuclear viral replication compartments (RC during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70, the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42 degrees C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.

  13. Ensemble Structure of the Highly Flexible Complex Formed between Vesicular Stomatitis Virus Unassembled Nucleoprotein and its Phosphoprotein Chaperone.

    Science.gov (United States)

    Yabukarski, Filip; Leyrat, Cedric; Martinez, Nicolas; Communie, Guillaume; Ivanov, Ivan; Ribeiro, Euripedes A; Buisson, Marlyse; Gerard, Francine C; Bourhis, Jean-Marie; Jensen, Malene Ringkjøbing; Bernadó, Pau; Blackledge, Martin; Jamin, Marc

    2016-07-01

    Nucleocapsid assembly is an essential process in the replication of the non-segmented, negative-sense RNA viruses (NNVs). Unassembled nucleoprotein (N(0)) is maintained in an RNA-free and monomeric form by its viral chaperone, the phosphoprotein (P), forming the N(0)-P complex. Our earlier work solved the structure of vesicular stomatitis virus complex formed between an N-terminally truncated N (NΔ21) and a peptide of P (P60) encompassing the N(0)-binding site, but how the full-length P interacts with N(0) remained unknown. Here, we combine several experimental biophysical methods including size exclusion chromatography with detection by light scattering and refractometry, small-angle X-ray and neutron scattering and nuclear magnetic resonance spectroscopy with molecular dynamics simulation and computational modeling to characterize the NΔ21(0)-PFL complex formed with dimeric full-length P. We show that for multi-molecular complexes, simultaneous multiple-curve fitting using small-angle neutron scattering data collected at varying contrast levels provides additional information and can help refine structural ensembles. We demonstrate that (a) vesicular stomatitis virus PFL conserves its high flexibility within the NΔ21(0)-PFL complex and interacts with NΔ21(0) only through its N-terminal extremity; (b) each protomer of P can chaperone one N(0) client protein, leading to the formation of complexes with stoichiometries 1N:P2 and 2N:P2; and (c) phosphorylation of residues Ser60, Thr62 and Ser64 provides no additional interactions with N(0) but creates a metal binding site in PNTR. A comparison with the structures of Nipah virus and Ebola virus N(0)-P core complex suggests a mechanism for the control of nucleocapsid assembly that is common to all NNVs. PMID:27107640

  14. Chemical chaperones reduce ionizing radiation-induced endoplasmic reticulum stress and cell death in IEC-6 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Sang; Lee, Hae-June; Lee, Yoon-Jin [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Jeong, Jae-Hoon [Division of Radiotherapy, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Kang, Seongman [Division of Life Sciences, Korea University, Seoul 136-701 (Korea, Republic of); Lim, Young-Bin, E-mail: yblim@kirams.re.kr [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

    2014-07-25

    Highlights: • UPR activation precedes caspase activation in irradiated IEC-6 cells. • Chemical ER stress inducers radiosensitize IEC-6 cells. • siRNAs that targeted ER stress responses ameliorate IR-induced cell death. • Chemical chaperons prevent cell death in irradiated IEC-6 cells. - Abstract: Radiotherapy, which is one of the most effective approaches to the treatment of various cancers, plays an important role in malignant cell eradication in the pelvic area and abdomen. However, it also generates some degree of intestinal injury. Apoptosis in the intestinal epithelium is the primary pathological factor that initiates radiation-induced intestinal injury, but the mechanism by which ionizing radiation (IR) induces apoptosis in the intestinal epithelium is not clearly understood. Recently, IR has been shown to induce endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) signaling pathway in intestinal epithelial cells. However, the consequences of the IR-induced activation of the UPR signaling pathway on radiosensitivity in intestinal epithelial cells remain to be determined. In this study, we investigated the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhanced IR-induced caspase 3 activation and DNA fragmentation in intestinal epithelial cells. Knockdown of Xbp1 or Atf6 with small interfering RNA inhibited IR-induced caspase 3 activation. Treatment with chemical chaperones prevented ER stress and subsequent apoptosis in IR-exposed intestinal epithelial cells. Our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Furthermore, inhibiting ER stress may be an effective strategy to prevent IR-induced intestinal injury.

  15. From flexibility to function: Molecular dynamics simulations of conformational changes in chaperones and photoreceptors

    NARCIS (Netherlands)

    K. Singhal

    2016-01-01

    Proteins are uniquely-shaped macromolecules that function as biological machines, and regulate a living cell’s behavior. Crucial to protein function is the folding of the polypeptide chain into a unique well-defined three-dimensional conformation. In complex cell environments, the spontaneous unassi

  16. One out of four: HspL but no other small heat shock protein of Agrobacterium tumefaciens acts as efficient virulence-promoting VirB8 chaperone.

    Directory of Open Access Journals (Sweden)

    Yun-Long Tsai

    Full Text Available Alpha-crystallin-type small heat shock proteins (sHsps are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named hspL, hspC, hspAT1, and hspAT2 were identified in Agrobacterium tumefaciens, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS. HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of hspL but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the hspL deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of hspL and only modest increase of hspC, hspAT1, and hspAT2 at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays in vitro indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated in vivo, in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in A. tumefaciens. Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence

  17. Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid

    Directory of Open Access Journals (Sweden)

    Quyen Thi Dinh

    2012-03-01

    Full Text Available Abstracts Background The lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA and its chaperone (LipB from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems. Results To produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB, six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein and only a small fraction of the overexpressed lipase was

  18. Activities of virE1 and the VirE1 secretion chaperone in export of the multifunctional VirE2 effector via an Agrobacterium type IV secretion pathway.

    Science.gov (United States)

    Zhao, Z; Sagulenko, E; Ding, Z; Christie, P J

    2001-07-01

    Agrobacterium tumefaciens uses a type IV secretion system to deliver oncogenic nucleoprotein particles and effector proteins, such as the multifunctional VirE2 protein, to plant cells. In this study, we examined the function of virE1 and its product, the VirE1 secretion chaperone, in mediating VirE2 export. A nonpolar virE1 null mutant accumulated low levels of VirE2, and trans expression of virE1 in this mutant only partially restored VirE2 abundance. Deletion of virE1 did not affect transcription but decreased translation of virE2, as shown by analysis of lacZ transcriptional and translational fusions. VirE2 was stable for a prolonged period, more than 6 h, when it was expressed in cis with virE1, and it exhibited half-lives of about 2 h when it was expressed in trans with virE1 and less than 10 min when it was expressed in the absence of virE1, as shown by pulse-chase experiments. VirE1 stabilized VirE2 via an interaction with a domain near the N terminus of VirE2, as shown by analyses of VirE2 truncation and insertion mutants synthesized in A. tumefaciens. VirE1 self-association was demonstrated by using bacteriophage lambda cI repressor fusion and pull-down assays, and evidence of VirE1 homomultimerization in vivo was obtained by native polyacrylamide gel electrophoresis and gel filtration chromatography. A putative VirE1-VirE2 complex with a molecular mass of about 70 to 80 kDa was detected by gel filtration chromatography of extracts from wild-type cells, whereas higher-order VirE2 complexes or aggregates were detected in extracts from a virE1 mutant. Taken together, our findings show that virE1 contributes in several ways to VirE2 export:(i) virE1 regulates efficient virE2 translation in the context of expression from the native P(virE) promoter; (ii) the VirE1 secretion chaperone stabilizes VirE2, most probably via an interaction with an N-terminal domain; and (iii) VirE1 forms a VirE1-VirE2 complex with a predicted 2:1 stoichiometry that inhibits assembly

  19. The molecular chaperone TRiC/CCT binds to the Trp-Asp 40 (WD40) repeat protein WDR68 and promotes its folding, protein kinase DYRK1A binding, and nuclear accumulation.

    Science.gov (United States)

    Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

    2014-11-28

    Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID

  20. The role of auxin in temperature regulated hypocotyl elongation

    Energy Technology Data Exchange (ETDEWEB)

    Estelle, Mark [Univ. of California, San Diego, CA (United States)

    2015-10-02

    The major goal of this project was to determine how auxin mediates the response of Arabidopsis seedlings to increased ambient temperature. Previous studies have shown that the response is due, in part, to increased auxin biosynthesis via the IPA auxin biosynthetic pathway. This effect is related to increased transcription of genes that encode enzymes in this pathway. However, during the last year we have shown that transcription of key auxin regulated genes increases within minutes of a shift to elevated temperature. This response is probably to rapid to be explained by changes in the levels of auxin biosynthetic enzymes. Interestingly, we have recently discovered that temperature shift is associated with a rapid increase in the level of the auxin co-receptor TIR1. This change appears is the result of increased stability of the protein. At the same time, we have discovered that stability of TIR1 is dependent on the chaperone HSP9o and its co-chaperone SGT1. By using the specific HSP90 inhibitor GDA, we show that HSP90 is required for the temperature dependent change in TIR1 levels. We have also shown that HSP90 and SGT1 interact directly with TIR1. Our results also lead us to propose a new model in which the plant responds rapidly to changes in ambient temperature by directly regulating the TIR1/AFB receptor system, thus modulating the auxin signaling pathway.

  1. Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production.

    Science.gov (United States)

    Crowley, P J; Brady, L J

    2016-02-01

    The respective contributions of components of the protein translocation/maturation machinery to cell surface biogenesis in Streptococcus mutans are not fully understood. Here we used a genetic approach to characterize the effects of deletion of genes encoding the ribosome-associated chaperone RopA (Trigger Factor), the surface-localized foldase PrsA, and the membrane-localized chaperone insertases YidC1 and YidC2, both singly and in combination, on bacterial growth, chain length, self-aggregation, cell surface hydrophobicity, autolysis, and antigenicity of surface proteins P1 (AgI/II, PAc), WapA, GbpC, and GtfD. The single and double deletion mutants, as well as additional mutant strains lacking components of the signal recognition particle pathway, were also evaluated for their effects on mutacin production and genetic competence. PMID:26386361

  2. Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress▿

    OpenAIRE

    Sugimoto, Shinya; Yoshida, Hiroyuki; Mizunoe, Yoshimitsu; Tsuruno, Keigo; Nakayama, Jiro; Sonomoto, Kenji

    2006-01-01

    In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpBTha) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaralde...

  3. Molecular Events Involved in a Single Cycle of Ligand Transfer from an ATP Binding Cassette Transporter, LolCDE, to a Molecular Chaperone, LolA*

    OpenAIRE

    Taniguchi, Naohiro; Tokuda, Hajime

    2008-01-01

    An ATP binding cassette transporter LolCDE complex releases lipoproteins from the inner membrane of Escherichia coli in an ATP-dependent manner, leading to the formation of a complex between a lipoprotein and a periplasmic chaperone, LolA. LolA is proposed to undergo a conformational change upon the lipoprotein binding. The lipoprotein is then transferred from the LolA-lipoprotein complex to the outer membrane via LolB. Unlike most ATP binding cassette transporters med...

  4. High-Level Formation of Active Pseudomonas cepacia Lipase after Heterologous Expression of the Encoding Gene and Its Modified Chaperone in Escherichia coli and Rapid In Vitro Refolding

    OpenAIRE

    Quyen, Dinh Thi; Schmidt-Dannert, Claudia; Schmid, Rolf D.

    1999-01-01

    The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P. cepacia harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible λPRL promoter. However, no overexpression of the lipase chape...

  5. High level accumulation of soluble diphtheria toxin mutant (CRM197) with co-expression of chaperones in recombinant Escherichia coli.

    Science.gov (United States)

    Mahamad, Pornpimol; Boonchird, Chuenchit; Panbangred, Watanalai

    2016-07-01

    CRM197 is the diphtheria toxin mutant used in many conjugate vaccines. A fusion CRM197 (fCRM197) containing all the tags conferred by the pET32a vector was produced as a soluble protein in Escherichia coli co-expressing several chaperone proteins in conjunction with low temperature cultivation. Trigger factor (Tf) enhanced formation of soluble fCRM197 (150.69 ± 8.95 μg/mL) to a greater degree than other chaperones when fCRM197 expression was induced at 25 °C for 12 h. However, prolonged cultivation resulted in a progressive reduction of fCRM197 accumulation. In contrast, at 15 °C cells, with or without Tf, fCRM197 accumulated to the highest level at 48 h (153.70 ± 13.14 μg/mL and 150.07 ± 8.13 μg/mL, respectively). Transmission electron microscopy (TEM) demonstrated that the formation of inclusion protein as well as cell lysis was reduced in cultures grown at 15 °C. Cell viability was substantially reduced in cells expressing Tf, compared to cultures without Tf, when fCRM197 was induced at 25 °C. The viability of Tf-expressing cells was enhanced when cultured at 15 °C. Both purified fCRM197 and CRM197 efficiently digested lambda DNA (λDNA) at 37 °C (92.78 and 97.45 %, respectively). Digestion efficiency of fCRM197 and CRM197 was reduced at 25 °C (80.80 and 62.73 %, respectively) and at 15 °C (7.34 and 24.79 %, respectively). These results demonstrating nuclease activity, enhanced cell lysis, and reduced cell viability are consistent with the finding of lower fCRM197 yield when cultivation and induction times were prolonged at 25 °C. The present work provides a procedure for the high-level production of soluble fCRM197 using E. coli as a heterologous host. PMID:27020286

  6. The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene.

    Directory of Open Access Journals (Sweden)

    Ana Paço

    Full Text Available The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials. The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants

  7. In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli.

    Science.gov (United States)

    Sugimoto, Shinya; Saruwatari, Kozue; Higashi, Chihana; Tsuruno, Keigo; Matsumoto, Shunsuke; Nakayama, Jiro; Sonomoto, Kenji

    2008-03-01

    In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli. PMID:18323638

  8. Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

    Directory of Open Access Journals (Sweden)

    Lai Yiu-Kay

    2012-11-01

    Full Text Available Abstract Background There are few studies that have examined the potential of RNA inference (RNAi to increase protein production in the baculovirus expression vector system (BEVS. Spodoptera frugiperda (fall armyworm (Sf-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated. Results In the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase and extracellular (secreted alkaline phosphatase [SEAP] recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study. Conclusion The data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.

