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Sample records for j-type co-chaperone hscb

  1. Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU.

    Science.gov (United States)

    Silberg, Jonathan J; Tapley, Tim L; Hoff, Kevin G; Vickery, Larry E

    2004-12-24

    The ATPase activity of HscA, a specialized hsp70 molecular chaperone from Escherichia coli, is regulated by the iron-sulfur cluster assembly protein IscU and the J-type co-chaperone HscB. IscU behaves as a substrate for HscA, and HscB enhances the binding of IscU to HscA. To better understand the mechanism by which HscB and IscU regulate HscA, we examined binding of HscB to the different conformational states of HscA and the effects of HscB and IscU on the kinetics of the individual steps of the HscA ATPase reaction cycle. Affinity sensor studies revealed that whereas IscU binds both ADP (R-state) and ATP (T-state) HscA complexes, HscB interacts only with an ATP-bound state. Studies of ATPase activity under single-turnover and rapid mixing conditions showed that both IscU and HscB interact with the low peptide affinity T-state of HscA (HscA++.ATP) and that both modestly accelerate (3-10-fold) the rate-determining steps in the HscA reaction cycle, k(hyd) and k(T-->R). When present together, IscU and HscB synergistically stimulate both k(hyd) (approximately = 500-fold) and k(T-->R) (approximately = 60-fold), leading to enhanced formation of the HscA.ADP-IscU complex (substrate capture). Following ADP/ATP exchange, IscU also stimulates k(R-->T) (approximately = 50-fold) and thereby accelerates the rate at which the low peptide affinity HscA++.ATP T-state is regenerated. Because HscA nucleotide exchange is fast, the overall rate of the chaperone cycle in vivo will be determined by the availability of the IscU-HscB substrate-co-chaperone complex.

  2. Human, Social, Cultural Behavior (HSCB) Modeling Workshop I: Characterizing the Capability Needs for HSCB Modeling

    Science.gov (United States)

    2008-07-01

    The expectations correspond to different roles individuals perform SocialConstructionis Social constructionism is a school of thought Peter L...HUMAN, SOCIAL , CULTURAL BEHAVIOR (HSCB) MODELING WORKSHOP I: CHARACTERIZING THE CAPABILITY NEEDS FOR HSCB MODELING FINAL REPORT... Social , Cultural Behavior (HSCB) Modeling Workshop I: Characterizing the Capability Needs for HSCB Modeling 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c

  3. A new tessera into the interactome of the isc operon:A novel interaction between HscB and IscS

    Directory of Open Access Journals (Sweden)

    Annalisa Pastore

    2016-09-01

    Full Text Available Iron sulfur clusters are essential universal prosthetic groups which can be formed inorganically but, in biology, are bound to proteins and produced enzymatically. Most of the components of the machine that produces the clusters are conserved throughout evolution. In bacteria, they are encoded in the isc operon. Previous reports provide information on the role of specific components but a clear picture of how the whole machine works is still missing. We have carried out a study of the effects of the co-chaperone HscB from the model system E. coli. We document a previously undetected weak interaction between the chaperone HscB and the desulfurase IscS, one of the two main players of the machine. The binding site involves a region of HscB in the longer stem of the approximately L-shaped molecules, whereas the interacting surface of IscS overlaps with the surface previously involved in binding other proteins, such as ferredoxin and frataxin. Our findings provide an entirely new perspective to our comprehension of the role of HscB and propose this protein as a component of the IscS complex.

  4. Association of HSP70 and its co-chaperones with Alzheimer's disease

    NARCIS (Netherlands)

    L. Broer (Linda); M.A. Ikram (Arfan); M. Schuur (Maaike); A.L. DeStefano (Anita); J.C. Bis (Joshua); F. Liu (Fan); F. Rivadeneira Ramirez (Fernando); A.G. Uitterlinden (André); A. Beiser (Alexa); W.T. Longstreth Jr; A. Hofman (Albert); Y.S. Aulchenko (Yurii); S. Seshadri (Sudha); A.L. Fitzpatrick (Annette); B.A. Oostra (Ben); M.M.B. Breteler (Monique); P. Tikka-Kleemola (Päivi)

    2011-01-01

    textabstractThe heat shock protein (HSP) 70 family has been implicated in the pathology of Alzheimer's disease (AD). In this study, we examined common genetic variations in the 80 genes encoding HSP70 and its co-chaperones. We conducted a study in a series of 462 patients and 5238 unaffected

  5. The FKBP51 Glucocorticoid Receptor Co-Chaperone: Regulation, Function, and Implications in Health and Disease.

    Science.gov (United States)

    Fries, Gabriel R; Gassen, Nils C; Rein, Theo

    2017-12-05

    Among the chaperones and co-chaperones regulating the glucocorticoid receptor (GR), FK506 binding protein (FKBP) 51 is the most intensely investigated across different disciplines. This review provides an update on the role of the different co-chaperones of Hsp70 and Hsp90 in the regulation of GR function. The development leading to the focus on FKBP51 is outlined. Further, a survey of the vast literature on the mechanism and function of FKBP51 is provided. This includes its structure and biochemical function, its regulation on different levels-transcription, post-transcription, and post-translation-and its function in signaling pathways. The evidence portraying FKBP51 as a scaffolding protein organizing protein complexes rather than a chaperone contributing to the folding of individual proteins is collated. Finally, FKBP51's involvement in physiology and disease is outlined, and the promising efforts in developing drugs targeting FKBP51 are discussed.

  6. The Role of Co-chaperones in Synaptic Proteostasis and Neurodegenerative Disease

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    Erica L. Gorenberg

    2017-05-01

    Full Text Available Synapses must be preserved throughout an organism's lifespan to allow for normal brain function and behavior. Synapse maintenance is challenging given the long distances between the termini and the cell body, reliance on axonal transport for delivery of newly synthesized presynaptic proteins, and high rates of synaptic vesicle exo- and endocytosis. Hence, synapses rely on efficient proteostasis mechanisms to preserve their structure and function. To this end, the synaptic compartment has specific chaperones to support its functions. Without proper synaptic chaperone activity, local proteostasis imbalances lead to neurotransmission deficits, dismantling of synapses, and neurodegeneration. In this review, we address the roles of four synaptic chaperones in the maintenance of the nerve terminal, as well as their genetic links to neurodegenerative disease. Three of these are Hsp40 co-chaperones (DNAJs: Cysteine String Protein alpha (CSPα; DNAJC5, auxilin (DNAJC6, and Receptor-Mediated Endocytosis 8 (RME-8; DNAJC13. These co-chaperones contain a conserved J domain through which they form a complex with heat shock cognate 70 (Hsc70, enhancing the chaperone's ATPase activity. CSPα is a synaptic vesicle protein known to chaperone the t-SNARE SNAP-25 and the endocytic GTPase dynamin-1, thereby regulating synaptic vesicle exocytosis and endocytosis. Auxilin binds assembled clathrin cages, and through its interactions with Hsc70 leads to the uncoating of clathrin-coated vesicles, a process necessary for the regeneration of synaptic vesicles. RME-8 is a co-chaperone on endosomes and may have a role in clathrin-coated vesicle endocytosis on this organelle. These three co-chaperones maintain client function by preserving folding and assembly to prevent client aggregation, but they do not break down aggregates that have already formed. The fourth synaptic chaperone we will discuss is Heat shock protein 110 (Hsp110, which interacts with Hsc70, DNAJAs, and

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

    International Nuclear Information System (INIS)

    Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H.; Prodromou, Chrisostomos

    2015-01-01

    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) 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

  8. 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.

  9. UBL/BAG-domain co-chaperones cause cellular stress upon overexpression through constitutive activation of Hsf1

    DEFF Research Database (Denmark)

    Poulsen, Esben Guldahl; Kampmeyer, Caroline; Kriegenburg, Franziska

    2017-01-01

    of molecular chaperones and other stress-relieving proteins. Here, we show that the fission yeast Schizosaccharomyces pombe orthologues of human BAG-1, Bag101, and Bag102, are Hsp70 co-chaperones that associate with 26S proteasomes. Only a subgroup of Hsp70-type chaperones, including Ssa1, Ssa2, and Sks2...

  10. Co-chaperone p23 regulates C. elegans Lifespan in Response to Temperature.

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    Makoto Horikawa

    2015-04-01

    Full Text Available Temperature potently modulates various physiologic processes including organismal motility, growth rate, reproduction, and ageing. In ectotherms, longevity varies inversely with temperature, with animals living shorter at higher temperatures. Thermal effects on lifespan and other processes are ascribed to passive changes in metabolic rate, but recent evidence also suggests a regulated process. Here, we demonstrate that in response to temperature, daf-41/ZC395.10, the C. elegans homolog of p23 co-chaperone/prostaglandin E synthase-3, governs entry into the long-lived dauer diapause and regulates adult lifespan. daf-41 deletion triggers constitutive entry into the dauer diapause at elevated temperature dependent on neurosensory machinery (daf-10/IFT122, insulin/IGF-1 signaling (daf-16/FOXO, and steroidal signaling (daf-12/FXR. Surprisingly, daf-41 mutation alters the longevity response to temperature, living longer than wild-type at 25°C but shorter than wild-type at 15°C. Longevity phenotypes at 25°C work through daf-16/FOXO and heat shock factor hsf-1, while short lived phenotypes converge on daf-16/FOXO and depend on the daf-12/FXR steroid receptor. Correlatively daf-41 affected expression of DAF-16 and HSF-1 target genes at high temperature, and nuclear extracts from daf-41 animals showed increased occupancy of the heat shock response element. Our studies suggest that daf-41/p23 modulates key transcriptional changes in longevity pathways in response to temperature.

  11. Breaking BAG: The Co-Chaperone BAG3 in Health and Disease.

    Science.gov (United States)

    Behl, Christian

    2016-08-01

    Human BAG (Bcl-2-associated athanogene) proteins form a family of antiapoptotic proteins that currently consists of six members (BAG1-6) all sharing the BAG protein domain from which the name arises. Via this domain, BAG proteins bind to the heat shock protein 70 (Hsp70), thereby acting as a co-chaperone regulating the activity of Hsp70. In addition to their antiapoptotic activity, all human BAG proteins have distinct functions in health and disease, and BAG3 in particular is the focus of many investigations. BAG3 has a modular protein domain composition offering the possibility for manifold interactions with other proteins. Various BAG3 functions are implicated in disorders including cancer, myopathies, and neurodegeneration. The discovery of its role in selective autophagy and the description of BAG3-mediated selective macroautophagy as an adaptive mechanism to maintain cellular homeostasis, under stress as well as during aging, make BAG3 a highly interesting target for future pharmacological interventions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. The co-chaperones Fkbp4/5 control Argonaute2 expression and facilitate RISC assembly.