  9. Characterization and Structure of a Zn[superscript 2+] and [2Fe-2S]-containing Copper Chaperone from Archaeoglobus fulgidus

    Energy Technology Data Exchange (ETDEWEB)

    Sazinsky, Matthew H.; LeMoine, Benjamin; Orofino, Maria; Davydov, Roman; Bencze, Krisztina Z.; Stemmler, Timothy L.; Hoffman, Brian M.; Arguello, Jose M.; Rosenzweig, Amy C. (Worcester); (WSU-MED); (NWU)

    2010-03-08

    Bacterial CopZ proteins deliver copper to P{sub 1B}-type Cu{sup +}-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8 {angstrom} resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu{sup +} to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu{sup 2+} to Cu{sup +}. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking.

  10. Market, Regulation, Market, Regulation

    DEFF Research Database (Denmark)

    Frankel, Christian; Galland, Jean-Pierre

    2015-01-01

    barriers to trade in Europe, realized the free movement of products by organizing progressively several orders of markets and regulation. Based on historical and institutional documents, on technical publications, and on interviews, this article relates how the European Commission and the Member States had......This paper focuses on the European Regulatory system which was settled both for opening the Single Market for products and ensuring the consumers' safety. It claims that the New Approach and Standardization, and the Global Approach to conformity assessment, which suppressed the last technical...... alternatively recourse to markets and to regulations, at the three main levels of the New Approach Directives implementation. The article focuses also more specifically on the Medical Devices sector, not only because this New Approach sector has long been controversial in Europe, and has recently been concerned...

  11. Aqueous Extract of Paeonia lactiflora and Paeoniflorin as Aggregation Reducers Targeting Chaperones in Cell Models of Spinocerebellar Ataxia 3

    Directory of Open Access Journals (Sweden)

    Kuo-Hsuan Chang

    2013-01-01

    Full Text Available Spinocerebellar ataxia (SCA types 1, 2, 3, 6, 7, and 17 as well as Huntington’s disease are a group of neurodegenerative disorders caused by expanded CAG repeats encoding a long polyglutamine (polyQ tract in the respective proteins. Evidence has shown that the accumulation of intranuclear and cytoplasmic misfolded polyQ proteins leads to apoptosis and cell death. Thus suppression of aggregate formation is expected to inhibit a wide range of downstream pathogenic events in polyQ diseases. In this study, we established a high-throughput aggregation screening system using 293 ATXN3/Q75-GFP cells and applied this system to test the aqueous extract of Paeonia lactiflora (P. lactiflora and its constituents. We found that the aggregation can be significantly prohibited by P. lactiflora and its active compound paeoniflorin. Meanwhile, P. lactiflora and paeoniflorin upregulated HSF1 and HSP70 chaperones in the same cell models. Both of them further reduced the aggregation in neuronal differentiated SH-SY5Y ATXN3/Q75-GFP cells. Our results demonstrate how P. lactiflora and paeoniflorin are likely to work on polyQ-aggregation reduction and provide insight into the possible working mechanism of P. lactiflora in SCA3. We anticipate our paper to be a starting point for screening more potential herbs for the treatment of SCA3 and other polyQ diseases.

  12. The molecular chaperone Hsp70 activates protein phosphatase 5 (PP5) by binding the tetratricopeptide repeat (TPR) domain.

    Science.gov (United States)

    Connarn, Jamie N; Assimon, Victoria A; Reed, Rebecca A; Tse, Eric; Southworth, Daniel R; Zuiderweg, Erik R P; Gestwicki, Jason E; Sun, Duxin

    2014-01-31

    Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development.

  13. Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA

    Directory of Open Access Journals (Sweden)

    Bogen Bjarne

    2010-02-01

    Full Text Available Abstract Background Efficient expression systems exist for antibody (Ab molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs. Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc TCRs. Results The effect of 1 over-expression of the periplasmic chaperon FkpA, 2 culture conditions and 3 molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Vαβ domain orientation was far superior to the Vβα domain orientation regarding monomeric yield of functionally folded molecules. Conclusion The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1 high yield recovery sufficient for biophysical characterization and 2 high throughput screening of such molecules following molecular engineering.

  14. Chaperone-like activity of β-casein and its effect on residual in vitro activity of horseradish peroxidase

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria; Olsen, Karsten; Sørensen, Jens Christian;

    2014-01-01

    proteins. Incubating HRP (0.1 mg mL-1) for 10 min at 72 °C resulted in residual activity of 59 ± 5%, while addition of 1 mg mL-1 β-casein resulted in increase in residual activity up to 85 ± 1%. Increased residual activity is not merely attributed to an effect of higher total protein concentration......In this study, the residual activity horseradish peroxidase was used as a novel marker of chaperone-like activity of β-casein under elevated temperature. It was shown that β-casein does affect residual activity of horseradish peroxidase (HRP) depending on the concentration and molar ratio between......, as similar experiment with bovine serum albumin resulted in residual activity of horseradish peroxidase that was significantly lower than without any addition. The effect of β-casein on HRP disappears when pH is below the isoelectric point of β-casein. It was also proven by light scattering studies that β...

  15. A cytosolic chaperone complexes with dynamic membrane J-proteins and mobilizes a nonenveloped virus out of the endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Christopher Paul Walczak

    2014-03-01

    Full Text Available Nonenveloped viruses undergo conformational changes that enable them to bind to, disrupt, and penetrate a biological membrane leading to successful infection. We assessed whether cytosolic factors play any role in the endoplasmic reticulum (ER membrane penetration of the nonenveloped SV40. We find the cytosolic SGTA-Hsc70 complex interacts with the ER transmembrane J-proteins DnaJB14 (B14 and DnaJB12 (B12, two cellular factors previously implicated in SV40 infection. SGTA binds directly to SV40 and completes ER membrane penetration. During ER-to-cytosol transport of SV40, SGTA disengages from B14 and B12. Concomitant with this, SV40 triggers B14 and B12 to reorganize into discrete foci within the ER membrane. B14 must retain its ability to form foci and interact with SGTA-Hsc70 to promote SV40 infection. Our results identify a novel role for a cytosolic chaperone in the membrane penetration of a nonenveloped virus and raise the possibility that the SV40-induced foci represent cytosol entry sites.

  16. Mis16 Independently Recognizes Histone H4 and the CENP-ACnp1-Specific Chaperone Scm3sp.

    Science.gov (United States)

    An, Sojin; Kim, Hanseong; Cho, Uhn-Soo

    2015-10-01

    CENP-A is a centromere-specific histone H3 variant that is required for kinetochore assembly and accurate chromosome segregation. For it to function properly, CENP-A must be specifically localized to centromeres. In fission yeast, Scm3sp and the Mis18 complex, composed of Mis16, Eic1, and Mis18, function as a CENP-A(Cnp1)-specific chaperone and a recruiting factor, respectively, and together ensure accurate delivery of CENP-A(Cnp1) to centromeres. Although how Scm3sp specifically recognizes CENP-A(Cnp1) has been revealed recently, the recruiting mechanism of CENP-A(Cnp1) via the Mis18 complex remains unknown. In this study, we have determined crystal structures of Schizosaccharomyces japonicus Mis16 alone and in complex with the helix 1 of histone H4 (H4α1). Crystal structures followed by mutant analysis and affinity pull-downs have revealed that Mis16 recognizes both H4α1 and Scm3sp independently within the CENP-A(Cnp1)/H4:Scm3sp complex. This observation suggests that Mis16 gains CENP-A(Cnp1) specificity by recognizing both Scm3sp and histone H4. Our studies provide insights into the molecular mechanisms underlying specific recruitment of CENP-A(Cnp1)/H4:Scm3sp into centromeres. PMID:26343758

  17. Mammalian ribosomal and chaperone protein RPS3A counteracts α-synuclein aggregation and toxicity in a yeast model system.

    Science.gov (United States)

    De Graeve, Stijn; Marinelli, Sarah; Stolz, Frank; Hendrix, Jelle; Vandamme, Jurgen; Engelborghs, Yves; Van Dijck, Patrick; Thevelein, Johan M

    2013-11-01

    Accumulation of aggregated forms of αSyn (α-synuclein) into Lewy bodies is a known hallmark associated with neuronal cell death in Parkinson's disease. When expressed in the yeast Saccharomyces cerevisiae, αSyn interacts with the plasma membrane, forms inclusions and causes a concentration-dependent growth defect. We have used a yeast mutant, cog6Δ, which is particularly sensitive to moderate αSyn expression, for screening a mouse brain-specific cDNA library in order to identify mammalian proteins that counteract αSyn toxicity. The mouse ribosomal and chaperone protein RPS3A was identified as a suppressor of αSyn [WT (wild-type) and A53T] toxicity in yeast. We demonstrated that the 50 N-terminal amino acids are essential for this function. The yeast homologues of RPS3A were not effective in suppressing the αSyn-induced growth defect, illustrating the potential of our screening system to identify modifiers that would be missed using yeast gene overexpression as the first screening step. Co-expression of mouse RPS3A delayed the formation of αSyn-GFP inclusions in the yeast cells. The results of the present study suggest that the recently identified extraribosomal chaperonin function of RPS3A also acts on the neurodegeneration-related protein αSyn and reveal a new avenue for identifying promising candidate mammalian proteins involved in αSyn functioning.

  18. Purification and in vitro chaperone activity of a class I small heat-shock protein abundant in recalcitrant chestnut seeds.

    Science.gov (United States)

    Collada, C; Gomez, L; Casado, R; Aragoncillo, C

    1997-09-01

    A 20-kD protein has been purified from cotyledons of recalcitrant (desiccation-sensitive) chestnut (Castanea sativa) seeds, where it accumulates at levels comparable to those of major seed storage proteins. This protein, termed Cs smHSP 1, forms homododecameric complexes under nondenaturing conditions and appears to be homologous to cytosolic class I small heat-shock proteins (smHSPs) from plant sources. In vitro evidence has been obtained that the isolated protein can function as a molecular chaperone; it increases, at stoichiometric levels, the renaturation yields of chemically denatured citrate synthase and also prevents the irreversible thermal inactivation of this enzyme. Although a role in desiccation tolerance has been hypothesized for seed smHSPs, this does not seem to be the case for Cs smHSP 1. We have investigated the presence of immunologically related proteins in orthodox and recalcitrant seeds of 13 woody species. Our results indicate that the presence of Cs smHSP 1-like proteins, even at high levels, is not enough to confer desiccation tolerance, and that the amount of these proteins does not furnish a reliable criterion to identify desiccation-sensitive seeds. Additional proteins or mechanisms appear necessary to keep the viability of orthodox seeds upon shedding.

  19. The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones

    Science.gov (United States)

    Liu, Wallace H; Roemer, Sarah C; Zhou, Yeyun; Shen, Zih-Jie; Dennehey, Briana K; Balsbaugh, Jeremy L; Liddle, Jennifer C; Nemkov, Travis; Ahn, Natalie G; Hansen, Kirk C; Tyler, Jessica K; Churchill, Mair EA

    2016-01-01

    The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4)2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4)2 tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication. DOI: http://dx.doi.org/10.7554/eLife.18023.001 PMID:27690308

  20. Lens gene expression analysis reveals downregulation of the anti-apoptotic chaperone alphaA-crystallin during cavefish eye degeneration.

    Science.gov (United States)

    Strickler, Allen G; Byerly, Mardi S; Jeffery, William R

    2007-12-01

    We have conducted a survey of the expression patterns of five genes encoding three different classes of major lens proteins during eye degeneration in the blind cavefish Astyanax mexicanus. This species consists of two forms, an eyed surface-dwelling form (surface fish) and a blind cave-dwelling (cavefish) form. Cavefish form an optic primordium with a lens vesicle and optic cup. In contrast to surface fish, however, the cavefish lens does not differentiate fiber cells and undergoes massive apoptosis. The genes encoding the lens intrinsic membrane proteins MIP and MP19 and the divergent betaB1- and gammaM2-crystallins are expressed during cavefish lens development, although their levels are reduced because of a smaller lens, and the spatial distribution of their transcripts is modified because of the lack of differentiated fiber cells. In contrast, the alphaA-crystallin gene, which encodes a heat shock protein-related chaperone with antiapoptotic activity, is substantially downregulated in the developing cavefish lens. The results suggest that suppression of alphaA-crystallin antiapoptotic activity may be involved in cavefish eye degeneration.

  1. Proteomics displays cytoskeletal proteins and chaperones involvement in Hedyotis corymbosa-induced photokilling in skin cancer cells.

    Science.gov (United States)

    You, Bang-Jau; Wu, Yang-Chang; Wu, Chi-Yu; Bao, Bo-Ying; Chen, Mei-Yu; Chang, Yu-Hao; Lee, Hong-Zin

    2011-08-01

    Photodynamic therapy was found to be an effective therapy for local malignant tumors. This study demonstrated that 80 μg/ml Hedyotis corymbosa extracts with 0.8 J/cm(2) fluence dose caused M21 skin cancer cell death. Photoactivated H. corymbosa-induced M21 cell death is a typical apoptosis that is accompanied by nuclear condensation, externalization of phosphatidylserine and the changes in protein expression of apoptosis-related proteins, such as Bcl-2 and caspase family members. This study applied 2D electrophoresis to analyse the proteins involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We found 12 proteins to be markedly changed. According to the results of protein sequence analysis of these altered protein spots, we identified that the expression of cytoskeletal proteins and chaperones were involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We further demonstrated that photoactivated H. corymbosa caused a significant effect on the cytoskeleton distribution and mitochondrial activity in M21 cells. Based on the above findings, this study characterized the effects and mechanisms of the photoactivated H. corymbosa-induced apoptosis in M21 skin cancer cells. PMID:21569101

  2. The therapeutic potential of antioxidants, ER chaperones, NO and H2S donors, and statins for treatment of preeclampsia

    Directory of Open Access Journals (Sweden)

    Tereza eCindrova-Davies

    2014-05-01

    Full Text Available Preeclampsia is a complex multifactorial disease. Placental oxidative stress, a result of deficient spiral artery remodeling, plays an important role in the pathophysiology of preeclampsia. Antiangiogenic factors secreted from malperfused placenta are instrumental in mediating maternal endothelial dysfunction and consequent symptoms of preeclampsia; the mechanism is likely to involve increased ET-1 secretion and reduced NO bioavailability. Therapeutic interventions so far remain only experimental and there is no established remedy for the treatment of preeclampsia. This review concentrates on the evidence for the therapeutic potential of antioxidants, ER chaperones, NO and H2S donors, and statins. These compounds display pleitropic antioxidant, anti-inflammatory, and pro-angiogenic effects in animal and in vitro studies. Although clinical trials on the use of antioxidant vitamins in pregnancy proved largely unsuccessful, the scope for their use still exists given the beneficial cardioprotective effects of antioxidant-rich Mediterranean diet, periconceptual vitamin use and the synergistic effect of vitamin C and l-arginine. Encouraging clinical evidence exists for the use of NO donors, and a clinical trial is underway testing the effect of statins in treatment of preeclampsia. H2S recently emerged as a novel therapeutic agent for cardiovascular disease, and its beneficial effects were also tested in animal models of preeclampsia. It is risky to prescribe any medication to pregnant women on a large scale, and any future therapeutic intervention has to be well tested and safe. Many of the compounds discussed could be potential candidates.

  3. Copper chaperone Atox1 interacts with the metal-binding domain of Wilson's disease protein in cisplatin detoxification.

    Science.gov (United States)

    Dolgova, Nataliya V; Nokhrin, Sergiy; Yu, Corey H; George, Graham N; Dmitriev, Oleg Y

    2013-08-15

    Human copper transporters ATP7B (Wilson's disease protein) and ATP7A (Menkes' disease protein) have been implicated in tumour resistance to cisplatin, a widely used anticancer drug. Cisplatin binds to the copper-binding sites in the N-terminal domain of ATP7B, and this binding may be an essential step of cisplatin detoxification involving copper ATPases. In the present study, we demonstrate that cisplatin and a related platinum drug carboplatin produce the same adduct following reaction with MBD2 [metal-binding domain (repeat) 2], where platinum is bound to the side chains of the cysteine residues in the CxxC copper-binding motif. This suggests the same mechanism for detoxification of both drugs by ATP7B. Platinum can also be transferred to MBD2 from copper chaperone Atox1, which was shown previously to bind cisplatin. Binding of the free cisplatin and reaction with the cisplatin-loaded Atox1 produce the same protein-bound platinum intermediate. Transfer of platinum along the copper-transport pathways in the cell may serve as a mechanism of drug delivery to its target in the cell nucleus, and explain tumour-cell resistance to cisplatin associated with the overexpression of copper transporters ATP7B and ATP7A.

  4. Histone chaperone spt16 promotes redeposition of the original h3-h4 histones evicted by elongating RNA polymerase.

    Science.gov (United States)

    Jamai, Adil; Puglisi, Andrea; Strubin, Michel

    2009-08-14

    Nucleosomes are surprisingly dynamic structures in vivo, showing transcription-independent exchange of histones H2A-H2B genome-wide and exchange of H3-H4 mainly within the promoters of transcribed genes. In addition, nucleosomes are disrupted in front of and reassembled behind the elongating RNA polymerase. Here we show that inactivation of histone chaperone Spt16 in yeast results in rapid loss of H2B and H3 from transcribed genes but also from inactive genes. In all cases, histone loss is blocked by a transcription inhibitor, indicating a transcription-dependent event. Thus, nucleosomes are efficiently evicted by the polymerase but do not reform in the absence of Spt16. Yet exchange of nucleosomal H2B with free histones occurs normally, and, unexpectedly, incorporation of new H3 increases at all loci tested. This points to Spt16 restoring normal nucleosome structure by redepositing the displaced H3-H4 histones, thereby preventing incorporation of new histones and perhaps changes in histone modification patterns associated with ongoing transcription.

  5. Clp chaperones and proteases are central in stress survival, virulence and antibiotic resistance of Staphylococcus aureus

    DEFF Research Database (Denmark)

    Frees, Dorte; Gerth, Ulf; Ingmer, Hanne

    2014-01-01

    Intracellular proteolysis carried out by energy-dependent proteases is one of the most conserved biological processes. In all cells proteolysis maintains and shapes the cellular proteome by ridding the cell of damaged proteins and by regulating abundance of functional proteins such as regulatory...... proteins. The ATP-dependent ClpP protease is highly conserved among eubacteria and in the chloroplasts and mitochondria of eukaryotic cells. In the serious human pathogen, Staphylococcus aureus inactivation of clpP rendered the bacterium avirulent emphasizing the central role of proteolysis in virulence....... The contribution of the Clp proteins to virulence is likely to occur at multiple levels. First of all, both Clp ATPases and the Clp protease are central players in stress responses required to cope with the adverse conditions met in the host. The ClpP protease has a dual role herein, as it both eliminates stress...

  6. Chaperone-Targeting Cytotoxin and Endoplasmic Reticulum Stress-Inducing Drug Synergize to Kill Cancer Cells

    Directory of Open Access Journals (Sweden)

    Joseph M. Backer

    2009-11-01

    Full Text Available Diverse physiological and therapeutic insults that increase the amount of unfolded or misfolded proteins in the endoplasmic reticulum (ER induce the unfolded protein response, an evolutionarily conserved protective mechanism that manages ER stress. Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP is an ER-resident protein that plays a central role in the ER stress response and is the only known substrate of the proteolytic A subunit (SubA of a novel bacterial AB5 toxin. Here, we report that an engineered fusion protein, epidermal growth factor (EGF-SubA, combining EGF and SubA, is highly toxic to growing and confluent epidermal growth factor receptor-expressing cancer cells, and its cytotoxicity is mediated by a remarkably rapid cleavage of GRP78/BiP. Systemic delivery of EGF-SubA results in a significant inhibition of human breast and prostate tumor xenografts in mouse models. Furthermore, EGF-SubA dramatically increases the sensitivity of cancer cells to the ER stress-inducing drug thapsigargin, and vice versa, demonstrating the first example of mechanism-based synergism in the action of a cytotoxin and an ER-targeting drug.

  7. Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

    Directory of Open Access Journals (Sweden)

    Nannenga Brent L

    2011-12-01

    Full Text Available Abstract Background Membrane proteins (MPs populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. Results Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells, toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host. Conclusions Our system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

  8. Impact of the lectin chaperone calnexin on the stress response, virulence and proteolytic secretome of the fungal pathogen Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Margaret V Powers-Fletcher

    Full Text Available Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER that is part of a quality control system that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but had no impact on growth rates in either minimal or complex media at 37°C. However, the ΔclxA mutant was growth impaired at temperatures above 42°C and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol. In contrast to wild type A. fumigatus, ΔclxA hyphae were unable to grow when transferred to starvation medium. In addition, depleting the medium of cations by chelation prevented ΔclxA from sustaining polarized hyphal growth, resulting in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the ΔclxA mutant remained virulent in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses encountered in the host environment.

  9. Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

    Science.gov (United States)

    Harris, Julia M; Nguyen, Phil P; Patel, Milan J; Sporn, Zachary A; Hines, Justin K

    2014-07-01

    Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have

  10. BiPPred: Combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP.

    Science.gov (United States)

    Schneider, Markus; Rosam, Mathias; Glaser, Manuel; Patronov, Atanas; Shah, Harpreet; Back, Katrin Christiane; Daake, Marina Angelika; Buchner, Johannes; Antes, Iris

    2016-10-01

    Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc.

  11. The Salmonella type III effector SspH2 specifically exploits the NLR co-chaperone activity of SGT1 to subvert immunity.

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    Amit P Bhavsar

    Full Text Available To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone.

  12. Regulation of apoptotic signal transduction pathways by the heat shock proteins

    Institute of Scientific and Technical Information of China (English)

    LI; Zhengyu; ZHAO; Xia; WEI; Yuquan

    2004-01-01

    The study about apoptotic signal transductions has become a project to reveal the molecular mechanisms of apoptosis. Heat shock proteins (hsps), which play an important role in cell growth and apoptosis, have attracted great attentions. A lot of researches have showed there is a hsps superfamily including hsp90, hsp70, hsp60 and hsp27, etc., which regulates the biological behaviors of cells, particularly apoptotic signal transduction in Fas pathway, JNK/SAPK pathway and caspases pathway at different levels, partly by the function of molecular chaperone.

  13. Chaperone functions common to nonhomologous Epstein-Barr virus gL and Varicella-Zoster virus gL proteins.

    OpenAIRE

    Li, Q.; Buranathai, C.; Grose, C; Hutt-Fletcher, L M

    1997-01-01

    Herpesviruses encode the complex-forming, essential glycoproteins gH and gL. Maturation and transport of gH are dependent on coexpression of its chaperone, gL. The gL proteins of alpha herpesviruses and gamma herpesviruses do not have a significant percentage of amino acid sequence homology. Yet, as we report herein, the diverse gL glycoproteins of Epstein-Barr virus (EBV) and varicella-zoster virus (VZV) were functionally interchangeable, although membrane expression and maturation of gH wer...

  14. The Escherichia Coli Hfq Protein: An Unattended DNA-Transactions Regulator.

    Science.gov (United States)

    Cech, Grzegorz M; Szalewska-Pałasz, Agnieszka; Kubiak, Krzysztof; Malabirade, Antoine; Grange, Wilfried; Arluison, Veronique; Węgrzyn, Grzegorz

    2016-01-01

    The Hfq protein was discovered in Escherichia coli as a host factor for bacteriophage Qβ RNA replication. Subsequent studies indicated that Hfq is a pleiotropic regulator of bacterial gene expression. The regulatory role of Hfq is ascribed mainly to its function as an RNA-chaperone, facilitating interactions between bacterial non-coding RNA and its mRNA target. Thus, it modulates mRNA translation and stability. Nevertheless, Hfq is able to interact with DNA as well. Its role in the regulation of DNA-related processes has been demonstrated. In this mini-review, it is discussed how Hfq interacts with DNA and what is the role of this protein in regulation of DNA transactions. Particularly, Hfq has been demonstrated to be involved in the control of ColE1 plasmid DNA replication, transposition, and possibly also transcription. Possible mechanisms of these Hfq-mediated regulations are described and discussed. PMID:27517037

  15. Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

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    Takashi Onikubo

    2015-03-01

    Full Text Available Nucleoplasmin (Npm is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here, we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity toward the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction, leading to histone sequestration in the egg.

  16. RIC-3 phosphorylation enables dual regulation of excitation and inhibition of Caenorhabditis elegans muscle.

    Science.gov (United States)

    Safdie, Gracia; Liewald, Jana F; Kagan, Sarah; Battat, Emil; Gottschalk, Alexander; Treinin, Millet

    2016-10-01

    Brain function depends on a delicate balance between excitation and inhibition. Similarly, Caenorhabditis elegans motor system function depends on a precise balance between excitation and inhibition, as C. elegans muscles receive both inhibitory, GABAergic and excitatory, cholinergic inputs from motor neurons. Here we show that phosphorylation of the ER-resident chaperone of nicotinic acetylcholine receptors, RIC-3, leads to increased muscle excitability. RIC-3 phosphorylation at Ser-164 depends on opposing functions of the phosphatase calcineurin (TAX-6), and of the casein kinase II homologue KIN-10. Effects of calcineurin down-regulation and of phosphorylated RIC-3 on muscle excitability are mediated by GABAA receptor inhibition. Thus RIC-3 phosphorylation enables effects of this chaperone on GABAA receptors in addition to nAChRs. This dual effect provides coordinated regulation of excitation and inhibition and enables fine-tuning of the excitation-inhibition balance. Moreover, regulation of inhibitory GABAA signaling by calcineurin, a calcium- and calmodulin-dependent phosphatase, enables homeostatic balancing of excitation and inhibition.