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    Martinez, Natalia J; Chang, Hao-Ming; Borrajo, Jacob de Riba; Gregory, Richard I

    2013-11-01

    Argonaute2 (Ago2) protein and associated microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC) for target messenger RNA cleavage and post-transcriptional gene silencing. Although Ago2 is essential for RISC activity, the mechanism of RISC assembly is not well understood, and factors controlling Ago2 protein expression are largely unknown. A role for the Hsc70/Hsp90 chaperone complex in loading small RNA duplexes into the RISC has been demonstrated in cell extracts, and unloaded Ago2 is unstable and degraded by the lysosome in mammalian cells. Here we identify the co-chaperones Fkbp4 and Fkbp5 as Ago2-associated proteins in mouse embryonic stem cells. Pharmacological inhibition of this interaction using FK506 or siRNA-mediated Fkbp4/5 depletion leads to decreased Ago2 protein levels. We find FK506 treatment inhibits, whereas Fkbp4/5 overexpression promotes, miRNA-mediated stabilization of Ago2 expression. Simultaneous treatment with a lysosome inhibitor revealed the accumulation of unloaded Ago2 complexes in FK506-treated cells. We find that, consistent with unloaded miRNAs being unstable, FK506 treatment also affects miRNA abundance, particularly nascent miRNAs. Our results support a role for Fkbp4/5 in RISC assembly.

  13. Comparison of the carboxy-terminal DP-repeat region in the co-chaperones Hop and Hip.

    Science.gov (United States)

    Nelson, Gregory M; Huffman, Holly; Smith, David F

    2003-01-01

    Functional steroid receptor complexes are assembled and maintained by an ordered pathway of interactions involving multiple components of the cellular chaperone machinery. Two of these components, Hop and Hip, serve as co-chaperones to the major heat shock proteins (Hsps), Hsp70 and Hsp90, and participate in intermediate stages of receptor assembly. In an effort to better understand the functions of Hop and Hip in the assembly process, we focused on a region of similarity located near the C-terminus of each co-chaperone. Contained within this region is a repeated sequence motif we have termed the DP repeat. Earlier mutagenesis studies implicated the DP repeat of either Hop or Hip in Hsp70 binding and in normal assembly of the co-chaperones with progesterone receptor (PR) complexes. We report here that the DP repeat lies within a protease-resistant domain that extends to or is near the C-terminus of both co-chaperones. Point mutations in the DP repeats render the C-terminal regions hypersensitive to proteolysis. In addition, a Hop DP mutant displays altered proteolytic digestion patterns, which suggest that the DP-repeat region influences the folding of other Hop domains. Although the respective DP regions of Hop and Hip share sequence and structural similarities, they are not functionally interchangeable. Moreover, a double-point mutation within the second DP-repeat unit of Hop that converts this to the sequence found in Hip disrupts Hop function; however, the corresponding mutation in Hip does not alter its function. We conclude that the DP repeats are important structural elements within a C-terminal domain, which is important for Hop and Hip function.

  14. The Hsp90 co-chaperones Sti1, Aha1, and P23 regulate adaptive responses to antifungal azoles

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    Xiaokui Gu

    2016-10-01

    Full Text Available Heat Shock Protein 90 (Hsp90 is essential for tumor progression in humans and drug resistance in fungi. However, the roles of its many co-chaperones in antifungal resistance are unknown. In this study, by susceptibility test of Neurospora crassa mutants lacking each of 18 Hsp90/Calcineurin system member genes (including 8 Hsp90 co-chaperone genes to antifungal drugs and other stresses, we demonstrate that the Hsp90 co-chaperones Sti1 (Hop1 in yeast, Aha1, and P23 (Sba1 in yeast were required for the basal resistance to antifungal azoles and heat stress. Deletion of any of them resulted in hypersensitivity to azoles and heat. Liquid chromatography–mass spectrometry (LC-MS analysis showed that the toxic sterols eburicol and 14α-methyl-3,6-diol were significantly accumulated in the sti1 and p23 deletion mutants after ketoconazole treatment, which has been shown before to led to cell membrane stress. At the transcriptional level, Aha1, Sti1, and P23 positively regulate responses to ketoconazole stress by erg11 and erg6, key genes in the ergosterol biosynthetic pathway. Aha1, Sti1, and P23 are highly conserved in fungi, and sti1 and p23 deletion also increased the susceptibility to azoles in Fusarium verticillioides. These results indicate that Hsp90-cochaperones Aha1, Sti1, and P23 are critical for the basal azole resistance and could be potential targets for developing new antifungal agents.

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

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    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. Summary of human social, cultural, behavioral (HSCB) modeling for information fusion panel discussion

    Science.gov (United States)

    Blasch, Erik; Salerno, John; Kadar, Ivan; Yang, Shanchieh J.; Fenstermacher, Laurie; Endsley, Mica; Grewe, Lynne

    2013-05-01

    During the SPIE 2012 conference, panelists convened to discuss "Real world issues and challenges in Human Social/Cultural/Behavioral modeling with Applications to Information Fusion." Each panelist presented their current trends and issues. The panel had agreement on advanced situation modeling, working with users for situation awareness and sense-making, and HSCB context modeling in focusing research activities. Each panelist added different perspectives based on the domain of interest such as physical, cyber, and social attacks from which estimates and projections can be forecasted. Also, additional techniques were addressed such as interest graphs, network modeling, and variable length Markov Models. This paper summarizes the panelists discussions to highlight the common themes and the related contrasting approaches to the domains in which HSCB applies to information fusion applications.

  17. Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

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    Hicham Mahboubi

    2015-12-01

    Full Text Available Background. Chaperones and their co-factors are components of a cellular network; they collaborate to maintain proteostasis under normal and harmful conditions. In particular, hsp70 family members and their co-chaperones are essential to repair damaged proteins. Co-chaperones are present in different subcellular compartments, where they modulate chaperone activities.Methods and Results. Our studies assessed the relationship between hsc70 and its co-factor HspBP1 in human cancer cells. HspBP1 promotes nucleotide exchange on hsc70, but has also chaperone-independent functions. We characterized the interplay between hsc70 and HspBP1 by quantitative confocal microscopy combined with automated image analyses and statistical evaluation. Stress and the recovery from insult changed significantly the subcellular distribution of hsc70, but had little effect on HspBP1. Single-cell measurements and regression analysis revealed that the links between the chaperone and its co-factor relied on (i the physiological state of the cell and (ii the subcellular compartment. As such, we identified a linear relationship and strong correlation between hsc70 and HspBP1 distribution in control and heat-shocked cells; this correlation changed in a compartment-specific fashion during the recovery from stress. Furthermore, we uncovered significant stress-induced changes in the colocalization between hsc70 and HspBP1 in the nucleus and cytoplasm.Discussion. Our quantitative approach defined novel properties of the co-chaperone HspBP1 as they relate to its interplay with hsc70. We propose that changes in cell physiology promote chaperone redistribution and thereby stimulate chaperone-independent functions of HspBP1.

  18. Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy.

    Science.gov (United States)

    Fang, Xi; Bogomolovas, Julius; Wu, Tongbin; Zhang, Wei; Liu, Canzhao; Veevers, Jennifer; Stroud, Matthew J; Zhang, Zhiyuan; Ma, Xiaolong; Mu, Yongxin; Lao, Dieu-Hung; Dalton, Nancy D; Gu, Yusu; Wang, Celine; Wang, Michael; Liang, Yan; Lange, Stephan; Ouyang, Kunfu; Peterson, Kirk L; Evans, Sylvia M; Chen, Ju

    2017-08-01

    Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

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

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    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.

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

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    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.

  1. The Hsc/Hsp70 co-chaperone network controls antigen aggregation and presentation during maturation of professional antigen presenting cells.

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    Nadja Kettern

    Full Text Available The maturation of mouse macrophages and dendritic cells involves the transient deposition of ubiquitylated proteins in the form of dendritic cell aggresome-like induced structures (DALIS. Transient DALIS formation was used here as a paradigm to study how mammalian cells influence the formation and disassembly of protein aggregates through alterations of their proteostasis machinery. Co-chaperones that modulate the interplay of Hsc70 and Hsp70 with the ubiquitin-proteasome system (UPS and the autophagosome-lysosome pathway emerged as key regulators of this process. The chaperone-associated ubiquitin ligase CHIP and the ubiquitin-domain protein BAG-1 are essential for DALIS formation in mouse macrophages and bone-marrow derived dendritic cells (BMDCs. CHIP also cooperates with BAG-3 and the autophagic ubiquitin adaptor p62 in the clearance of DALIS through chaperone-assisted selective autophagy (CASA. On the other hand, the co-chaperone HspBP1 inhibits the activity of CHIP and thereby attenuates antigen sequestration. Through a modulation of DALIS formation CHIP, BAG-1 and HspBP1 alter MHC class I mediated antigen presentation in mouse BMDCs. Our data show that the Hsc/Hsp70 co-chaperone network controls transient protein aggregation during maturation of professional antigen presenting cells and in this way regulates the immune response. Similar mechanisms may modulate the formation of aggresomes and aggresome-like induced structures (ALIS in other mammalian cell types.

  2. Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1.

    Science.gov (United States)

    Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A

    2015-04-10

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70∆EEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. The Salmonella type III effector SspH2 specifically exploits the NLR co-chaperone activity of SGT1 to subvert immunity.

    Directory of Open Access Journals (Sweden)

    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.

  4. 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

    International Nuclear Information System (INIS)

    Pearson, Joel D; Mohammed, Zubair; Bacani, Julinor T C; Lai, Raymond; Ingham, Robert J

    2012-01-01

    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. 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. 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 be due to a decrease in NPM-ALK levels or the

  5. Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensible in vivo and conserved in evolution

    Science.gov (United States)

    Ciesielski, Szymon; Schilke, Brenda; Osipiuk, Jerzy; Bigelow, Lance; Mulligan, Rory; Majewska, Julia; Joachimiak, Andrzej; Marszalek, Jaroslaw; Craig, Elizabeth A.; Dutkiewicz, Rafal

    2012-01-01

    The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70’s ATPase activity. However, in apparent contradiction, we previously reported that an 8 fold decrease in Jac1’s affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, a total of eight surface exposed residues were determined to play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype and a reduction in the activity of Fe-S cluster containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensible role in the essential process of mitochondrial Fe-S cluster biogenesis. PMID:22306468

  6. Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensable in vivo and conserved in evolution.