  17. HSP90 regulates temperature-dependent seedling growth in Arabidopsis by stabilizing the auxin co-receptor F-box protein TIR1

    Science.gov (United States)

    Wang, Renhou; Zhang, Yi; Kieffer, Martin; Yu, Hong; Kepinski, Stefan; Estelle, Mark

    2016-01-01

    Recent studies have revealed that a mild increase in environmental temperature stimulates the growth of Arabidopsis seedlings by promoting biosynthesis of the plant hormone auxin. However, little is known about the role of other factors in this process. In this report, we show that increased temperature promotes rapid accumulation of the TIR1 auxin co-receptor, an effect that is dependent on the molecular chaperone HSP90. In addition, we show that HSP90 and the co-chaperone SGT1 each interact with TIR1, confirming that TIR1 is an HSP90 client. Inhibition of HSP90 activity results in degradation of TIR1 and interestingly, defects in a range of auxin-mediated growth processes at lower as well as higher temperatures. Our results indicate that HSP90 and SGT1 integrate temperature and auxin signalling in order to regulate plant growth in a changing environment. PMID:26728313

  18. HSP90 regulates temperature-dependent seedling growth in Arabidopsis by stabilizing the auxin co-receptor F-box protein TIR1.

    Science.gov (United States)

    Wang, Renhou; Zhang, Yi; Kieffer, Martin; Yu, Hong; Kepinski, Stefan; Estelle, Mark

    2016-01-01

    Recent studies have revealed that a mild increase in environmental temperature stimulates the growth of Arabidopsis seedlings by promoting biosynthesis of the plant hormone auxin. However, little is known about the role of other factors in this process. In this report, we show that increased temperature promotes rapid accumulation of the TIR1 auxin co-receptor, an effect that is dependent on the molecular chaperone HSP90. In addition, we show that HSP90 and the co-chaperone SGT1 each interact with TIR1, confirming that TIR1 is an HSP90 client. Inhibition of HSP90 activity results in degradation of TIR1 and interestingly, defects in a range of auxin-mediated growth processes at lower as well as higher temperatures. Our results indicate that HSP90 and SGT1 integrate temperature and auxin signalling in order to regulate plant growth in a changing environment. PMID:26728313

  19. Characterization of molten globule PopB in absence and presence of its chaperone PcrH.

    Science.gov (United States)

    Dey, Supratim; Basu, Abhishek; Datta, Saumen

    2012-06-01

    The TTSS encoding "translocator operon" of Pseudomonas aeruginosa consists of a major translocator protein PopB, minor translocator protein PopD and their cognate chaperone PcrH. Far-UV CD spectra and secondary structure prediction servers predict an α-helical model for PopB, PcrH and PopB-PcrH complex. PopB itself forms a single species of higher order oligomer (15 mer) as seen from AUC, but in complex with PcrH, both monomeric (1:1) and oligomeric form exist. PopB has large solvent-exposed hydrophobic patches and exists as an unordered molten globule in its native state, but on forming complex with PcrH it gets transformed into an ordered molten globule. Tryptophan fluorescence spectrum indicates that PopB interacts with the first TPR region of dimeric PcrH to form a stable PopB-PcrH complex that has a partial rigid structure with a large hydrodynamic radius and few tertiary contacts. The pH-dependent studies of PopB, PcrH and complex by ANS fluorescence, urea induced unfolding and thermal denaturation experiments prove that PcrH not only provides structural support to the ordered molten globule PopB in complex but also undergoes conformational change to assist PopB to pass through the needle complex of TTSS and form pores in the host cell membrane. ITC experiments show a strong affinity (K(d) ~ 0.37 μM) of PopB for PcrH at pH 7.8, which reduces to ~0.68 μM at pH 5.8. PcrH also loses its rigid tertiary structure at pH 5 and attains a molten globule conformation. This indicates that the decrease in pH releases PopB molecules and thus triggers the TTSS activation mechanism for the formation of a functional translocon. PMID:22585368

  20. alpha-Crystallin protein cognates in eggs of the moth, Plodia interpunctella: possible chaperones for the follicular epithelium yolk protein.

    Science.gov (United States)

    Shirk, P D; Broza, R; Hemphill, M; Perera, O P

    1998-03-01

    alpha-Crystallin protein cognates were found in germ cells of the Indianmeal moth, Plodia interpunctella (Shirk and Zimowska, 1997). A cDNA clone of 674 bp with a single open reading frame was isolated for a 25,000 molecular weight polypeptide member of this family, alpha CP25, and a single transcript of approximately 700 bp was found in the ovary of vitellogenic females. Both the DNA sequence and predicted amino acid sequence showed considerable homology with the embryonic lethal gene, l(2)efl, in Drosophila melanogaster. In addition to the sequence for l(2)efl, the predicted amino acid sequence for acp25 also showed significant sequence similarly with the alpha-crystallin A chain polypeptides from the lenses of vertebrae eyes. An N-terminal hydrophobic aggregation site and a C-terminal protective binding site common to alpha-crystallin proteins were present in the predicted acp25 and l(2)efl amino acid sequences, while only the C-terminal protective binding site was present in the small heat shock protein sequences from D. melanogaster. This evidence suggests that although the alpha-crystallin protein cognates in P. interpunctella evolved from a gene common with small heat shock protein genes, the amino acid sequence has converged on a structure similar to that of alpha-crystallin proteins. Native immunoblot analysis showed that the alpha-crystallin proteins formed high molecular weight complexes with the follicular epithelium yolk protein (FEYP) but not vitellin in yolk. An electroblot binding assay was used to show that the germ-cell alpha-crystallins of P. interpunctella bind specifically with the FEYP and that the binding was reversible in the presence of ATP or low pH. This evidence in conjunction with the evidence that the alpha-crystallins and FEYP form a stable complex that co-purifies from native egg proteins suggests that the alpha-cystallin cognates function as chaperones for the follicular epithelium yolk proteins in the embryos of P. interpunctella.

  1. Redox Regulation in Amyotrophic Lateral Sclerosis

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    Sonam Parakh

    2013-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a neurodegenerative disease that results from the death of upper and lower motor neurons. Due to a lack of effective treatment, it is imperative to understand the underlying mechanisms and processes involved in disease progression. Regulations in cellular reduction/oxidation (redox processes are being increasingly implicated in disease. Here we discuss the possible involvement of redox dysregulation in the pathophysiology of ALS, either as a cause of cellular abnormalities or a consequence. We focus on its possible role in oxidative stress, protein misfolding, glutamate excitotoxicity, lipid peroxidation and cholesterol esterification, mitochondrial dysfunction, impaired axonal transport and neurofilament aggregation, autophagic stress, and endoplasmic reticulum (ER stress. We also speculate that an ER chaperone protein disulphide isomerase (PDI could play a key role in this dysregulation. PDI is essential for normal protein folding by oxidation and reduction of disulphide bonds, and hence any disruption to this process may have consequences for motor neurons. Addressing the mechanism underlying redox regulation and dysregulation may therefore help to unravel the molecular mechanism involved in ALS.

  2. Leptin and Fasting Regulate Rat Gastric Glucose-Regulated Protein 58

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    Susana B. Bravo

    2011-01-01

    Full Text Available The stomach secretes a wide range of peptides with essential metabolic functions, and thereby plays an important role in the regulation of energy homeostasis. Disulfide isomerase glucose-regulated protein 58 (GRp58 is a molecular chaperone member of the endoplasmic reticulum (ER stress signaling pathway, which is a marker for human gastric cancer. Since GRp58 seems to be regulated by a phosphorylation/dephosphorylation pattern shift, we used the 2DE gel methodology and peptide mass fingerprinting-protein identification by means of MALDI-TOF mass spectrometry. We show that gastric mucosa GRp58 is dephosphorylated by fasting, and this effect is blunted when fasted rats are treated with leptin. Furthermore, we assessed the gene expression of GRp58 under different physiological settings known to be associated with energy homeostasis (fasting, leptin treatment and leptin deficiency. We found that intraperitoneal administration of leptin increases whereas leptin deficiency decreases GRp58 mRNA levels. However, GRp58 expression remains unchanged after fasting, indicating that leptin actions on GRp58 are no direct sensitivity to fasting. Dissection of the molecular pathways mediating the interactions between ER stress-related factors and nutrient availability, as well as their target genes, may open a new avenue for the study of obesity and other metabolic disorders.

  3. A structural comparison of Listeria monocytogenes protein chaperones PrsA1 and PrsA2 reveals molecular features required for virulence.

    Science.gov (United States)

    Cahoon, Laty A; Freitag, Nancy E; Prehna, Gerd

    2016-07-01

    Listeria monocytogenes is a Gram-positive environmental bacterium that lives within soil but transitions into a pathogen upon contact with a mammalian host. The transition of L. monocytogenes from soil dweller to cytosolic pathogen is dependent upon secreted virulence factors that mediate cell invasion and intracellular growth. PrsA1 and PrsA2 are secreted bacterial lipoprotein chaperones that contribute to the folding of proteins translocated across the bacterial membrane; PrsA2 is required for L. monocytogenes virulence, whereas the function of PrsA1 remains to be determined. We have solved an X-ray crystal structure of PrsA1 and have used this model to guide comparison structure-based mutagenesis studies with PrsA2. Targeted mutagenesis of PrsA2 demonstrates that oligomerization of PrsA2 as well as molecular features of the foldase domain are required for protein secretion and virulence, whereas a functional role was uncovered for PrsA1 in bacterial resistance to alcohol. Interestingly, PrsA2 membrane localization is not required for all PrsA2-dependent activities, suggesting that the lipoprotein retains function when released from the bacterial cell. PrsA chaperones are thus multifaceted proteins with distinct domains adapted to accommodate the functional needs of a diverse array of secreted substrates. PMID:27007641

  4. The human TPR protein TTC4 is a putative Hsp90 co-chaperone which interacts with CDC6 and shows alterations in transformed cells.

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    Gilles Crevel

    Full Text Available BACKGROUND: The human TTC4 protein is a TPR (tetratricopeptide repeat motif-containing protein. The gene was originally identified as being localized in a genomic region linked to breast cancer and subsequent studies on melanoma cell lines revealed point mutations in the TTC4 protein that may be associated with the progression of malignant melanoma. METHODOLOGY/PRINCIPLE FINDINGS: Here we show that TTC4 is a nucleoplasmic protein which interacts with HSP90 and HSP70, and also with the replication protein CDC6. It has significant structural and functional similarities with a previously characterised Drosophila protein Dpit47. We show that TTC4 protein levels are raised in malignant melanoma cell lines compared to melanocytes. We also see increased TTC4 expression in a variety of tumour lines derived from other tissues. In addition we show that TTC4 proteins bearing some of the mutations previously identified from patient samples lose their interaction with the CDC6 protein. CONCLUSIONS/SIGNIFICANCE: Based on these results and our previous work with the Drosophila Dpit47 protein we suggest that TTC4 is an HSP90 co-chaperone protein which forms a link between HSP90 chaperone activity and DNA replication. We further suggest that the loss of the interaction with CDC6 or with additional client proteins could provide one route through which TTC4 could influence malignant development of cells.

  5. The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans.

    Science.gov (United States)

    Ni, Weiming; Hutagalung, Alex H; Li, Shumin; Epstein, Henry F

    2011-09-15

    The UNC-45 family of molecular chaperones is expressed in metazoan organisms from Caenorhabditis elegans to humans. The UNC-45 protein is essential in C. elegans for early body-wall muscle cell development and A-band assembly. We show that the myosin-binding UCS domain of UNC-45 alone is sufficient to rescue lethal unc-45 null mutants arrested in embryonic muscle development and temperature-sensitive loss-of-function unc-45 mutants defective in worm A-band assembly. Removal of the Hsp90-binding TPR domain of UNC-45 does not affect rescue. Similar results were obtained with overexpression of the same fragments in wild-type nematodes when assayed for diminution of myosin accumulation and assembly. Titration experiments show that, on a per molecule basis, UCS has greater activity in C. elegans muscle in vivo than full-length UNC-45 protein, suggesting that UNC-45 is inhibited by either the TPR domain or its interaction with the general chaperone Hsp90. In vitro experiments with purified recombinant C. elegans Hsp90 and UNC-45 proteins show that they compete for binding to C. elegans myosin. Our in vivo genetic and in vitro biochemical experiments are consistent with a novel inhibitory role for Hsp90 with respect to UNC-45 action.

  6. Toxoplasma gondii: a bradyzoite-specific DnaK-tetratricopeptide repeat (DnaK-TPR) protein interacts with p23 co-chaperone protein.

    Science.gov (United States)

    Ueno, Akio; Dautu, George; Haga, Kaori; Munyaka, Biscah; Carmen, Gabriella; Kobayashi, Yoshiyasu; Igarashi, Makoto

    2011-04-01

    The DnaK-tetratricopeptide repeat (DnaK-TPR) gene (ToxoDB ID, TGME49_002020) is expressed predominantly at the bradyzoite stage. DnaK-TPR protein has a heat shock protein (DnaK) and tetratricopeptide repeat (TPR) domains with amino acid sequence similarity to the counterparts of other organisms (40.2-43.7% to DnaK domain and 41.1-66.0% to TPR domain). These findings allowed us to infer that DnaK-TPR protein is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. An immunofluorescence assay (IFA) revealed that DnaK-TPR protein was expressed in Toxoplasma gondii-encysted and in vitro-induced bradyzoites and distributed in the whole part of parasite cells. We conducted yeast two-hybrid screening to identify proteins interacting with DnaK-TPR protein, and demonstrated that DnaK-TPR protein interacts with p23 co-chaperone protein (Tgp23). It was expected that DnaK-TPR protein would have a function as a molecular chaperon in bradyzoite cells associated with Tgp23. Possible mechanisms for this gene are discussed.

  7. Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer

    Science.gov (United States)

    Godet, Julien; Kenfack, Cyril; Przybilla, Frédéric; Richert, Ludovic; Duportail, Guy; Mély, Yves

    2013-01-01

    The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (−)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11–55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger–dependent binding of NC to the G10 and G50 residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G10 and G50 the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway. PMID:23511968

  8. Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

    Science.gov (United States)

    Sung, Nuri; Lee, Jungsoon; Kim, Ji-Hyun; Chang, Changsoo; Joachimiak, Andrzej; Lee, Sukyeong; Tsai, Francis T. F.

    2016-01-01

    Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding. PMID:26929380

  9. Rearrangements of α-helical structures of FlgN chaperone control the binding affinity for its cognate substrates during flagellar type III export.

    Science.gov (United States)

    Kinoshita, Miki; Nakanishi, Yuki; Furukawa, Yukio; Namba, Keiichi; Imada, Katsumi; Minamino, Tohru

    2016-08-01

    The bacterial flagellar type III export chaperones not only act as bodyguards to protect their cognate substrates from aggregation and proteolysis in the cytoplasm but also ensure the order of export through their interactions with an export gate protein FlhA. FlgN chaperone binds to FlgK and FlgL with nanomolar affinity and transfers them to FlhA for their efficient and rapid transport for the formation of the hook-filament junction zone. However, it remains unknown how FlgN releases FlgK and FlgL at the FlhA export gate platform in a timely manner. Here, we have solved the crystal structure of Salmonella FlgN at 2.3 Å resolution and carried out structure-based functional analyses. FlgN consists of three α helices, α1, α2 and α3. Helix α1 adopts two distinct, extended and bent conformations through the conformational change of N-loop between α1 and α2. The N-loop deletion not only increases the probability of FlgN dimer formation but also abolish the interaction between FlgN and FlgK. Highly conserved Asn-92, Asn-95 and Ile-103 residues in helix α3 are involved in the strong interaction with FlgK. We propose that the N-loop coordinates helical rearrangements of FlgN with the association and dissociation of its cognate substrates during their export. PMID:27178222

  10. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Kamalika Roy [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Centre for Neuroscience, Indian Institute of Science, Bangalore 560012 (India); Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com [Biomedical Genomics Centre, PG Polyclinic Building, 5, Suburbun Hospital Road, Kolkata 700020 (India)

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  11. Telomerase Regulation

    OpenAIRE

    Cifuentes-Rojas, Catherine; Dorothy E Shippen

    2011-01-01

    The intimate connection between telomerase regulation and human disease is now well established. The molecular basis for telomerase regulation is highly complex and entails multiple layers of control. While the major target of enzyme regulation is the catalytic subunit TERT, the RNA subunit of telomerase is also implicated in telomerase control. In addition, alterations in gene dosage and alternative isoforms of core telomerase components have been described. Finally, telomerase localization,...

  12. Regulating Transplants

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Legislation to determine brain death is viewed as essential in controlling the organ transplant industry Organ transplant represents a very sensitive and complicated issue. Experts say the temporary administrative regulations recently promulgated by the Central Government are an important step, but relevant laws and regulations must follow. Among these, the

  13. Heat Shock Protein 90 Indirectly Regulates ERK Activity by Affecting Raf Protein Metabolism

    Institute of Scientific and Technical Information of China (English)

    Fei DOU; Liu-Di YUAN; Jing-Jing ZHU

    2005-01-01

    Extracellular signal-regulated protein kinase (ERK) has been implicated in the pathogenesis of several nerve system diseases. As more and more kinases have been discovered to be the client proteins of the molecular chaperone Hsp90, the use of Hsp90 inhibitors to reduce abnormal kinase activity is a new treatment strategy for nerve system diseases. This study investigated the regulation of the ERK pathway by Hsp90. We showed that Hsp90 inhibitors reduce ERK phosphorylation without affecting the total ERK protein level. Further investigation showed that Raf, the upstream kinase in the Ras-Raf-MEK-ERK pathway,forms a complex with Hsp90 and Hsp70. Treating cells with Hsp90 inhibitors facilitates Raf degradation,thereby down-regulating the activity of ERK.

  14. A Hsp40 chaperone protein interacts with and modulates the cellular distribution of the primase protein of human cytomegalovirus.

    Directory of Open Access Journals (Sweden)

    Yonggang Pei

    Full Text Available Genomic DNA replication is a universal and essential process for all herpesvirus including human cytomegalovirus (HCMV. HCMV UL70 protein, which is believed to encode the primase activity of the viral DNA replication machinery and is highly conserved among herpesviruses, needs to be localized in the nucleus, the site of viral DNA synthesis. No host factors that facilitate the nuclear import of UL70 have been reported. In this study, we provided the first direct evidence that UL70 specifically interacts with a highly conserved and ubiquitously expressed member of the heat shock protein Hsp40/DNAJ family, DNAJB6, which is expressed as two isoforms, a and b, as a result of alternative splicing. The interaction of UL70 with a common region of DNAJB6a and b was identified by both a two hybrid screen in yeast and coimmunoprecipitation in human cells. In transfected cells, UL70 was primarily co-localized with DNAJB6a in the nuclei and with DNAJB6b in the cytoplasm, respectively. The nuclear import of UL70 was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Furthermore, the level of viral DNA synthesis and progeny production was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Thus, DNAJB6a and b appear to enhance the nuclear import and cytoplasmic accumulation of UL70, respectively. Our results also suggest that the relative expression levels of DNAJB6 isoforms may play a key role in regulating the cellular localization of UL70, leading to modulation of HCMV DNA synthesis and lytic infection.

  15. The Role of Monocarboxylate Transporters and Their Chaperone CD147 in Lactate Efflux Inhibition and the Anticancer Effects of Terminalia chebula in Neuroblastoma Cell Line N2-A

    Science.gov (United States)

    Messeha, S. S.; Zarmouh, N. O.; Taka, E.; Gendy, S. G.; Shokry, G. R.; Kolta, M. G.; Soliman, K. F. A.

    2016-01-01

    Aims In the presence of oxygen, most of the synthesized pyruvate during glycolysis in the cancer cell of solid tumors is released away from the mitochondria to form lactate (Warburg Effect). To maintain cell homeostasis, lactate is transported across the cell membrane by monocarboxylate transporters (MCTs). The major aim of the current investigation is to identify novel compounds that inhibit lactate efflux that may lead to identifying effective targets for cancer treatment. Study Design In this study, 900 ethanol plant extracts were screened for their lactate efflux inhibition using neuroblastoma (N2-A) cell line. Additionally, we investigated the mechanism of inhibition for the most potent plant extract regarding monocarboxylate transporters expression, and consequences effects on viability, growth, and apoptosis. Methodology The potency of lactate efflux inhibition of ethanol plant extracts was evaluated in N2-A cells by measuring extracellular lactate levels. Caspase 3- activity and acridine orange/ethidium bromide staining were performed to assess the apoptotic effect. The antiproliferative effect was measured using WST assay. Western blotting was performed to quantify protein expression of MCTs and their chaperone CD147 in treated cells lysates. Results Terminalia chebula plant extract was the most potent lactate efflux inhibitor in N2-A cells among the 900 - tested plant extracts. The results obtained show that extract of Terminalia chebula fruits (TCE) significantly (P = 0.05) reduced the expression of the MCT1, MCT3, MCT4 and the chaperone CD147. The plant extract was more potent (IC50 of 3.59 ± 0.26 μg/ml) than the MCT standard inhibitor phloretin (IC50 76.54 ± 3.19 μg/ml). The extract also showed more potency and selective cytotoxicity in cancer cells than DI-TNC1 primary cell line (IC50 7.37 ± 0.28 vs. 17.35 ± 0.19 μg/ml). Moreover, TCE Inhibited N2-A cell growth (IG50 = 5.20 ± 0.30 μg/ml) and induced apoptosis at the 7.5 μg/ml concentration

  16. Ric-3 chaperone-mediated stable cell-surface expression of the neuronal a7 nicotinic acetylcholine receptor in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Ana Sofia VALLfiS; Ana M ROCCAMO; Francisco J BARRANTES

    2009-01-01

    Aim: Studies of the a7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of a7-type AChRs'11. The current work aims at obtaining and characterizing a cell line with high functional expression of the human a7 AChR.Methods: Ric-3 cDNA was incorporated into SHE-Pl-ha7 cells expressing the a7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [125I]a-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent a-bungarotoxin, anti-a7 antibody, and GFP-a7 were performed on the new clone.Results: The human a7-type AChR was stably expressed in a new cell line, which we coined SHE-PI-ha7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [125I]aBTX saturable binding with an apparent KD of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-PI-ha7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-a7 antibodies, hi contrast, chaperone-less SHE-PI-ha7 cells expressed only intracellular a.7 AChRs and failed to produce detectable single-channel currents.Conclusion: The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal a7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.

  17. Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

    Directory of Open Access Journals (Sweden)

    Julia M Harris

    2014-07-01

    Full Text Available Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion

  18. Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

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    Katarzyna Marta Bocian-Ostrzycka

    2015-10-01

    Full Text Available Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595 with a -propeller structure. The aim of presented work was to assess relations between HP0231 structure and function.We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C, a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in E. coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (Escherichia coli DsbA protein were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

  19. NOISE REGULATION

    OpenAIRE

    Cristina Voican; Constantin Stanescu

    2012-01-01

    Noise regulation includes statutes or guidelines relating to sound transmission established by national, state or provincial and municipal levels of government. After the watershed passage of the United States Noise Control Act of 1972, other local and state governments passed further regulations. Although the UK and Japan enacted national laws in 1960 and 1967 respectively, these laws were not at all comprehensive or fully enforceable as to address generally rising ambient noise, enforceable...

  20. Forma selvagem e formas mutantes da fenilalanina hidroxilase humana (hPAH) recombinante:avaliação de interacções com diferentes chaperones moleculares

    OpenAIRE

    Cristo, Inês Rodrigues Silva

    2008-01-01

    Os chaperones moleculares desempenham um papel crucial nos vários processos celulares relacionados com a aquisição e manutenção da conformação proteica, sendo por isso peças importantes na patogénese das doenças conformacionais, nomeadamente da fenilcetonúria (PKU), uma doença genética causada por uma deficiente actividade da fenilalanina hidroxilase (hPAH). Embora seja presentemente reconhecido que a maior parte das proteínas hPAH mutantes apresentam alterações na sua conformação, são ainda ...

  1. The aggregation and neurotoxicity of TDP-43 and its ALS-associated 25 kDa fragment are differentially affected by molecular chaperones in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jenna M Gregory

    Full Text Available Almost all cases of sporadic amyotrophic lateral sclerosis (ALS, and some cases of the familial form, are characterised by the deposition of TDP-43, a member of a family of heteronuclear ribonucleoproteins (hnRNP. Although protein misfolding and deposition is thought to be a causative feature of many of the most prevalent neurodegenerative diseases, a link between TDP-43 aggregation and the dysfunction of motor neurons has yet to be established, despite many correlative neuropathological studies. We have investigated this relationship in the present study by probing the effect of altering TDP-43 aggregation behaviour in vivo by modulating the levels of molecular chaperones in a Drosophila model. More specifically, we quantify the effect of either pharmacological upregulation of the heat shock response or specific genetic upregulation of a small heat shock protein, CG14207, on the neurotoxicity of both TDP-43 and of its disease associated 25 kDa fragment (TDP-25 in a Drosophila model. Inhibition of the aggregation of TDP-43 by either method results in a partial reduction of its neurotoxic effects on both photoreceptor and motor neurons, whereas inhibition of the aggregation of TDP-25 results not only in a complete suppression of its toxicity but also its clearance from the brain in both neuronal subtypes studied. The results demonstrate, therefore, that aggregation plays a crucial role in mediating the neurotoxic effects of both full length and truncated TDP-43, and furthermore reveal that the in vivo propensity of these two proteins to aggregate and their susceptibility to molecular chaperone mediated clearance are quite distinct.

  2. Binding of 3,4,5,6-Tetrahydroxyazepanes to the Acid-[beta]-glucosidase Active Site: Implications for Pharmacological Chaperone Design for Gaucher Disease

    Energy Technology Data Exchange (ETDEWEB)

    Orwig, Susan D.; Tan, Yun Lei; Grimster, Neil P.; Yu, Zhanqian; Powers, Evan T.; Kelly, Jeffery W.; Lieberman, Raquel L. (Scripps); (GIT)

    2013-03-07

    Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-{beta}-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, such as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.