    Science.gov (United States)

    Ciesielski, Szymon J; Schilke, Brenda A; Osipiuk, Jerzy; Bigelow, Lance; Mulligan, Rory; Majewska, Julia; Joachimiak, Andrzej; Marszalek, Jaroslaw; Craig, Elizabeth A; Dutkiewicz, Rafal

    2012-03-16

    The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70's ATPase activity. However, in apparent contradiction, we previously reported that an 8-fold decrease in Jac1's affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here, we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, we determined that a total of eight surface-exposed residues play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype, and a reduction in the activity of Fe-S cluster-containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensable role in the essential process of mitochondrial Fe-S cluster biogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. 1H, 15N and 13C resonance assignments of the J-domain of co-chaperone Sis1 from Saccharomyces cerevisiae.

    Science.gov (United States)

    Pinheiro, Glaucia M S; Amorim, Gisele C; Iqbal, Anwar; Ramos, C H I; Almeida, Fabio C L

    2018-04-30

    Protein folding in the cell is usually aided by molecular chaperones, from which the Hsp70 (Hsp = heat shock protein) family has many important roles, such as aiding nascent folding and participating in translocation. Hsp70 has ATPase activity which is stimulated by binding to the J-domain present in co-chaperones from the Hsp40 family. Hsp40s have many functions, as for instance the binding to partially folded proteins to be delivered to Hsp70. However, the presence of the J-domain characterizes Hsp40s or, by this reason, as J-proteins. The J-domain alone can stimulate Hsp70 ATPase activity. Apparently, it also maintains the same conformation as in the whole protein although structural information on full J-proteins is still missing. This work reports the 1 H, 15 N and 13 C resonance assignments of the J-domain of a Hsp40 from Saccharomyces cerevisiae, named Sis1. Secondary structure and order parameter prediction from chemical shifts are also reported. Altogether, the data show that Sis1 J-domain is highly structured and predominantly formed by α-helices, results that are in very good agreement with those previously reported for the crystallographic structure.

  8. 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.

  9. Autoregulation of Co-Chaperone BAG3 Gene Transcription

    OpenAIRE

    Gentilella, Antonio; Khalili, Kamel

    2009-01-01

    The Bcl-2-associated athanogene, BAG, protein family through their BAG domain associates with the heat shock protein 70 (HSP-70) and modulates its chaperone activity. One member of this family, BAG3, appears to play an important role in protein homeostasis, as its expression promotes cell survival by preventing polyubiquitination of Hsp-70 client proteins. Expression of BAG3 is enhanced by a variety of stress-inducing agents. Here we describe a role for BAG3 to modulate transcription of its o...

  10. J-type Carbon Stars: A Dominant Source of {sup 14}N-rich Presolar SiC Grains of Type AB

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Nan; Nittler, Larry R.; Alexander, Conel M. O’D.; Wang, Jianhua [Department of Terrestrial Magnetism, Carnegie Institution for Science, Washington, DC 20015 (United States); Stephan, Thomas; Boehnke, Patrick; Davis, Andrew M.; Trappitsch, Reto; Pellin, Michael J., E-mail: nliu@carnegiescience.edu [Department of the Geophysical Sciences, The University of Chicago, Chicago, IL 60637 (United States)

    2017-07-20

    We report Mo isotopic data of 27 new presolar SiC grains, including 12 {sup 14}N-rich AB ({sup 14}N/{sup 15}N > 440, AB2) and 15 mainstream (MS) grains, and their correlated Sr and Ba isotope ratios when available. Direct comparison of the data for the MS grains, which came from low-mass asymptotic giant branch (AGB) stars with large s -process isotope enhancements, with the AB2 grain data demonstrates that AB2 grains show near-solar isotopic compositions and lack s -process enhancements. The near-normal Sr, Mo, and Ba isotopic compositions of AB2 grains clearly exclude born-again AGB stars, where the intermediate neutron-capture process ( i -process) takes place, as their stellar source. On the other hand, low-mass CO novae and early R- and J-type carbon stars show {sup 13}C and {sup 14}N excesses but no s -process enhancements and are thus potential stellar sources of AB2 grains. Because both early R-type carbon stars and CO novae are rare objects, the abundant J-type carbon stars (10%–15% of all carbon stars) are thus likely to be a dominant source of AB2 grains.

  11. J-type Carbon Stars: A Dominant Source of 14 N-rich Presolar SiC Grains of Type AB

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Nan; Stephan, Thomas; Boehnke, Patrick; Nittler, Larry R.; Alexander, Conel M. O’D.; Wang, Jianhua; Davis, Andrew M.; Trappitsch, Reto; Pellin, Michael J.

    2017-07-20

    We report Mo isotopic data of 27 new presolar SiC grains, including 12 N-14-rich AB (N-14/N-15 > 440, AB2) and 15 mainstream (MS) grains, and their correlated Sr and Ba isotope ratios when available. Direct comparison of the data for the MS grains, which came from low-mass asymptotic giant branch (AGB) stars with large s-process isotope enhancements, with the AB2 grain data demonstrates that AB2 grains show near-solar isotopic compositions and lack s-process enhancements. The near-normal Sr, Mo, and Ba isotopic compositions of AB2 grains clearly exclude born-again AGB stars, where the intermediate neutron-capture process (i-process) takes place, as their stellar source. On the other hand, low-mass CO novae and early R-and J-type carbon stars show C-13 and N-14 excesses but no s-process enhancements and are thus potential stellar sources of AB2 grains. Because both early R-type carbon stars and CO novae are rare objects, the abundant J-type carbon stars (10%-15% of all carbon stars) are thus likely to be a dominant source of AB2 grains.

  12. Hydronephrosis with ureteritis developed in C57BL/6N mice carrying the congenic region derived from MRL/MpJ-type chromosome 11.

    Science.gov (United States)

    Ichii, Osamu; Chihara, Masataka; Lee, Shin-Hyo; Nakamura, Teppei; Otsuka-Kanazawa, Saori; Horino, Taro; Elewa, Yaser Hosny Ali; Kon, Yasuhiro

    2017-03-01

    Inbred MRL/MpJ mice show several unique phenotypes in tissue regeneration processes and the urogenital and immune systems. Clarifying the genetic and molecular bases of these phenotypes requires the analysis of their genetic susceptibility locus. Herein, hydronephrosis development was incidentally observed in MRL/MpJ-derived chromosome 11 (D11Mit21-212)-carrying C57BL/6N-based congenic mice, which developed bilateral or unilateral hydronephrosis in both males and females with 23.5% and 12.5% prevalence, respectively. Histopathologically, papillary malformations of the transitional epithelium in the pelvic-ureteric junction seemed to constrict the ureter luminal entrance. Characteristically, eosinophilic crystals were observed in the lumen of diseased ureters. These ureters were surrounded by infiltrating cells mainly composed of numerous CD3 +  T-cells and B220 +  B-cells. Furthermore, several Iba-1 +  macrophages, Gr-1 +  granulocytes, mast cells and chitinase 3-like 3/Ym1 (an important inflammatory lectin)-positive cells were detected. Eosinophils also accumulated to these lesions in diseased ureters. Some B6.MRL-(D11Mit21-D11Mit212) mice had duplicated ureters. We determined >100 single nucleotide variants between C57BL/6N- and MRL/MpJ-type chromosome 11 congenic regions, which were associated with nonsynonymous substitution, frameshift or stopgain of coding proteins. In conclusion, B6.MRL-(D11Mit21-D11Mit212) mice spontaneously developed hydronephrosis due to obstructive uropathy with inflammation. Thus, this mouse line would be useful for molecular pathological analysis of obstructive uropathy in experimental medicine.

  13. The Role of the Co-Chaperone, CHIP, in Androgen Independent Prostate Cancer

    Science.gov (United States)

    2008-02-01

    C4-2 there was no evidence of lysosomal congregration, the hallmark of autophagy, rather vaculolization of cytoplasm and disintergration of cellular...RNA polymerase promoter at the 5′ end. Second-strand synthesis was followed by cRNA production incorporating a biotinylated base. Hybridization to

  14. Intracellular dynamics of the Hsp90 co-chaperone p23 is dictated by Hsp90

    International Nuclear Information System (INIS)

    Picard, Didier

    2006-01-01

    p23 is a component of the Hsp90 molecular chaperone machine. It binds and stabilizes the ATP-bound dimeric form of Hsp90. Since Hsp90 binds protein substrates in the ATP conformation, p23 has been proposed to stabilize Hsp90-substrate complexes. In addition, p23 can also function as a molecular chaperone by itself and even possesses an unrelated enzymatic activity. Whether it fulfills the latter functions in cells while bound to Hsp90 remains unknown and is difficult to extrapolate from cell-free biochemical experiments. Using the 'fluorescence recovery after photobleaching' (FRAP) technology, I have examined the dynamics of human p23, expressed as a fusion protein with the green fluorescent protein (GFP), in living human HeLa cells. GFP-p23 is distributed throughout the cell, and its mobility is identical in the cytoplasm and in the nucleus. When the Hsp90 interaction is disrupted either with the Hsp90 inhibitor geldanamycin or by introduction of point mutations into p23, the mobility of p23 is greatly accelerated. Under these conditions, its intracellular movement may be diffusion-controlled. In contrast, when wild-type p23 is able to bind Hsp90, a more complex FRAP behavior is observed, suggesting that it is quantitatively bound in Hsp90 complexes undergoing a multitude of other interactions

  15. Emerging roles of molecular chaperones and co-chaperones in selective autophagy : focus on BAG proteins

    NARCIS (Netherlands)

    Gamerdinger, Martin; Carra, Serena; Behl, Christian

    2011-01-01

    Macroautophagy is a catabolic process by which the cell degrades cytoplasmic components through the lysosomal machinery. While initially acknowledged as a rather unspecific bulk degradation process, growing lines of evidence indicate the selectivity of macroautophagy pathways in the removal of

  16. Alterations in the steroid hormone receptor co-chaperone FKBPL are associated with male infertility: a case-control study

    LENUS (Irish Health Repository)

    Sunnotel, Olaf

    2010-03-08

    Abstract Background Male infertility is a common cause of reproductive failure in humans. In mice, targeted deletions of the genes coding for FKBP6 or FKBP52, members of the FK506 binding protein family, can result in male infertility. In the case of FKBP52, this reflects an important role in potentiating Androgen Receptor (AR) signalling in the prostate and accessory glands, but not the testis. In infertile men, no mutations of FKBP52 or FKBP6 have been found so far, but the gene for FKBP-like (FKBPL) maps to chromosome 6p21.3, an area linked to azoospermia in a group of Japanese patients. Methods To determine whether mutations in FKBPL could contribute to the azoospermic phenotype, we examined expression in mouse and human tissues by RNA array blot, RT-PCR and immunohistochemistry and sequenced the complete gene from two azoospermic patient cohorts and matching control groups. FKBPL-AR interaction was assayed using reporter constructs in vitro. Results FKBPL is strongly expressed in mouse testis, with expression upregulated at puberty. The protein is expressed in human testis in a pattern similar to FKBP52 and also enhanced AR transcriptional activity in reporter assays. We examined sixty patients from the Japanese patient group and found one inactivating mutation and one coding change, as well as a number of non-coding changes, all absent in fifty-six controls. A second, Irish patient cohort of thirty showed another two coding changes not present in thirty proven fertile controls. Conclusions Our results describe the first alterations in the gene for FKBPL in azoospermic patients and indicate a potential role in AR-mediated signalling in the testis.