  3. Overproduction of the Escherichia coli Chaperones GroEL-GroES in Rhodococcus ruber Improves the Activity and Stability of Cell Catalysts Harboring a Nitrile Hydratase.

    Science.gov (United States)

    Tian, Yuxuan; Chen, Jie; Yu, Huimin; Shen, Zhongyao

    2016-02-01

    Three combinations of molecular chaperones from Escherichia coli (i.e., DnaK-DnaJ-GrpEGroEL- GroES, GroEL-GroES, and DnaK-DnaJ-GrpE) were overproduced in E. coli BL21, and their in vitro stabilizing effects on a nitrile hydratase (NHase) were assessed. The optimal gene combination, E. coli groEL-groES (ecgroEL-ES), was introduced into Rhodococcus ruber TH3. A novel engineered strain, R. ruber TH3G was constructed with the native NHase gene on its chromosome and the heterologous ecgroEL-ES genes in a shuttle plasmid. In R. ruber TH3G, NHase activity was enhanced 37.3% compared with the control, TH3. The in vivo stabilizing effect of ecGroEL-ES on the NHase was assessed using both acrylamide immersion and heat shock experiments. The inactivation behavior of the in vivo NHase after immersion in a solution of dynamically increased concentrations of acrylamide was particularly evident. When the acrylamide concentration was increased to 500 g/l (50%), the remaining NHase activity in TH3G was 38%, but in TH3, activity was reduced to 10%. Reactivation of the in vivo NHases after varying degrees of inactivation was further assessed. The activity of the reactivated NHase was more than 2-fold greater in TH3G than in TH3. The hydration synthesis of acrylamide catalyzed by the in vivo NHase was performed with continuous acrylonitrile feeding. The final concentration of acrylamide was 640 g/l when catalyzed by TH3G, compared with 490 g/l acrylamide by TH3. This study is the first to show that the chaperones ecGroEL-ES work well in Rhodococcus and simultaneously possess protein-folding assistance functions and the ability to stabilize and reactivate the native NHases. PMID:26562693

  4. Cloning, molecular characterization, and expression analysis of the unc45 myosin chaperone b(unc45b)gene of grass carp (Ctenopharyngodon idellus).

    Science.gov (United States)

    Hu, Jing; Guo, Ting; Pan, Wen-Qian; Gan, Tian; Wei, Jing; Wang, Jun-Peng; Leng, Xiang-Jun; Li, Xiao-Qin

    2016-06-01

    Unc45 myosin chaperone b(unc45b)gene is a molecular chaperone that mediates the folding, assembly and accumulation of thick-filament myosin in the formation of sarcomere, which plays an important role in the development of striated muscle and the stability of sarcomere. In this study, the complete cDNA sequence of unc45b gene of grass carp was obtained by rapid amplification of cDNA ends (RACE), and the characteristics of the unc45b protein predicted from gene sequence was analyzed by bioinformatics methods. The differential expression pattern in tissues was also detected by quantitative real-time PCR. The results showed that the full-length of unc45b gene of grass carp is 3163 bp, which contains a 60 bp 5'UTR, a 298 bp 3'UTR, and a 2865 bp open reading frame (ORF) encoding a 934 amino acid peptide. The deduced unc45b protein exhibits a homology of 92, 86, 86 % with the protein of zebrafish (Danio rerio), channel catfish (Ietalurus punctatus) and tilapia (Oreochromis niloticus) respectively, and the protein contains UCS myosin head binding domain and TPR peptide repeat domain. The protein is a hydrophilic and non-secretory protein with a molecular mass and isoeletronic point of 103,699.8 and 7.39 Da. The structural elements of the protein includes α-helixes and loops, and the unc45b gene highly expresses in skeletal muscle and heart in grass carp. This study laid a foundation for further research in explaining the myofibril accumulation in crisped grass carp. PMID:27334505

  5. A platform to view huntingtin exon 1 aggregation flux in the cell reveals divergent influences from chaperones hsp40 and hsp70.

    Science.gov (United States)

    Ormsby, Angelique R; Ramdzan, Yasmin M; Mok, Yee-Foong; Jovanoski, Kristijan D; Hatters, Danny M

    2013-12-27

    Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell. PMID:24196953

  6. A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70*

    Science.gov (United States)

    Ormsby, Angelique R.; Ramdzan, Yasmin M.; Mok, Yee-Foong; Jovanoski, Kristijan D.; Hatters, Danny M.

    2013-01-01

    Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell. PMID:24196953

  7. Safety and pharmacodynamic effects of a pharmacological chaperone on α-galactosidase A activity and globotriaosylceramide clearance in Fabry disease: report from two phase 2 clinical studies

    Directory of Open Access Journals (Sweden)

    Germain Dominique P

    2012-11-01

    Full Text Available Abstract Background Fabry disease (FD is a genetic disorder resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A, which leads to globotriaosylceramide (GL-3 accumulation in multiple tissues. We report on the safety and pharmacodynamics of migalastat hydrochloride, an investigational pharmacological chaperone given orally at 150 mg every-other-day. Methods Two open-label uncontrolled phase 2 studies of 12 and 24 weeks (NCT00283959 and NCT00283933 in 9 males with FD were combined. At multiple time points, α-Gal A activity and GL-3 levels were quantified in blood cells, kidney and skin. GL-3 levels were also evaluated through skin and renal histology. Results Compared to baseline, increased α-Gal A activity of at least 50% was demonstrated in blood, skin and kidney in 6 of 9 patients. Patients’ increased α-Gal A activities paralleled the α-Gal A increases observed in vitro in HEK-293 cells transfected with the corresponding mutant form of the enzyme. The same 6 patients who demonstrated increases of α-Gal A activity also had GL-3 reduction in skin, urine and/or kidney, and had α-Gal A mutations that responded in transfected cells incubated with the drug. The 3 patients who did not show a consistent response in vivo had α-Gal A mutations that did not respond to migalastat HCl in transfected cells. Migalastat HCl was well tolerated. Conclusions Migalastat HCl is a candidate pharmacological chaperone that provides a novel genotype-specific treatment for FD. It enhanced α-Gal A activity and resulted in GL-3 substrate decrease in patients with responsive GLA mutations. Phase 3 studies are ongoing. Trial registration Clinicaltrial.gov: NCT00283959 and NCT00283933

  8. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea

    Directory of Open Access Journals (Sweden)

    Petitjean Céline

    2012-11-01

    Full Text Available Abstract Background In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants. Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Results Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. Conclusions We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

  9. Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ.

    Science.gov (United States)

    Sonamoto, Rie; Kii, Isao; Koike, Yuka; Sumida, Yuto; Kato-Sumida, Tomoe; Okuno, Yukiko; Hosoya, Takamitsu; Hagiwara, Masatoshi

    2015-01-01

    The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer's disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases. PMID:26234946

  10. 5-Azacytidine-induced protein 2 (AZI2) regulates bone mass by fine-tuning osteoclast survival.

    Science.gov (United States)

    Maruyama, Kenta; Fukasaka, Masahiro; Uematsu, Satoshi; Takeuchi, Osamu; Kondo, Takeshi; Saitoh, Tatsuya; Martino, Mikaël M; Akira, Shizuo

    2015-04-10

    5-Azacytidine-induced protein 2 (AZI2) is a TNF receptor (TNFR)-associated factor family member-associated NF-κB activator-binding kinase 1-binding protein that regulates the production of IFNs. A previous in vitro study showed that AZI2 is involved in dendritic cell differentiation. However, the roles of AZI2 in immunity and its pleiotropic functions are unknown in vivo. Here we report that AZI2 knock-out mice exhibit normal dendritic cell differentiation in vivo. However, we found that adult AZI2 knock-out mice have severe osteoporosis due to increased osteoclast longevity. We revealed that the higher longevity of AZI2-deficient osteoclasts is due to an augmented activation of proto-oncogene tyrosine-protein kinase Src (c-Src), which is a critical player in osteoclast survival. We found that AZI2 inhibits c-Src activity by regulating the activation of heat shock protein 90 (Hsp90), a chaperone involved in c-Src dephosphorylation. Furthermore, we demonstrated that AZI2 indirectly inhibits c-Src by interacting with the Hsp90 co-chaperone Cdc37. Strikingly, administration of a c-Src inhibitor markedly prevented bone loss in AZI2 knock-out mice. Together, these findings indicate that AZI2 regulates bone mass by fine-tuning osteoclast survival. PMID:25691576

  11. 5-Azacytidine-induced Protein 2 (AZI2) Regulates Bone Mass by Fine-tuning Osteoclast Survival*

    Science.gov (United States)

    Maruyama, Kenta; Fukasaka, Masahiro; Uematsu, Satoshi; Takeuchi, Osamu; Kondo, Takeshi; Saitoh, Tatsuya; Martino, Mikaël M.; Akira, Shizuo

    2015-01-01

    5-Azacytidine-induced protein 2 (AZI2) is a TNF receptor (TNFR)-associated factor family member-associated NF-κB activator-binding kinase 1-binding protein that regulates the production of IFNs. A previous in vitro study showed that AZI2 is involved in dendritic cell differentiation. However, the roles of AZI2 in immunity and its pleiotropic functions are unknown in vivo. Here we report that AZI2 knock-out mice exhibit normal dendritic cell differentiation in vivo. However, we found that adult AZI2 knock-out mice have severe osteoporosis due to increased osteoclast longevity. We revealed that the higher longevity of AZI2-deficient osteoclasts is due to an augmented activation of proto-oncogene tyrosine-protein kinase Src (c-Src), which is a critical player in osteoclast survival. We found that AZI2 inhibits c-Src activity by regulating the activation of heat shock protein 90 (Hsp90), a chaperone involved in c-Src dephosphorylation. Furthermore, we demonstrated that AZI2 indirectly inhibits c-Src by interacting with the Hsp90 co-chaperone Cdc37. Strikingly, administration of a c-Src inhibitor markedly prevented bone loss in AZI2 knock-out mice. Together, these findings indicate that AZI2 regulates bone mass by fine-tuning osteoclast survival. PMID:25691576

  12. NORM regulations

    Energy Technology Data Exchange (ETDEWEB)

    Gray, P. [ed.

    1997-02-01

    The author reviews the question of regulation for naturally occuring radioactive material (NORM), and the factors that have made this a more prominent concern today. Past practices have been very relaxed, and have often involved very poor records, the involvment of contractors, and the disposition of contaminated equipment back into commercial service. The rationale behind the establishment of regulations is to provide worker protection, to exempt low risk materials, to aid in scrap recycling, to provide direction for remediation and to examine disposal options. The author reviews existing regulations at federal and state levels, impending legislation, and touches on the issue of site remediation and potential liabilities affecting the release of sites contaminated by NORM.

  13. Transcription regulation of HYPK by Heat Shock Factor 1.

    Directory of Open Access Journals (Sweden)

    Srijit Das

    Full Text Available HYPK (Huntingtin Yeast Partner K was originally identified by yeast two-hybrid assay as an interactor of Huntingtin, the protein mutated in Huntington's disease. HYPK was characterized earlier as an intrinsically unstructured protein having chaperone-like activity in vitro and in vivo. HYPK has the ability of reducing rate of aggregate formation and subsequent toxicity caused by mutant Huntingtin. Further investigation revealed that HYPK is involved in diverse cellular processes and required for normal functioning of cells. In this study we observed that hyperthermia increases HYPK expression in human and mouse cells in culture. Expression of exogenous Heat Shock Factor 1 (HSF1, upon heat treatment could induce HYPK expression, whereas HSF1 knockdown reduced endogenous as well as heat-induced HYPK expression. Putative HSF1-binding site present in the promoter of human HYPK gene was identified and validated by reporter assay. Chromatin immunoprecipitation revealed in vivo interaction of HSF1 and RNA polymerase II with HYPK promoter sequence. Additionally, acetylation of histone H4, a known epigenetic marker of inducible HSF1 binding, was observed in response to heat shock in HYPK gene promoter. Overexpression of HYPK inhibited cells from lethal heat-induced death whereas knockdown of HYPK made the cells susceptible to lethal heat shock-induced death. Apart from elevated temperature, HYPK was also upregulated by hypoxia and proteasome inhibition, two other forms of cellular stress. We concluded that chaperone-like protein HYPK is induced by cellular stress and under transcriptional regulation of HSF1.

  14. S-nitrosylation of the Mitochondrial Chaperone TRAP1 Sensitizes Hepatocellular Carcinoma Cells to Inhibitors of Succinate Dehydrogenase

    DEFF Research Database (Denmark)

    Rizza, Salvatore; Montagna, Costanza; Cardaci, Simone;

    2016-01-01

    S-nitrosoglutathione reductase (GSNOR) represents the best-documented denitrosylase implicated in regulating the levels of proteins posttranslationally modified by nitric oxide on cysteine residues by S-nitrosylation. GSNOR controls a diverse array of physiologic functions, including cellular gro...

  15. Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA

    OpenAIRE

    Wang, Ping; Eshaq, Randa S.; Meshul, Charles K.; Moore, Cynthia; Hood, Rebecca L; Leidenheimer, Nancy J.

    2015-01-01

    GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endo...