  17. Alterations in the steroid hormone receptor co-chaperone FKBPL are associated with male infertility: a case-control study

    Directory of Open Access Journals (Sweden)

    Barton David

    2010-03-01

    Full Text Available Abstract Background Male infertility is a common cause of reproductive failure in humans. In mice, targeted deletions of the genes coding for FKBP6 or FKBP52, members of the FK506 binding protein family, can result in male infertility. In the case of FKBP52, this reflects an important role in potentiating Androgen Receptor (AR signalling in the prostate and accessory glands, but not the testis. In infertile men, no mutations of FKBP52 or FKBP6 have been found so far, but the gene for FKBP-like (FKBPL maps to chromosome 6p21.3, an area linked to azoospermia in a group of Japanese patients. Methods To determine whether mutations in FKBPL could contribute to the azoospermic phenotype, we examined expression in mouse and human tissues by RNA array blot, RT-PCR and immunohistochemistry and sequenced the complete gene from two azoospermic patient cohorts and matching control groups. FKBPL-AR interaction was assayed using reporter constructs in vitro. Results FKBPL is strongly expressed in mouse testis, with expression upregulated at puberty. The protein is expressed in human testis in a pattern similar to FKBP52 and also enhanced AR transcriptional activity in reporter assays. We examined sixty patients from the Japanese patient group and found one inactivating mutation and one coding change, as well as a number of non-coding changes, all absent in fifty-six controls. A second, Irish patient cohort of thirty showed another two coding changes not present in thirty proven fertile controls. Conclusions Our results describe the first alterations in the gene for FKBPL in azoospermic patients and indicate a potential role in AR-mediated signalling in the testis.

  18. Residues Phe103 and Phe149 are critical for the co-chaperone activity of Bacillus licheniformis GrpE.

    Science.gov (United States)

    Lin, Min-Guan; Chi, Meng-Chun; Chen, Bo-En; Wang, Tzu-Fan; Lo, Huei-Fen; Lin, Long-Liu

    2015-01-01

    A tryptophan-free Bacillus licheniformis nucleotide exchange factor (BlGrpE) and its Trp mutants (F70W, F103W, F149W, F70/103W, F70/149W, F103/149W and F70/103/149W) were over-expressed and purified to near homogeneity. Simultaneous addition of B. licheniformis DnaJ, NR-peptide and individual variants synergistically stimulated the ATPase activity of a recombinant DnaK (BlDnaK) from the same bacterium by 3.1-14.7-fold, which are significantly lower than the synergistic stimulation (18.9-fold) of BlGrpE. Protein-protein interaction analysis revealed that Trp mutants relevant to amino acid positions 103 and 149 lost the ability to bind BlDnaK. Circular dichroism measurements indicate that F70W displayed a comparable level of secondary structure to that of BlGrpE, and the wild-type protein and the Trp mutants as well all experienced a reversible behavior of thermal denaturation. Guanidine hydrochloride (GdnHCl)-induced unfolding transition for BlGrpE was calculated to be 1.25 M corresponding to ΔG(N-U) of 4.29 kcal/mol, whereas the unfolding transitions of mutant proteins were in the range of 0.77-1.31 M equivalent to ΔG(N-U) of 2.41-4.14 kcal/mol. Taken together, the introduction of tryptophan residue, especially at positions 103 and 149, into the primary structure of BlGrpE has been proven to be detrimental to structural integrity and proper function of the protein. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Enhancements for a Dynamic Data Warehousing and Mining System for Large-scale HSCB Data

    Science.gov (United States)

    2016-07-20

    Development of YouTube Analytics and UI ................................................... 2 1.1.1 YouTube Top K Statistics Computation and View...2 1.1.2 YouTube Timeline View ............................................................................... 3...1.1.3 YouTube Media Gallery View ..................................................................... 3 1.1.4 YouTube Named Entity Recognition (NER

  20. Enhancements for a Dynamic Data Warehousing and Mining System for Large-scale HSCB Data

    Science.gov (United States)

    2016-06-20

    seamlessly. A representative UI is shown in Figure 1. The UI has the same look and feel with other social media searches, Page | 4...released it part of Scraawl. In particular, we have developed two APIs for continuous (Streaming) and recent YouTube data searches, and designed a...allows searching on recent YouTube videos, and the resulting data will be saved in the configured database . Both APIs make use of the (i

  1. Enhancements for a Dynamic Data Warehousing and Mining System for Large-Scale HSCB Data

    Science.gov (United States)

    2016-07-20

    geolocation information about YouTube videos. In particular, we show geo-coded and geo-referenced YouTube posts. Geo-coded posts are those which...the Next Reporting Period In the next report period, we will focus on the following tasks:  We will develop an API for news feed collection.  We will deliver Scraawl 1.17.

  2. 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.

  3. Co-chaperone BAG2 Determines the Pro-oncogenic Role of Cathepsin B in Triple-Negative Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kyung-Min Yang

    2017-12-01

    Full Text Available Summary: Triple-negative breast cancer (TNBC is considered incurable with currently available treatments, highlighting the need for therapeutic targets and predictive biomarkers. Here, we report a unique role for Bcl-2-associated athanogene 2 (BAG2, which is significantly overexpressed in TNBC, in regulating the dual functions of cathepsin B as either a pro- or anti-oncogenic enzyme. Silencing BAG2 suppresses tumorigenesis and lung metastasis and induces apoptosis by increasing the intracellular mature form of cathepsin B, whereas BAG2 expression induces metastasis by blocking the auto-cleavage processing of pro-cathepsin B via interaction with the propeptide region. BAG2 regulates pro-cathepsin B/annexin II complex formation and facilitates the trafficking of pro-cathespin-B-containing TGN38-positive vesicles toward the cell periphery, leading to the secretion of pro-cathepsin B, which induces metastasis. Collectively, our results uncover BAG2 as a regulator of the oncogenic function of pro-cathepsin B and a potential diagnostic and therapeutic target that may reduce the burden of metastatic breast cancer. : The mechanisms controlling the pro- and anti-oncogenic roles of cathepsin B are unclear. Yang et al. find that BAG2 is a regulator of the dual functions of its client protein, CTSB, facilitating the progression of TNBC. Keywords: BAG2, cathepsin B, TNBC, tumorigenesis, metastasis, breast cancer, TGN38

  4. Linear ion-trap mass spectrometric characterization of human pituitary nitrotyrosine-containing proteins

    Science.gov (United States)

    Zhan, Xianquan; Desiderio, Dominic M.

    2007-01-01

    The nitric oxide-mediated Tyr-nitration of endogenous proteins is associated with several pathological and physiological processes. In order to investigate the presence - and potential roles - of Tyr-nitration in the human pituitary, a large-format two-dimensional gel separation plus a Western blot against a specific anti-3-nitrotyrosine antibody were used to separate and detect nitroproteins from a human pituitary proteome. The nitroproteins were subjected to in-gel trypsin digestion, and high-sensitivity vacuum matrix-assisted laser desorption/ionization (vMALDI) linear ion-trap tandem mass spectrometry was used to analyze the tryptic peptides. Those MS/MS data were used to determine the amino acid sequence and the specific nitration site of each tryptic nitropeptide, and were matched to corresponding proteins with Bioworks TuboSEQUEST software. Compared to our previous study, 16 new nitrotyrosine-immunoreactive positive Western blot spots were found within the area pI 3.0-10 and Mr 10-100 kDa. Four new nitroproteins were discovered: the stanniocalcin 1 precursor--involved in calcium and phosphate metabolism; mitochondrial co-chaperone protein HscB, which might act as a co-chaperone in iron-sulfur cluster assembly in mitochrondria; progestin and adipoQ receptor family member III--a seven-transmembrane receptor; proteasome subunit alpha type 2--involved in an ATP/ubiquitin-dependent non-lysosomal proteolytic pathway. Those data demonstrate that nitric oxide-mediated Tyr-nitration might participate in various biochemical, metabolic, and pathological processes in the human pituitary.

  5. Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels.

    Science.gov (United States)

    Hantouche, Christine; Williamson, Brittany; Valinsky, William C; Solomon, Joshua; Shrier, Alvin; Young, Jason C

    2017-02-10

    Cardiac long QT syndrome type 2 is caused by mutations in the human ether a go-go-related gene (hERG) potassium channel, many of which cause misfolding and degradation at the endoplasmic reticulum instead of normal trafficking to the cell surface. The Hsc70/Hsp70 chaperones assist the folding of the hERG cytosolic domains. Here, we demonstrate that the Hsp70 nucleotide exchange factor Bag1 promotes hERG degradation by the ubiquitin-proteasome system at the endoplasmic reticulum to regulate hERG levels and channel activity. Dissociation of hERG complexes containing Hsp70 and the E3 ubiquitin ligase CHIP requires the interaction of Bag1 with Hsp70, but this does not involve the Bag1 ubiquitin-like domain. The interaction with Bag1 then shifts hERG degradation to the membrane-anchored E3 ligase TRC8 and its E2-conjugating enzyme Ube2g2, as determined by siRNA screening. TRC8 interacts through the transmembrane region with hERG and decreases hERG functional expression. TRC8 also mediates degradation of the misfolded hERG-G601S disease mutant, but pharmacological stabilization of the mutant structure prevents degradation. Our results identify TRC8 as a previously unknown Hsp70-independent quality control E3 ligase for hERG. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. DTIC Review: Human, Social, Cultural and Behavior Modeling. Volume 9, Number 1 (CD-ROM)

    National Research Council Canada - National Science Library

    2008-01-01

    ...: Human, Social, Cultural and Behavior (HSCB) models are designed to help understand the structure, interconnections, dependencies, behavior, and trends associated with any collection of individuals...

  7. Sequence Classification: 893601 [

    Lifescience Database Archive (English)

    Full Text Available the mitochondrial import receptor Tom70p for preprotein delivery; interacts with co-chaperones Cns1p, Cpr6p, Cpr7p, and Sti1p; Hsp82p || http://www.ncbi.nlm.nih.gov/protein/6325016 ...

  8. Tombusvirus-yeast interactions identify conserved cell-intrinsic viral restriction factors

    Directory of Open Access Journals (Sweden)

    Zsuzsanna eSasvari

    2014-08-01

    Full Text Available To combat viral infections, plants possess innate and adaptive immune pathways, such as RNA silencing, R gene and recessive gene-mediated resistance mechanisms. However, it is likely that additional cell-intrinsic restriction factors (CIRF are also involved in limiting plant virus replication. This review discusses novel CIRFs with antiviral functions, many of them RNA-binding proteins or affecting the RNA binding activities of viral replication proteins. The CIRFs against tombusviruses have been identified in yeast (Saccharomyces cerevisiae, which is developed as an advanced model organism. Grouping of the identified CIRFs based on their known cellular functions and subcellular localization in yeast reveals that TBSV replication is limited by a wide variety of host gene functions. Yeast proteins with the highest connectivity in the network map include the well-characterized Xrn1p 5’-3’ exoribonuclease, Act1p actin protein and Cse4p centromere protein. The protein network map also reveals an important interplay between the pro-viral Hsp70 cellular chaperone and the antiviral co-chaperones, and possibly key roles for the ribosomal or ribosome-associated factors. We discuss the antiviral functions of selected CIRFs, such as the RNA binding nucleolin, ribonucleases, WW-domain proteins, single- and multi-domain cyclophilins, TPR-domain co-chaperones and cellular ion pumps. These restriction factors frequently target the RNA-binding region in the viral replication proteins, thus interfering with the recruitment of the viral RNA for replication and the assembly of the membrane-bound viral replicase. Although many of the characterized CIRFs act directly against TBSV, we propose that the TPR-domain co-chaperones function as guardians of the cellular Hsp70 chaperone system, which is subverted efficiently by TBSV for viral replicase assembly in the absence of the TPR-domain co-chaperones.

  9. Histone chaperone networks shaping chromatin function

    DEFF Research Database (Denmark)

    Hammond, Colin; Strømme, Caroline Bianchi; Huang, Hongda

    2017-01-01

    and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone...... chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin....

  10. Primary fibroblasts from CSP? mutation carriers recapitulate hallmarks of the adult onset neuronal ceroid lipofuscinosis

    OpenAIRE

    Benitez, Bruno A.; Sands, Mark S.

    2017-01-01

    Mutations in the co- chaperone protein, CSP?, cause an autosomal dominant, adult-neuronal ceroid lipofuscinosis (AD-ANCL). The current understanding of CSP? function exclusively at the synapse fails to explain the autophagy-lysosome pathway (ALP) dysfunction in cells from AD-ANCL patients. Here, we demonstrate unexpectedly that primary dermal fibroblasts from pre-symptomatic mutation carriers recapitulate in vitro features found in the brains of AD-ANCL patients including auto-fluorescent sto...

  11. Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Adélle Burger

    2014-01-01

    Full Text Available The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70 is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.

  12. 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.

  13. WW domain of BAG3 is required for the induction of autophagy in glioma cells

    OpenAIRE

    Merabova, Nana; Sariyer, Ilker Kudret; Saribas, A Sami; Knezevic, Tijana; Gordon, Jennifer; Weaver, Michael; Landry, Jacques; Khalili, Kamel

    2015-01-01

    Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co-chaperone family of proteins that associate with Hsp70 through a conserved BAG domain positioned near the C-terminus of the protein. Expression of BAG3 is induced by a variety of e...

  14. BAG3 promotes stem cell-like phenotype in breast cancer by upregulation of CXCR4 via interaction with its transcript

    OpenAIRE

    Liu, Bao-Qin; Zhang, Song; Li, Si; An, Ming-Xin; Li, Chao; Yan, Jing; Wang, Jia-Mei; Wang, Hua-Qin

    2017-01-01

    BAG3 is an evolutionarily conserved co-chaperone expressed at high levels and has a prosurvival role in many tumor types. The current study reported that BAG3 was induced under specific floating culture conditions that enrich breast cancer stem cell (BCSC)-like cells in spheres. Ectopic BAG3 overexpression increased CD44+/CD24? CSC subpopulations, first-generation and second-generation mammosphere formation, indicating that BAG3 promotes CSC self-renewal and maintenance in breast cancer. We f...

  15. Hsp90: Friends, clients and natural foes.

    Science.gov (United States)

    Verma, Sharad; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-08-01

    Hsp90, a homodimeric ATPase, is responsible for the correct folding of a number of newly synthesized polypeptides in addition to the correct folding of denatured/misfolded client proteins. It requires several co-chaperones and other partner proteins for chaperone activity. Due to the involvement of Hsp90-dependent client proteins in a variety of oncogenic signaling pathways, Hsp90 inhibition has emerged as one of the leading strategies for anticancer chemotherapeutics. Most of Hsp90 inhibitors blocks the N terminal ATP binding pocket and prevents the conformational changes which are essential for the loading of co-chaperones and client proteins. Several other inhibitors have also been reported which disrupt chaperone cycle in ways other than binding to N terminal ATP binding pocket. The Hsp90 inhibition is associated with heat shock response, mediated by HSF-1, to overcome the loss of Hsp90 and sustain cell survival. This review is an attempt to give an over view of all the important players of chaperone cycle. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  16. How to orient the functional GroEL-SR1 mutant for atomic force microscopy investigations

    International Nuclear Information System (INIS)

    Schiener, Jens; Witt, Susanne; Hayer-Hartl, Manajit; Guckenberger, Reinhard

    2005-01-01

    We present high-resolution atomic force microscopy (AFM) imaging of the single-ring mutant of the chaperonin GroEL (SR-EL) from Escherichia coli in buffer solution. The native GroEL is generally unsuitable for AFM scanning as it is easily being bisected by forces exerted by the AFM tip. The single-ring mutant of GroEL with its simplified composition, but unaltered capability of binding substrates and the co-chaperone GroES, is a more suited system for AFM studies. We worked out a scheme to systematically investigate both the apical and the equatorial faces of SR-EL, as it binds in a preferred orientation to hydrophilic mica and hydrophobic highly ordered pyrolytic graphite. High-resolution topographical imaging and the interaction of the co-chaperone GroES were used to assign the orientations of SR-EL in comparison with the physically bisected GroEL. The usage of SR-EL facilitates single molecule studies on the folding cycle of the GroE system using AFM

  17. Sequence analyses reveal that a TPR-DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR-DP domains and prokaryotic GerD proteins.

    Science.gov (United States)

    Hernández Torres, Jorge; Papandreou, Nikolaos; Chomilier, Jacques

    2009-05-01

    The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR-DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR-DP domains.

  18. Sequence analyses reveal that a TPR–DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR–DP domains and prokaryotic GerD proteins

    Science.gov (United States)

    Papandreou, Nikolaos; Chomilier, Jacques

    2008-01-01

    The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR–DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR–DP domains. Electronic supplementary material The online version of this article (doi:10.1007/s12192-008-0083-8) contains supplementary material, which is available to authorized users. PMID:18987995

  19. Complex formation of CdSe/ZnS/TOPO nanocrystal vs. molecular chaperone in aqueous solution by hydrophobic interaction

    International Nuclear Information System (INIS)

    Horiuchi, Hiromi; Iwami, Noriya; Tachibana, Fumi; Ohtaki, Akashi; Iizuka, Ryo; Zako, Tamotsu; Oda, Masaru; Yohda, Masafumi; Tani, Toshiro

    2007-01-01

    Feasibilities to stabilize CdSe/ZnS/trioctylphosphineoxide (TOPO) nanocrystals (quantum dots, QDs) in aqueous solutions with prefoldin macromolecules in their bioactive states are reported. Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. As a protein folding intermediate is captured within its central cavity, so CdSe/ZnS/TOPO QDs would also be included within this cavity. It is also found the QDs can be much more dispersed in aqueous solutions and suspended for certain period of time by adding trace amount of t-butanol in the buffer prior to the mixing of the QDs mother solution. While biochemical procedures are evaluated with ordinary fluorescence measurements, possible complex formations are also evaluated with TIRFM single-molecule detection techniques

  20. Cellular Handling of Protein Aggregates by Disaggregation Machines.

    Science.gov (United States)

    Mogk, Axel; Bukau, Bernd; Kampinga, Harm H

    2018-01-18

    Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Review: The HSP90 molecular chaperone-an enigmatic ATPase.

    Science.gov (United States)

    Pearl, Laurence H

    2016-08-01

    The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. © 2016 The Authors. Biopolymers Published by Wiley Periodicals, Inc.

  2. Dual inhibition of chaperoning process by taxifolin: molecular dynamics simulation study.

    Science.gov (United States)

    Verma, Sharad; Singh, Amit; Mishra, Abha

    2012-07-01

    Hsp90 (heat shock protein 90), a molecular chaperone, stabilizes more than 200 mutated and over expressed oncogenic proteins in cancer development. Cdc37 (cell division cycle protein 37), a co-chaperone of Hsp90, has been found to facilitate the maturation of protein kinases by acting as an adaptor and load these kinases onto the Hsp90 complex. Taxifolin (a natural phytochemical) was found to bind at ATP-binding site of Hsp90 and stabilized the inactive "open" or "lid-up" conformation as evidenced by molecular dynamic simulation. Furthermore, taxifolin was found to bind to interface of Hsp90 and Cdc37 complex and disrupt the interaction of residues of both proteins which were essential for the formation of active super-chaperone complex. Thus, taxifolin was found to act as an inhibitor of chaperoning process and may play a potential role in the cancer chemotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Hsp90 molecular chaperone: structure, functions and participation in cardio-vascular pathologies

    Directory of Open Access Journals (Sweden)

    Kroupskaya I. V.

    2009-10-01

    Full Text Available The review is devoted to the analysis of structural and functional properties of molecular chaperon Hsp90. Hsp90 is a representative of highly widespread family of heat shock proteins. The protein is found in eubacteria and all branches of eukarya, but it is apparently absent in archaea. It is one of key regulators of numerous signalling pathways, cell growth and development, apoptosis, induction of autoimmunity, and progression of heart failure. The full functional activity of Hsp90 shows up in a complex with other molecular chaperones and co-chaperones. Molecular interactions between chaperones, different signalling proteins and protein-partners are highly crucial for the normal functioning of signalling pathways and their destruction causes an alteration in the cell physiology up to its death.