  16. Plasmodium falciparum Hop (PfHop Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity.

    Directory of Open Access Journals (Sweden)

    Tawanda Zininga

    Full Text Available Heat shock proteins (Hsps play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70 is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90 facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop. We previously characterised the Hop protein from Plasmodium falciparum (PfHop. We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR analyses. The interaction of the two proteins was further validated by co-immunoprecipitation studies. We observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP or ADP. However, ADP appears to promote the association of the two proteins better than ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted with the C-terminus of PfHsp90. Finally, we

  17. VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium.

    Science.gov (United States)

    Deng, W; Chen, L; Peng, W T; Liang, X; Sekiguchi, S; Gordon, M P; Comai, L; Nester, E W

    1999-03-01

    export-competent state. Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems. We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system. PMID:10209751

  18. SseA is a chaperone for the SseB and SseD translocon components of the Salmonella pathogenicity-island-2-encoded type III secretion system.

    Science.gov (United States)

    Ruiz-Albert, Javier; Mundy, Rosanna; Yu, Xiu-Jun; Beuzón, Carmen R; Holden, David W

    2003-05-01

    The type III secretion system (TTSS) encoded by the Salmonella pathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages and for systemic infection in mice. Many TTSS secreted proteins, including effectors and components of the translocon, require chaperones which promote their stability, prevent their premature interactions or facilitate their secretion. In this study, the function of the first gene (sseA) of one of the SPI-2 operons (sseA-G) was investigated. This operon includes genes that encode translocon components (SseB, SseC and SseD), translocated proteins (SseF and SseG) and putative chaperones (SscA and SscB). sseA encodes a 12.5 kDa protein with a C-terminal region with the potential to form a coiled-coil structure, but no sequence similarity to other proteins. Mutation of sseA results in severe virulence attenuation and an intracellular replication defect. It is shown here that SseA is not a secreted protein, but is required for SPI-2-dependent translocation of two effector proteins (SifA and PipB). Furthermore, the translocon components SseB and SseD were not detected in an sseA mutant strain. By using a yeast two-hybrid assay and column binding experiments, it is demonstrated that SseA interacts directly with SseB and SseD. These results indicate that SseA is a chaperone for SseB and SseD. The inability of an sseA mutant to assemble the SPI-2 TTSS translocon accounts for its high level of virulence attenuation in vivo. To the authors' knowledge, this is the first chaperone described for the SPI-2 TTSS.

  19. The aqueous extract of Glycyrrhiza inflata can upregulate unfolded protein response-mediated chaperones to reduce tau misfolding in cell models of Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Chang KH

    2016-02-01

    Full Text Available Kuo-Hsuan Chang,1,* I-Cheng Chen,1,* Hsuan-Yuan Lin,2 Hsuan-Chiang Chen,2 Chih-Hsin Lin,1 Te-Hsien Lin,2 Yu-Ting Weng,1 Chih-Ying Chao,1 Yih-Ru Wu,1 Jung-Yaw Lin,2 Guey-Jen Lee-Chen,2 Chiung-Mei Chen1 1Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 2Department of Life Science, National Taiwan Normal University, Taipei, Taiwan, Republic of China *These authors contributed equally to this work Background: Alzheimer’s disease (AD and several neurodegenerative disorders known as tauopathies are characterized by misfolding and aggregation of tau protein. Although several studies have suggested the potential of traditional Chinese medicine (TCM as treatment for neurodegenerative diseases, the role of TCM in treating AD and tauopathies have not been well explored.Materials and methods: Tau protein was coupled to the DsRed fluorophore by fusing a pro-aggregation mutant of repeat domain of tau (ΔK280 tauRD with DsRed. The ΔK280 tauRD-DsRed fusion gene was then used to generate Tet-On 293 and SH-SY5Y cell clones as platforms to test the efficacy of 39 aqueous extracts of TCM in reducing tau misfolding and in neuroprotection.Results: Seven TCM extracts demonstrated a significant reduction in tau misfolding and reactive oxidative species with low cytotoxicity in the ΔK280 tauRD-DsRed 293 cell model. Glycyrrhiza inflata and Panax ginseng also demonstrated the potential to improve neurite outgrowth in the ΔK280 tauRD-DsRed SH-SY5Y neuronal cell model. G. inflata further rescued the upregulation of ERN2 (pro-apoptotic and downregulation of unfolded-protein-response-mediated chaperones ERP44, DNAJC3, and SERP1 in ΔK280 tauRD-DsRed 293 cells.Conclusion: This in vitro study provides evidence that G. inflata may be a novel therapeutic for AD and tauopathies. Future applications of G. inflata on animal models of AD and tauopathies are warranted to corroborate its effect of reducing misfolding and potential

  20. Translational regulation of gene expression by an anaerobically induced small non-coding RNA in Escherichia coli

    DEFF Research Database (Denmark)

    Boysen, Anders; Møller-Jensen, Jakob; Kallipolitis, Birgitte H.;

    2010-01-01

    Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post-transcriptional regu......Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post......S, possesses signatures of base-pairing RNAs, and we show by employing global proteomic and transcriptomic profiling that the expression of multiple genes is negatively regulated by the sRNA. Intriguingly, many of these genes encode enzymes with "aerobic" functions or enzymes linked to oxidative stress....... Furthermore, in previous work most of the potential target genes have been shown to be repressed by FNR through an undetermined mechanism. Collectively, our results provide insight into the mechanism by which FNR negatively regulates genes such as sodA, sodB, cydDC, and metE, thereby demonstrating...

  1. Cytokines, Chaperones and Neuroinflammatory Responses in Heroin-Related Death: What Can We Learn from Different Patterns of Cellular Expression?

    Directory of Open Access Journals (Sweden)

    Vittorio Fineschi

    2013-09-01

    Full Text Available Heroin (3,6-diacetylmorphine has various effects on the central nervous system with several neuropathological alterations including hypoxic-ischemic brain damage from respiratory depressing effects and neuroinflammatory response. Both of these mechanisms induce the release of cytokines, chemokines and other inflammatory mediators by the activation of many cell types such as leucocytes and endothelial and glial cells, especially microglia, the predominant immunocompetent cell type within the central nervous system. The aim of this study is to clarify the correlation between intravenous heroin administration in heroin related death and the neuroinflammatory response. We selected 45 cases among autopsies executed for heroin-related death (358 total cases; immunohistochemical studies and Western blotting analyses were used to investigate the expression of brain markers such as tumor necrosis factor-α, oxygen-regulated protein 150, (interleukins IL-1β, IL-6, IL-8, IL-10, IL-15, cyclooxygenase-2, heat shock protein 70, and CD68 (MAC387. Findings demonstrated that morphine induces inflammatory response and cytokine release. In particular, oxygen-regulated protein 150, cyclooxygenase-2, heat shock protein 70, IL-6 and IL-15 cytokines were over-expressed with different patterns of cellular expression.

  2. Nuclear regulation

    International Nuclear Information System (INIS)

    Today, 112 nuclear power plants, 22 facilities that support these plants, 54 reactors used in research, and approximately 23,000 organizations hold licenses from either the Nuclear Regulator Commission or various states to use radioactive material; other facilities are operated by various government agencies. Eventually most of these facilities will be decommissioned, which involves removing the radioactive material and terminating the license. NRC needs to ensure that licensees appropriately decontaminate their facilities because, under current regulations, NRC cannot specifically require additional cleanup once it terminates a license. This paper presents a GAO report on NRC's decommissioning procedures. In two of eight cases GAO reviewed, NRC fully or partially released sites for unrestricted use where radioactive contamination was higher than its guidelines allowed; in the other cases, NRC's information was inadequate or incomplete. Further, NRC lacks information on the types and amounts of radioactive waste buried on-site. At five sites reviewed by GAO, groundwater has been found to be contaminated by radioactive waste

  3. Sequential steps of macroautophagy and chaperone-mediated autophagy are involved in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori.

    Science.gov (United States)

    Shiba, Hajime; Yabu, Takeshi; Sudayama, Makoto; Mano, Nobuhiro; Arai, Naoto; Nakanishi, Teruyuki; Hosono, Kuniaki

    2016-04-15

    To elucidate the degradation process of the posterior silk gland during metamorphosis of the silkworm ITALIC! Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP) and after cocoon formation (CO) were used to analyze macroautophagy, chaperone-mediated autophagy (CMA) and the adenosine triphosphate (ATP)-dependent ubiquitin proteasome. Immediately after entering metamorphosis stage PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form II at all stages. The levels of ubiquitinated proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the posterior silk gland during metamorphosis.

  4. A deleterious mutation in DNAJC6 encoding the neuronal-specific clathrin-uncoating co-chaperone auxilin, is associated with juvenile parkinsonism.

    Directory of Open Access Journals (Sweden)

    Simon Edvardson

    Full Text Available Parkinson disease is caused by neuronal loss in the substantia nigra which manifests by abnormality of movement, muscle tone, and postural stability. Several genes have been implicated in the pathogenesis of Parkinson disease, but the underlying molecular basis is still unknown for ∼70% of the patients. Using homozygosity mapping and whole exome sequencing we identified a deleterious mutation in DNAJC6 in two patients with juvenile parkinsonism. The mutation was associated with abnormal transcripts and marked reduced DNAJC6 mRNA level. DNAJC6 encodes the HSP40 Auxilin, a protein which is selectively expressed in neurons and confers specificity to the ATPase activity of its partner Hcs70 in clathrin uncoating. In Auxilin null mice it was previously shown that the abnormally increased retention of assembled clathrin on vesicles and in empty cages leads to impaired synaptic vesicle recycling and perturbed clathrin mediated endocytosis. Endocytosis function, studied by transferring uptake, was normal in fibroblasts from our patients, likely because of the presence of another J-domain containing partner which co-chaperones Hsc70-mediated uncoating activity in non-neuronal cells. The present report underscores the importance of the endocytic/lysosomal pathway in the pathogenesis of Parkinson disease and other forms of parkinsonism.

  5. Investigation of the complex I assembly chaperones B17.2L and NDUFAF1 in a cohort of CI deficient patients.

    Science.gov (United States)

    Vogel, Rutger O; van den Brand, Mariël A M; Rodenburg, Richard J; van den Heuvel, Lambert P W J; Tsuneoka, Makoto; Smeitink, Jan A M; Nijtmans, Leo G J

    2007-06-01

    Dysfunction of complex I (NADH:ubiquinone oxidoreductase; CI), the largest enzyme of the oxidative phosphorylation (OXPHOS) system, often results in severe neuromuscular disorders and early childhood death. Mutations in its seven mitochondrial and 38 nuclear DNA-encoded structural components can only partly explain these deficiencies. Recently, CI assembly chaperones NDUFAF1 and B17.2L were linked to CI deficiency, but it is still unclear by which mechanism. To better understand their requirement during assembly we have studied their presence in CI subcomplexes in a cohort of CI deficient patients using one- and two-dimensional blue-native PAGE. This analysis revealed distinct differences between their associations to subcomplexes in different patients. B17.2L occurred in a 830 kDa subcomplex specifically in patients with mutations in subunits NDUFV1 and NDUFS4. Contrasting with this seemingly specific requirement, the previously described NDUFAF1 association to 500-850 kDa intermediates did not appear to be related to the nature and severity of the CI assembly defect. Surprisingly, even in the absence of assembly intermediates in a patient harboring a mutation in translation elongation factor G1 (EFG1), NDUFAF1 remained associated to the 500-850 kDa subcomplexes. These findings illustrate the difference in mechanism between B17.2L and NDUFAF1 and suggest that the involvement of NDUFAF1 in the assembly process could be indirect rather than direct via the binding to assembly intermediates.

  6. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    Science.gov (United States)

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. PMID:27006402

  7. DnaK dependence of mutant ethanol oxidoreductases evolved for aerobic function and protective role of the chaperone against protein oxidative damage in Escherichia coli

    Science.gov (United States)

    Echave, Pedro; Esparza-Cerón, M. Angel; Cabiscol, Elisa; Tamarit, Jordi; Ros, Joaquim; Membrillo-Hernández, Jorge; Lin, E. C. C.

    2002-01-01

    The adhE gene of Escherichia coli encodes a multifunctional ethanol oxidoreductase (AdhE) that catalyzes successive reductions of acetyl-CoA to acetaldehyde and then to ethanol reversibly at the expense of NADH. Mutant JE52, serially selected for acquired and improved ability to grow aerobically on ethanol, synthesized an AdhEA267T/E568K with two amino acid substitutions that sequentially conferred improved catalytic properties and stability. Here we show that the aerobic growth ability on ethanol depends also on protection of the mutant AdhE against metal-catalyzed oxidation by the chaperone DnaK (a member of the Hsp70 family). No DnaK protection of the enzyme is evident during anaerobic growth on glucose. Synthesis of DnaK also protected E. coli from H2O2 killing under conditions when functional AdhE is not required. Our results therefore suggest that, in addition to the known role of protecting cells against heat stress, DnaK also protects numerous kinds of proteins from oxidative damage. PMID:11917132

  8. Ribosomal P3 protein AtP3B of Arabidopsis acts as both protein and RNA chaperone to increase tolerance of heat and cold stresses.