  4. E. coli Fis protein insulates the cbpA gene from uncontrolled transcription.

    Science.gov (United States)

    Chintakayala, Kiran; Singh, Shivani S; Rossiter, Amanda E; Shahapure, Rajesh; Dame, Remus T; Grainger, David C

    2013-01-01

    The Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and co-chaperone. It is expressed at high levels as cells enter stationary phase. Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. We show that Fis, the dominant growth-phase nucleoid protein, prevents CbpA expression in growing cells. Regulation by Fis involves an unusual "insulation" mechanism. Thus, Fis protects cbpA from the effects of a distal promoter, located in an adjacent gene. In stationary phase, when Fis levels are low, CbpA binds the E. coli chromosome with a preference for the intrinsically curved Ter macrodomain. Disruption of the cbpA gene prompts dramatic changes in DNA topology. Thus, our work identifies a novel role for Fis and incorporates CbpA into the growing network of factors that mediate bacterial chromosome structure.

  5. Expression of Anti-apoptotic Protein BAG3 in Human Sebaceous Gland Carcinoma of the Eyelid.

    Science.gov (United States)

    Yunoki, Tatsuya; Tabuchi, Yoshiaki; Hayashi, Atsushi

    2017-04-01

    Bcl-2-associated athanogene 3 (BAG3), a co-chaperone of heat shock protein 70 (HSP70), has been shown to play a role in anti-apoptosis of various malignant tumors. In this study, the expression of BAG3 was examined in human sebaceous gland carcinoma of the eyelid. The expression of BAG3 was evaluated by immunohistochemistry of surgical samples from 5 patients with sebaceous gland carcinoma in the eyelid. BAG3 was positive diffusely in the cytoplasm in all patients. The average positive rate of BAG3 was 73.0±26.0% in tumor cells of all patients. BAG3 was highly expressed in sebaceous gland carcinoma of the eyelid. BAG3 may play an important role in the pathogenesis and progression of sebaceous gland carcinoma of the eyelid. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  6. Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

    Science.gov (United States)

    Martínez-Alonso, Mónica; Villaverde, Antonio

    2010-01-01

    Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

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

    Energy Technology Data Exchange (ETDEWEB)

    Grover, Abhinav [Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology (IIT) Delhi, Hauz Khas, New Delhi 110016 (India); Shandilya, Ashutosh [Supercomputing Facility for Bioinformatics and Computational Biology, Indian Institute of Technology (IIT) Delhi, Hauz Khas, New Delhi 110016 (India); Agrawal, Vibhuti; Pratik, Piyush; Bhasme, Divya; Bisaria, Virendra S. [Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology (IIT) Delhi, Hauz Khas, New Delhi 110016 (India); Sundar, Durai, E-mail: sundar@dbeb.iitd.ac.in [Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology (IIT) Delhi, Hauz Khas, New Delhi 110016 (India)

    2011-01-07

    Research highlights: {yields} Withaferin A and 17-DMAG synergistically inhibit the Hsp90-Cdc37 chaperone pair. {yields} Binding of WA to Cdc37 cleft suppresses its kinase binding activity. {yields} 17-DMAG binding to the association complex results in H-bonds with 60% clustering. {yields} 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.

  8. Arabidopsis AtDjA3 null mutant shows increased sensitivity to abscisic acid, salt, and osmotic stress in germination and postgermination stages

    Directory of Open Access Journals (Sweden)

    Silvia eSalas-Muñoz

    2016-02-01

    Full Text Available DnaJ proteins are essential co-chaperones involved in abiotic and biotic stress responses. Arabidopsis AtDjA3 gene encodes a molecular co-chaperone of 420 amino acids, which belongs to the J-protein family. In this study, we report the functional characterization of the AtDjA3 gene using the Arabidopsis knockout line designated j3 and the 35S::AtDjA3 overexpression lines. Loss of AtDjA3 function was associated with small seed production. In fact, j3 mutant seeds showed a reduction of 24% in seed weight compared to Col-0 seeds. Expression analysis showed that the AtDjA3 gene was modulated in response to NaCl, glucose, and abscisic acid. The j3 line had increased sensitivity to NaCl and glucose treatments in the germination and cotyledon development in comparison to parental Col-0. Furthermore, the j3 mutant line exhibited higher abscisic acid sensitivity in comparison to parental Col-0 and 35S::AtDjA3 overexpression lines. In addition, we examined the expression of ABI3 gene, which is a central regulator in ABA signalling, in j3 mutant and 35S::AtDjA3 overexpression lines. Under 5 μM ABA treatment at 24 h, j3 mutant seedlings displayed higher ABI3 expression, whereas in 35S::AtDjA3 overexpression lines, ABI3 gene expression was repressed. Taken together, these results demonstrate that the AtDjA3 gene is involved in seed development and abiotic stress tolerance.

  9. Interaction of the iron–sulfur cluster assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of Escherichia coli

    Science.gov (United States)

    Hoff, Kevin G.; Silberg, Jonathan J.; Vickery, Larry E.

    2000-01-01

    The iscU gene in bacteria is located in a gene cluster encoding proteins implicated in iron–sulfur cluster assembly and an hsc70-type (heat shock cognate) molecular chaperone system, iscSUA-hscBA. To investigate possible interactions between these systems, we have overproduced and purified the IscU protein from Escherichia coli and have studied its interactions with the hscA and hscB gene products Hsc66 and Hsc20. IscU and its iron–sulfur complex (IscU–Fe/S) stimulated the basal steady-state ATPase activity of Hsc66 weakly in the absence of Hsc20 but, in the presence of Hsc20, increased the ATPase activity up to 480-fold. Hsc20 also decreased the apparent Km for IscU stimulation of Hsc66 ATPase activity, and surface plasmon resonance studies revealed that Hsc20 enhances binding of IscU to Hsc66. Surface plasmon resonance and isothermal titration calorimetry further showed that IscU and Hsc20 form a complex, and Hsc20 may thereby aid in the targeting of IscU to Hsc66. These results establish a direct and specific role for the Hsc66/Hsc20 chaperone system in functioning with isc gene components for the assembly of iron–sulfur cluster proteins. PMID:10869428

  10. Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

    Directory of Open Access Journals (Sweden)

    Trevor M. Morey

    2017-12-01

    Full Text Available Choline acetyltransferase (ChAT synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic syndrome (CMS. One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues 14PKLPVPP20. We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A or mutation of residue Val18 (V18M enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID, co-immunoprecipitation and in situ proximity-ligation assay (PLA, we identified the heat shock proteins (HSPs HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP in situ, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms

  11. Role of the HSPA9/HSC20 chaperone pair in promoting directional human iron-sulfur cluster exchange involving monothiol glutaredoxin 5.

    Science.gov (United States)

    Olive, Joshua A; Cowan, J A

    2018-07-01

    Iron‑sulfur clusters are essential cofactors found across all domains of life. Their assembly and transfer are accomplished by highly conserved protein complexes and partners. In eukaryotes a [2Fe-2S] cluster is first assembled in the mitochondria on the iron‑sulfur cluster scaffold protein ISCU in tandem with iron, sulfide, and electron donors. Current models suggest that a chaperone pair interacts with a cluster-bound ISCU to facilitate cluster transfer to a monothiol glutaredoxin. In humans this protein is glutaredoxin 5 (GLRX5) and the cluster can then be exchanged with a variety of target apo proteins. By use of circular dichroism spectroscopy, the kinetics of cluster exchange reactivity has been evaluated for human GLRX5 with a variety of cluster donor and acceptor partners, and the role of chaperones determined for several of these. In contrast to the prokaryotic model, where heat-shock type chaperone proteins HscA and HscB are required for successful and efficient transfer of a [2Fe-2S] cluster from the ISCU scaffold to a monothiol glutaredoxin. However, in the human system the chaperone homologs, HSPA9 and HSC20, are not necessary for human ISCU to promote cluster transfer to GLRX5, and appear to promote the reverse transfer. Cluster exchange with the human iron‑sulfur cluster carrier protein NFU1 and ferredoxins (FDX's), and the role of chaperones, has also been evaluated, demonstrating in certain cases control over the directionality of cluster transfer. In contrast to other prokaryotic and eukaryotic organisms, NFU1 is identified as a more likely physiological donor of [2Fe-2S] cluster to human GLRX5 than ISCU. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. 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.

  13. Missing Links in Antibody Assembly Control

    Directory of Open Access Journals (Sweden)

    Tiziana Anelli

    2013-01-01

    Full Text Available Fidelity of the humoral immune response requires that quiescent B lymphocytes display membrane bound immunoglobulin M (IgM on B lymphocytes surface as part of the B cell receptor, whose function is to recognize an antigen. At the same time B lymphocytes should not secrete IgM until recognition of the antigen has occurred. The heavy chains of the secretory IgM have a C-terminal tail with a cysteine instead of a membrane anchor, which serves to covalently link the IgM subunits by disulfide bonds to form “pentamers” or “hexamers.” By virtue of the same cysteine, unassembled secretory IgM subunits are recognized and retained (via mixed disulfide bonds by members of the protein disulfide isomerase family, in particular ERp44. This so-called “thiol-mediated retention” bars assembly intermediates from prematurely leaving the cell and thereby exerts quality control on the humoral immune response. In this essay we discuss recent findings on how ERp44 governs such assembly control in a pH-dependent manner, shuttling between the cisGolgi and endoplasmic reticulum, and finally on how pERp1/MZB1, possibly as a co-chaperone of GRP94, may help to overrule the thiol-mediated retention in the activated B cell to give way to antibody secretion.

  14. The Stoichiometric Interaction of the Hsp90-Sgt1-Rar1 Complex by CD and SRCD Spectroscopy

    Directory of Open Access Journals (Sweden)

    Giuliano Siligardi

    2018-01-01

    Full Text Available While the molecular details by which Hsp90 interacts with Sgt1 and Rar1 were previously described the exact stoichiometric complex that is formed remains elusive. Several possibilities remain that include two asymmetric complexes, Sgt12-Hsp902-Rar12 (two molecules of Sgt1 and Rar1 and one Hsp90 dimer or Sgt12-Hsp902-Rar11 (with a single Rar1 molecule and an asymmetric complex (Sgt11-Hsp902-Rar11. The Hsp90-mediated activation of NLR receptors (Nucleotide-binding domain and Leucine-rich Repeat in the innate immunity of both plants and animals is dependent on the co-chaperone Sgt1 and in plants on Rar1, a cysteine- and histidine-rich domain (CHORD-containing protein. The exact stoichiometry of such a complex may have a direct impact on NLR protein oligomerization and thus ultimately on the mechanism by which NLRs are activated. CD spectroscopy was successfully used to determine the stoichiometry of a ternary protein complex among Hsp90, Sgt1, and Rar1 in the presence of excess ADP. The results indicated that a symmetric Sgt12-Hsp902-Rar11 complex was formed that could allow two NLR molecules to simultaneously bind. The stoichiometry of this complex has implications on, and might promote, the dimerization of NLR proteins following their activation.