    Science.gov (United States)

    Kang, Chang Ho; Lee, Young Mee; Park, Joung Hun; Nawkar, Ganesh M; Oh, Hun Taek; Kim, Min Gab; Lee, Soo In; Kim, Woe Yeon; Yun, Dae-Jin; Lee, Sang Yeol

    2016-07-01

    The P3 proteins are plant-specific ribosomal P-proteins; however, their molecular functions have not been characterized. In a screen for components of heat-stable high-molecular weight (HMW) complexes, we isolated the P3 protein AtP3B from heat-treated Arabidopsis suspension cultures. By size-exclusion chromatography (SEC), SDS-PAGE and native PAGE followed by immunoblotting with anti-AtP3B antibody, we showed that AtP3B was stably retained in HMW complexes following heat shock. The level of AtP3B mRNA increased in response to both high- and low-temperature stresses. Bacterially expressed recombinant AtP3B protein exhibited both protein and RNA chaperone activities. Knockdown of AtP3B by RNAi made plants sensitive to both high- and low-temperature stresses, whereas overexpression of AtP3B increased tolerance of both conditions. Together, our results suggest that AtP3B protects cells against both high- and low-temperature stresses. These findings provide novel insight into the molecular functions and in vivo roles of acidic ribosomal P-proteins, thereby expanding our knowledge of the protein production machinery. PMID:27004478

  9. UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly.

    Science.gov (United States)

    Hunziker, Mirjam; Barandun, Jonas; Petfalski, Elisabeth; Tan, Dongyan; Delan-Forino, Clémentine; Molloy, Kelly R; Kim, Kelly H; Dunn-Davies, Hywel; Shi, Yi; Chaker-Margot, Malik; Chait, Brian T; Walz, Thomas; Tollervey, David; Klinge, Sebastian

    2016-06-29

    Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes-UtpA and UtpB-interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5' end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5' ETS and U3 snoRNA as well as the 3' boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

  10. Serological detection of 'Candidatus Liberibacter asiaticus' in citrus, and identification by GeLC-MS/MS of a chaperone protein responding to cellular pathogens.

    Science.gov (United States)

    Ding, Fang; Duan, Yongping; Yuan, Qing; Shao, Jonathan; Hartung, John S

    2016-01-01

    We describe experiments with antibodies against 'Candidatus Liberibacter asiaticus used to detect the pathogen in infected plants. We used scFv selected to bind epitopes exposed on the surface of the bacterium in tissue prints, with secondary monoclonal antibodies directed at a FLAG epitope included at the carboxyl end of the scFv. Unexpectedly, the anti-FLAG secondary antibody produced positive results with CaLas diseased samples when the primary scFv were not used. The anti-FLAG monoclonal antibody (Mab) also identified plants infected with other vascular pathogens. We then identified a paralogous group of secreted chaperone proteins in the HSP-90 family that contained the amino acid sequence DDDDK identical to the carboxy-terminal sequence of the FLAG epitope. A rabbit polyclonal antibody against one of the same epitopes combined with a goat anti-rabbit secondary antibody produced very strong purple color in individual phloem cells, as expected for this pathogen. These results were entirely specific for CaLas-infected citrus. The simplicity, cost and ability to scale the tissue print assay makes this an attractive assay to complement PCR-based assays currently in use. The partial FLAG epitope may itself be useful as a molecular marker for the rapid screening of citrus plants for the presence of vascular pathogens. PMID:27381064

  11. CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA.

    Science.gov (United States)

    Liu, Wallace H; Roemer, Sarah C; Port, Alex M; Churchill, Mair E A

    2012-12-01

    Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1-H3/H4 or H3/H4-DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)(2) tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.

  12. The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase.

    Science.gov (United States)

    Cao, Hong; Schroeder, Barbara; Chen, Jing; Schott, Micah B; McNiven, Mark A

    2016-08-01

    Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments. PMID:27226592

  13. The SIRT1 modulators AROS and DBC1 regulate HSF1 activity and the heat shock response.

    Directory of Open Access Journals (Sweden)

    Rachel Raynes

    Full Text Available The heat shock response, the cellular response to protein damaging stress, is critical in maintaining proteostasis. The heat shock response is regulated by the transcription factor HSF1, which is activated upon heat shock and other stresses to induce the expression of molecular chaperones. SIRT1 has previously been shown to activate HSF1 by deacetylating it, leading to increased DNA binding ability. We have investigated how the heat shock response may be controlled by factors influencing SIRT1 activity. We found that heat shock results in an increase in the cellular NAD(+/NADH ratio and an increase in recruitment of SIRT1 to the hsp70 promoter. Furthermore, we found that the SIRT1 modulators AROS and DBC1 have an impact on hsp70 transcription, HSF1 acetylation status, and HSF1 recruitment to the hsp70 promoter. Therefore, AROS and DBC1 are now two new targets available for therapeutic regulation of the heat shock response.

  14. sRNA-Mediated Regulation of P-Fimbriae Phase Variation in Uropathogenic Escherichia coli

    DEFF Research Database (Denmark)

    Khandige, Surabhi; Kronborg, Tina; Uhlin, Bernt Eric;

    2015-01-01

    Uropathogenic Escherichia coli (UPEC) are capable of occupying physiologically distinct intracellular and extracellular niches within the urinary tract. This feat requires the timely regulation of gene expression and small RNAs (sRNAs) are known to mediate such rapid adjustments in response...... to changing environmental cues. This study aimed to uncover sRNA-mediated gene regulation in the UPEC strain UTI89, during infection of bladder epithelial cells. Hfq is an RNA chaperone known to facilitate and stabilize sRNA and target mRNA interactions with bacterial cells. The co......-immunoprecipitation and high throughput RNA sequencing of Hfq bound sRNAs performed in this study, revealed distinct sRNA profiles in UPEC in the extracellular and intracellular environments. Our findings emphasize the importance of studying regulatory sRNAs in a biologically relevant niche. This strategy also led...

  15. The posttranslocational chaperone lipoprotein PrsA is involved in both glycopeptide and oxacillin resistance in Staphylococcus aureus.

    Science.gov (United States)

    Jousselin, Ambre; Renzoni, Adriana; Andrey, Diego O; Monod, Antoinette; Lew, Daniel P; Kelley, William L

    2012-07-01

    Understanding in detail the factors which permit Staphylococcus aureus to counteract cell wall-active antibiotics is a prerequisite to elaborating effective strategies to prolong the usefulness of these drugs and define new targets for pharmacological intervention. Methicillin-resistant S. aureus (MRSA) strains are major pathogens of hospital-acquired and community-acquired infections and are most often treated with glycopeptides (vancomycin and teicoplanin) because of their resistance to most penicillins and a limited arsenal of clinically proven alternatives. In this study, we examined PrsA, a lipid-anchored protein of the parvulin PPIase family (peptidyl-prolyl cis/trans isomerase) found ubiquitously in all Gram-positive species, in which it assists posttranslocational folding at the outer surface of the cytoplasmic membrane. We show by both genetic and biochemical assays that prsA is directly regulated by the VraRS two-component sentinel system of cell wall stress. Disruption of prsA is tolerated by S. aureus, and its loss results in no detectable overt macroscopic changes in cell wall architecture or growth rate under nonstressed growth conditions. Disruption of prsA leads, however, to notable alterations in the sensitivity to glycopeptides and dramatically decreases the resistance of COL (MRSA) to oxacillin. Quantitative transcriptional analysis reveals that prsA and vraR are coordinately upregulated in a panel of stable laboratory and clinical glycopeptide-intermediate S. aureus (GISA) strains compared to their susceptible parents. Collectively, our results point to a role for prsA as a facultative facilitator of protein secretion or extracellular folding and provide a framework for understanding why prsA is a key element of the VraRS-mediated cell wall stress response. PMID:22526301

  16. Genome-wide cooperation by HAT Gcn5, remodeler SWI/SNF, and chaperone Ydj1 in promoter nucleosome eviction and transcriptional activation.

    Science.gov (United States)

    Qiu, Hongfang; Chereji, Răzvan V; Hu, Cuihua; Cole, Hope A; Rawal, Yashpal; Clark, David J; Hinnebusch, Alan G

    2016-02-01

    Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes.

  17. Using codon optimization, chaperone co-expression, and rational mutagenesis for production and NMR assignments of human eIF2α

    International Nuclear Information System (INIS)

    Producing a well behaved sample at high concentration is one of the main hurdles when starting a new project on an interesting protein. Especially when one attempts to overexpress a eukaryotic protein in bacteria, some difficulties are encountered, such as low expression level, low solubility, or even lack of folded structure. Overexpression in prokaryotic systems is highly desirable for cost-effective production of different isotope-labeled samples needed for NMR studies. Here we describe generally applicable methods for obtaining highly concentrated protein samples efficiently. This approach was developed as we tried to produce a NMR-suitable sample of the 35 kDa human translation initiation factor eIF2α, a protein that expresses poorly in E. coli and has very low solubility. First, an E. coli codon-optimized gene was synthesized on a thermal cycler, which increased the expression level by a factor of two. Second, we used co-expression of bacterial chaperone proteins, which largely increased the fraction of correctly folded protein found in the soluble phase. Third, we used rational mutagenesis guided by both the sequence alignment among homologues and the homology of one domain to a known fold for improving solubility and stability of the target protein by tenfold. Combining all these methods made it possible to produce from a one-liter preparation a 0.5 mM sample of human eIF2α that showed well-resolved NMR spectra and enabled nearly complete assignment of the protein. These methods may be generally useful for studies of other eukaryotic proteins that are otherwise difficult to express and exhibit poor solubility

  18. Gut microbiota imbalance and chaperoning system malfunction are central to ulcerative colitis pathogenesis and can be counteracted with specifically designed probiotics: a working hypothesis.

    Science.gov (United States)

    Bellavia, Maurizio; Tomasello, Giovanni; Romeo, Marcello; Damiani, Provvidenza; Lo Monte, Attilio I; Lozio, Luciano; Campanella, Claudia; Marino Gammazza, Antonella; Rappa, Francesca; Zummo, Giovanni; Cocchi, Massimo; Conway de Macario, Everly; Macario, Alberto J L; Cappello, Francesco

    2013-12-01

    In this work, we propose that for further studies of the physiopathology and treatment for inflammatory bowel diseases, an integral view of the conditions, including the triad of microbiota-heat shock proteins (HSPs)-probiotics, ought to be considered. Microbiota is the complex microbial flora that resides in the gut, affecting not only gut functions but also the health status of the whole body. Alteration in the microbiota's composition has been implicated in a variety of pathological conditions (e.g., ulcerative colitis, UC), involving both gut and extra-intestinal tissues and organs. Some of these pathologies are also associated with an altered expression of HSPs (chaperones) and this is the reason why they may be considered chaperonopathies. Probiotics, which are live microorganisms able to restore the correct, healthy equilibrium of microbiota composition, can ameliorate symptoms in patients suffering from UC and modulate expression levels of HSPs. However, currently probiotic therapy follows ex-adiuvantibus criteria, i.e., treatments with beneficial effects but whose mechanism of action is unknown, which should be changed so the probiotics needed in each case are predetermined on the basis of the patient's microbiota. Consequently, efforts are necessary to develop diagnostic tools for elucidating levels and distribution of HSPs and the microbiota composition (microbiota fingerprint) of each subject and, thus, guide specific probiotic therapy, tailored to meet the needs of the patient. Microbiota fingerprinting ought to include molecular biology techniques for sequencing highly conserved DNA, e.g., genes encoding 16S RNA, for species identification and, in addition, quantification of each relevant microbe.

  19. Constitutively-active androgen receptor variants function independently of the HSP90 chaperone but do not confer resistance to HSP90 inhibitors.

    Science.gov (United States)

    Gillis, Joanna L; Selth, Luke A; Centenera, Margaret M; Townley, Scott L; Sun, Shihua; Plymate, Stephen R; Tilley, Wayne D; Butler, Lisa M

    2013-05-01

    The development of lethal, castration resistant prostate cancer is associated with adaptive changes to the androgen receptor (AR), including the emergence of mutant receptors and truncated, constitutively active AR variants. AR relies on the molecular chaperone HSP90 for its function in both normal and malignant prostate cells, but the requirement for HSP90 in environments with aberrant AR expression is largely unknown. Here, we investigated the efficacy of three HSP90 inhibitors, 17-AAG, HSP990 and AUY922, against clinically-relevant AR missense mutants and truncated variants. HSP90 inhibition effectively suppressed the signaling of wild-type AR and all AR missense mutants tested. By contrast, two truncated AR variants, AR-V7 and ARv567es, exhibited marked resistance to HSP90 inhibitors. Supporting this observation, nuclear localization of the truncated AR variants was not affected by HSP90 inhibition and AR variant:HSP90 complexes could not be detected in prostate cancer cells. Interestingly, HSP90 inhibition resulted in accumulation of AR-V7 and ARv567es in both cell lines and human tumor explants. Despite the apparent independence of AR variants from HSP90 and their treatment-associated induction, the growth of cell lines with endogenous or enforced expression of AR-V7 or ARv567es remained highly sensitive to AUY922. This study demonstrates that functional AR variant signaling does not confer resistance to HSP90 inhibition, yields insight into the interaction between AR and HSP90 and provides further impetus for the clinical application of HSP90 inhibitors in advanced prostate cancer.

  20. Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair.

    Science.gov (United States)

    Gursoy-Yuzugullu, Ozge; Ayrapetov, Marina K; Price, Brendan D

    2015-06-16

    The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4-Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.