  15. Progranulin Recruits HSP70 to β-Glucocerebrosidase and Is Therapeutic Against Gaucher Disease.

    Science.gov (United States)

    Jian, Jinlong; Tian, Qing-Yun; Hettinghouse, Aubryanna; Zhao, Shuai; Liu, Helen; Wei, Jianlu; Grunig, Gabriele; Zhang, Wujuan; Setchell, Kenneth D R; Sun, Ying; Overkleeft, Herman S; Chan, Gerald L; Liu, Chuan-Ju

    2016-11-01

    Gaucher disease (GD), the most common lysosomal storage disease, is caused by mutations in GBA1 encoding of β-glucocerebrosidase (GCase). Recently it was reported that progranulin (PGRN) insufficiency and deficiency associated with GD in human and mice, respectively. However the underlying mechanisms remain unknown. Here we report that PGRN binds directly to GCase and its deficiency results in aggregation of GCase and its receptor LIMP2. Mass spectrometry approaches identified HSP70 as a GCase/LIMP2 complex-associated protein upon stress, with PGRN as an indispensable adaptor. Additionally, 98 amino acids of C-terminal PGRN, referred to as Pcgin, are required and sufficient for the binding to GCase and HSP70. Pcgin effectively ameliorates the disease phenotype in GD patient fibroblasts and animal models. These findings not only demonstrate that PGRN is a co-chaperone of HSP70 and plays an important role in GCase lysosomal localization, but may also provide new therapeutic interventions for lysosomal storage diseases, in particular GD. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  16. RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation.

    LENUS (Irish Health Repository)

    Walsh, Naomi

    2011-07-28

    We previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70\\/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered. In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours. Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

  17. Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2

    Science.gov (United States)

    Rao, Feng; Cha, Jiyoung; Xu, Jing; Xu, Risheng; Vandiver, M. Scott; Tyagi, Richa; Tokhunts, Robert; Koldobskiy, Michael A.; Fu, Chenglai; Barrow, Roxanne; Wu, Mingxuan; Fiedler, Dorothea; Barrow, James C.; Snyder, Solomon H.

    2014-01-01

    The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) co-chaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks) of which IP6K2 has been implicated in p53-associated cell death. In the present study we report a novel apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine-15 to activate the cell death program in human cancer cells and in murine B cells. PMID:24657168

  18. Phylogeny-dominant classification of J-proteins in Arabidopsis thaliana and Brassica oleracea.

    Science.gov (United States)

    Zhang, Bin; Qiu, Han-Lin; Qu, Dong-Hai; Ruan, Ying; Chen, Dong-Hong

    2018-04-05

    Hsp40s or DnaJ/J-proteins are evolutionarily conserved in all organisms as co-chaperones of molecular chaperone HSP70s that mainly participate in maintaining cellular protein homeostasis, such as protein folding, assembly, stabilization, and translocation under normal conditions as well as refolding and degradation under environmental stresses. It has been reported that Arabidopsis J-proteins are classified into four classes (types A-D) according to domain organization, but their phylogenetic relationships are unknown. Here, we identified 129 J-proteins in the world-wide popular vegetable Brassica oleracea, a close relative of the model plant Arabidopsis, and also revised the information of Arabidopsis J-proteins based on the latest online bioresources. According to phylogenetic analysis with domain organization and gene structure as references, the J-proteins from Arabidopsis and B. oleracea were classified into 15 main clades (I-XV) separated by a number of undefined small branches with remote relationship. Based on the number of members, they respectively belong to multigene clades, oligo-gene clades, and mono-gene clades. The J-protein genes from different clades may function together or separately to constitute a complicated regulatory network. This study provides a constructive viewpoint for J-protein classification and an informative platform for further functional dissection and resistant genes discovery related to genetic improvement of crop plants.

  19. The AAA+ proteins Pontin and Reptin enter adult age: from understanding their basic biology to the identification of selective inhibitors.

    Science.gov (United States)

    Matias, Pedro M; Baek, Sung Hee; Bandeiras, Tiago M; Dutta, Anindya; Houry, Walid A; Llorca, Oscar; Rosenbaum, Jean

    2015-01-01

    Pontin and Reptin are related partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They are implicated in multiple and seemingly unrelated processes encompassing the regulation of gene transcription, the remodeling of chromatin, DNA damage sensing and repair, and the assembly of protein and ribonucleoprotein complexes, among others. The 2nd International Workshop on Pontin and Reptin took place at the Instituto de Tecnologia Química e Biológica António Xavier in Oeiras, Portugal on October 10-12, 2014, and reported significant new advances on the mechanisms of action of these two AAA+ ATPases. The major points under discussion were related to the mechanisms through which these proteins regulate gene transcription, their roles as co-chaperones, and their involvement in pathophysiology, especially in cancer and ciliary biology and disease. Finally, they may become anticancer drug targets since small chemical inhibitors were shown to produce anti-tumor effects in animal models.

  20. Relationship of the FKBP5 C/T polymorphism with dysfunctional attitudes predisposing to depression.

    Science.gov (United States)

    Suzuki, Akihito; Matsumoto, Yoshihiko; Sadahiro, Ryoichi; Enokido, Masanori; Goto, Kaoru; Otani, Koichi

    2014-08-01

    FK506-binding protein 51 (FKBP5) is a co-chaperone of the glucocorticoid receptor, and plays an important role in the negative feedback regulation of the hypothalamic-pituitary-adrenal axis. The C/T single nucleotide polymorphism in the intron 2 of the FKBP5 gene affects cortisol secretion, and has been implicated in the pathophysiology of depression. In this study, the relationship of the FKBP5 C/T polymorphism with dysfunctional attitudes predisposing to depression was examined. The subjects were 300 healthy Japanese. The FKBP5 genotypes were determined by a real-time PCR and cycling probe technology for SNP typing. Dysfunctional attitudes were assessed by the 24-item version of the Dysfunctional Attitude Scale (DAS-24), which has the Achievement, Self-control, and Dependency subscales. DAS-24 total scores were significantly higher in the group with the T allele than in that without this allele (p=0.001). Regarding the subscales, scores of the Achievement (p=0.003) and Self-control (p=0.009) subscales, but not those of the Dependency subscale, were significantly higher in the former group than in the latter group. The present study suggests that the FKBP5 C/T polymorphism is implicated in formation of dysfunctional attitudes, especially those about achievement and self-control. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Type I J-domain NbMIP1 proteins are required for both Tobacco mosaic virus infection and plant innate immunity.

    Directory of Open Access Journals (Sweden)

    Yumei Du

    Full Text Available Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV and Tobacco mosaic virus (TMV by recognizing the viral movement protein (MP. Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s associate with tobamovirus MP, Tm-2² and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-2²-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for Tm-2²-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.

  2. Progranulin Recruits HSP70 to β-Glucocerebrosidase and Is Therapeutic Against Gaucher Disease

    Directory of Open Access Journals (Sweden)

    Jinlong Jian

    2016-11-01

    Full Text Available Gaucher disease (GD, the most common lysosomal storage disease, is caused by mutations in GBA1 encoding of β-glucocerebrosidase (GCase. Recently it was reported that progranulin (PGRN insufficiency and deficiency associated with GD in human and mice, respectively. However the underlying mechanisms remain unknown. Here we report that PGRN binds directly to GCase and its deficiency results in aggregation of GCase and its receptor LIMP2. Mass spectrometry approaches identified HSP70 as a GCase/LIMP2 complex-associated protein upon stress, with PGRN as an indispensable adaptor. Additionally, 98 amino acids of C-terminal PGRN, referred to as Pcgin, are required and sufficient for the binding to GCase and HSP70. Pcgin effectively ameliorates the disease phenotype in GD patient fibroblasts and animal models. These findings not only demonstrate that PGRN is a co-chaperone of HSP70 and plays an important role in GCase lysosomal localization, but may also provide new therapeutic interventions for lysosomal storage diseases, in particular GD.

  3. Hop/STI1 modulates retinal proliferation and cell death independent of PrPC

    International Nuclear Information System (INIS)

    Arruda-Carvalho, Maithe; Njaine, Brian; Silveira, Mariana S.; Linden, Rafael; Chiarini, Luciana B.

    2007-01-01

    Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP C ). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP C dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 (α-STI1) blocked both ganglion cell and NBL cell death independent of PrP C . cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while α-STI1 increased proliferation in the developing retina, both independent of PrP C . We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP C

  4. Signaling induced by hop/STI-1 depends on endocytosis

    International Nuclear Information System (INIS)

    Americo, Tatiana A.; Chiarini, Luciana B.; Linden, Rafael

    2007-01-01

    The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP C ), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP C and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling induced by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrP C by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases

  5. Whey protein hydrolysate enhances HSP90 but does not alter HSP60 and HSP25 in skeletal muscle of rats.

    Directory of Open Access Journals (Sweden)

    Carolina Soares Moura

    Full Text Available Whey protein hydrolysate (WPH intake has shown to increase HSP70 expression. The aim of the present study was to investigate whether WPH intake would also influences HSP90, HSP60 and HSP25 expression, as well as associated parameters. Forty-eight male Wistar rats were divided into sedentary (unstressed and exercised (stressed groups, and were fed with three different sources of protein: whey protein (WP, whey protein hydrolysate (WPH and casein (CAS as a control, based on the AIN93G diet for 3 weeks. WPH intake increased HSP90 expression in both sedentary and exercised animals compared to WP or CAS, however no alteration was found from exercise or diet to HSP60 or HSP25. Co-chaperone Aha1 and p-HSF1 were also increased in the exercised animals fed with WPH in comparison with WP or CAS, consistent with enhanced HSP90 expression. VEGF and p-AKT were increased in the WPH exercised group. No alteration was found in BCKDH, PI3-Kinase (p85, GFAT, OGT or PGC for diet or exercise. The antioxidant system GPx, catalase and SOD showed different responses to diet and exercise. The data indicate that WPH intake enhanced factors related to cell survival, such as HSP90 and VEGF, but does not alter HSP60 or HSP25 in rat skeletal muscle.

  6. The AAA+ proteins Pontin and Reptin enter adult age: from understanding their basic biology to the identification of selective inhibitors

    Science.gov (United States)

    Matias, Pedro M.; Baek, Sung Hee; Bandeiras, Tiago M.; Dutta, Anindya; Houry, Walid A.; Llorca, Oscar; Rosenbaum, Jean

    2015-01-01

    Pontin and Reptin are related partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They are implicated in multiple and seemingly unrelated processes encompassing the regulation of gene transcription, the remodeling of chromatin, DNA damage sensing and repair, and the assembly of protein and ribonucleoprotein complexes, among others. The 2nd International Workshop on Pontin and Reptin took place at the Instituto de Tecnologia Química e Biológica António Xavier in Oeiras, Portugal on October 10–12, 2014, and reported significant new advances on the mechanisms of action of these two AAA+ ATPases. The major points under discussion were related to the mechanisms through which these proteins regulate gene transcription, their roles as co-chaperones, and their involvement in pathophysiology, especially in cancer and ciliary biology and disease. Finally, they may become anticancer drug targets since small chemical inhibitors were shown to produce anti-tumor effects in animal models. PMID:25988184

  7. Combined x-ray crystallography and computational modeling approach to investigate the Hsp90 C-terminal peptide binding to FKBP51.

    Science.gov (United States)

    Kumar, Rajnish; Moche, Martin; Winblad, Bengt; Pavlov, Pavel F

    2017-10-27

    FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.

  8. TTT and PIKK Complex Genes Reverted to Single Copy Following Polyploidization and Retain Function Despite Massive Retrotransposition in Maize.

    Science.gov (United States)

    Garcia, Nelson; Messing, Joachim

    2017-01-01

    The TEL2, TTI1, and TTI2 proteins are co-chaperones for heat shock protein 90 (HSP90) to regulate the protein folding and maturation of phosphatidylinositol 3-kinase-related kinases (PIKKs). Referred to as the TTT complex, the genes that encode them are highly conserved from man to maize. TTT complex and PIKK genes exist mostly as single copy genes in organisms where they have been characterized. Members of this interacting protein network in maize were identified and synteny analyses were performed to study their evolution. Similar to other species, there is only one copy of each of these genes in maize which was due to a loss of the duplicated copy created by ancient allotetraploidy. Moreover, the retained copies of the TTT complex and the PIKK genes tolerated extensive retrotransposon insertion in their introns that resulted in increased gene lengths and gene body methylation, without apparent effect in normal gene expression and function. The results raise an interesting question on whether the reversion to single copy was due to selection against deleterious unbalanced gene duplications between members of the complex as predicted by the gene balance hypothesis, or due to neutral loss of extra copies. Uneven alteration of dosage either by adding extra copies or modulating gene expression of complex members is being proposed as a means to investigate whether the data supports the gene balance hypothesis or not.

  9. Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands.

    Science.gov (United States)

    Sattin, Sara; Tao, Jiahui; Vettoretti, Gerolamo; Moroni, Elisabetta; Pennati, Marzia; Lopergolo, Alessia; Morelli, Laura; Bugatti, Antonella; Zuehlke, Abbey; Moses, Mike; Prince, Thomas; Kijima, Toshiki; Beebe, Kristin; Rusnati, Marco; Neckers, Len; Zaffaroni, Nadia; Agard, David A; Bernardi, Anna; Colombo, Giorgio

    2015-09-21

    Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 Å from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules' effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. 2.4 Å resolution crystal structure of human TRAP1 NM , the Hsp90 paralog in the mitochondrial matrix

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Nuri; Lee, Jungsoon; Kim, Ji-Hyun; Chang, Changsoo; Tsai, Francis T. F.; Lee, Sukyeong

    2016-07-13

    TRAP1 is an organelle-specific Hsp90 paralog that is essential for neoplastic growth. As a member of the Hsp90 family, TRAP1 is presumed to be a general chaperone facilitating the late-stage folding of Hsp90 client proteins in the mitochondrial matrix. Interestingly, TRAP1 cannot replace cytosolic Hsp90 in protein folding, and none of the known Hsp90 co-chaperones are found in mitochondria. Thus, the three-dimensional structure of TRAP1 must feature regulatory elements that are essential to the ATPase activity and chaperone function of TRAP1. Here, the crystal structure of a human TRAP1NMdimer is presented, featuring an intact N-domain and M-domain structure, bound to adenosine 5'-β,γ-imidotriphosphate (ADPNP). The crystal structure together with epitope-mapping results shows that the TRAP1 M-domain loop 1 contacts the neighboring subunit and forms a previously unobserved third dimer interface that mediates the specific interaction with mitochondrial Hsp70.

  11. An RNA aptamer specific to Hsp70-ATP conformation inhibits its ATPase activity independent of Hsp40.

    Science.gov (United States)

    Thirunavukarasu, Deepak; Shi, Hua

    2015-04-01

    The highly conserved and ubiquitous molecular chaperone heat shock protein 70 (Hsp70) plays a critical role in protein homeostasis (proteostasis). Controlled by its ATPase activity, Hsp70 cycles between two conformations, Hsp70-ATP and Hsp70-ADP, to bind and release its substrate. Chemical tools with distinct modes of action, especially those capable of modulating the ATPase activity of Hsp70, are being actively sought after in the mechanistic dissection of this system. Here, we report a conformation-specific RNA aptamer that binds only to Hsp70-ATP but not to Hsp70-ADP. We have refined this aptamer and demonstrated its inhibitory effect on Hsp70's ATPase activity. We have also shown that this inhibitory effect on Hsp70 is independent of its interaction with the Hsp40 co-chaperone. As Hsp70 is increasingly being recognized as a drug target in a number of age related diseases such as neurodegenerative, protein misfolding diseases and cancer, this aptamer is potentially useful in therapeutic applications. Moreover, this work also demonstrates the feasibility of using aptamers to target ATPase activity as a general therapeutic strategy.

  12. The effects of amino acid composition of glutamine-rich domains on amyloid formation and fragmentation.

    Directory of Open Access Journals (Sweden)

    Alexander I Alexandrov

    Full Text Available Fragmentation of amyloid polymers by the chaperone Hsp104 allows them to propagate as prions in yeast. The factors which determine the frequency of fragmentation are unclear, though it is often presumed to depend on the physical strength of prion polymers. Proteins with long polyglutamine stretches represent a tractable model for revealing sequence elements required for polymer fragmentation in yeast, since they form poorly fragmented amyloids. Here we show that interspersion of polyglutamine stretches with various amino acid residues differentially affects the in vivo formation and fragmentation of the respective amyloids. Aromatic residues tyrosine, tryptophan and phenylalanine strongly stimulated polymer fragmentation, leading to the appearance of oligomers as small as dimers. Alanine, methionine, cysteine, serine, threonine and histidine also enhanced fragmentation, while charged residues, proline, glycine and leucine inhibited polymerization. Our data indicate that fragmentation frequency primarily depends on the recognition of fragmentation-promoting residues by Hsp104 and/or its co-chaperones, rather than on the physical stability of polymers. This suggests that differential exposure of such residues to chaperones defines prion variant-specific differences in polymer fragmentation efficiency.

  13. 2.4 Å resolution crystal structure of human TRAP1NM, the Hsp90 paralog in the mitochondrial matrix.

    Science.gov (United States)

    Sung, Nuri; Lee, Jungsoon; Kim, Ji Hyun; Chang, Changsoo; Tsai, Francis T F; Lee, Sukyeong

    2016-08-01

    TRAP1 is an organelle-specific Hsp90 paralog that is essential for neoplastic growth. As a member of the Hsp90 family, TRAP1 is presumed to be a general chaperone facilitating the late-stage folding of Hsp90 client proteins in the mitochondrial matrix. Interestingly, TRAP1 cannot replace cytosolic Hsp90 in protein folding, and none of the known Hsp90 co-chaperones are found in mitochondria. Thus, the three-dimensional structure of TRAP1 must feature regulatory elements that are essential to the ATPase activity and chaperone function of TRAP1. Here, the crystal structure of a human TRAP1NM dimer is presented, featuring an intact N-domain and M-domain structure, bound to adenosine 5'-β,γ-imidotriphosphate (ADPNP). The crystal structure together with epitope-mapping results shows that the TRAP1 M-domain loop 1 contacts the neighboring subunit and forms a previously unobserved third dimer interface that mediates the specific interaction with mitochondrial Hsp70.

  14. Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1.

    Science.gov (United States)

    Dobriyal, Neha; Tripathi, Prerna; Sarkar, Susrita; Tak, Yogesh; Verma, Amit K; Sahi, Chandan

    2017-05-01

    J proteins are obligate co-chaperones of Hsp70s. Via their signature J domain, all J proteins interact with their partner Hsp70s and stimulate their weak ATPase activity, which is vital for Hsp70 functions. The dependency of J proteins on their J domain is such that mutations in critical amino acids in the J domain often results into a null phenotype for a particular J protein. Here, we show that the J domain of Djp1, a cytosolic J protein important for peroxisomal protein import in Saccharomyces cerevisiae, is partially dispensable. A complete deletion of Djp1 J domain resulted into only partial loss in peroxisomal protein import function. Instead, the C-terminal domain of Djp1 was found to be essential for proper localization of the peroxisomal targeted GFP-PTS1. Furthermore, we show that Caj1, another cytosolic J protein, also has some role in peroxisomal protein import. Caj1 was found to be partially redundant with Djp1 as cells lacking both Djp1 and Caj1 resulted into a much more severe defect in GFP-PTS1 localization. Based on these results, we propose that dispensability of J domains could be attributed to genetic redundancy between different J proteins sharing common structural topology and cellular localization.

  15. Molecular characterization of DnaJ 5 homologs in silkworm Bombyx mori and its expression during egg diapause.

    Science.gov (United States)

    Sirigineedi, Sasibhushan; Vijayagowri, Esvaran; Murthy, Geetha N; Rao, Guruprasada; Ponnuvel, Kangayam M

    2014-12-01

    A comparison of the cDNA sequences (1 056 bp) of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog of B. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of Hsp40 gene, and its role in egg diapause induction in B. mori. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  16. Targeting HSP70 and GRP78 in canine osteosarcoma cells in combination with doxorubicin chemotherapy.

    Science.gov (United States)

    Asling, Jonathan; Morrison, Jodi; Mutsaers, Anthony J

    2016-11-01

    Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

  17. Interactive effects of attachment and FKBP5 genotype on school-aged children's emotion regulation and depressive symptoms.

    Science.gov (United States)

    Borelli, Jessica L; Smiley, Patricia A; Rasmussen, Hannah F; Gómez, Anthony; Seaman, Lauren C; Nurmi, Erika L

    2017-05-15

    Attachment insecurity is influenced by both environmental and genetic factors, but few studies have examined the effects of gene-environment interactions. In the context of environmental stress, a functional variant in the glucocorticoid receptor co-chaperone FKBP5 gene has been repeatedly shown to increase risk for psychiatric illness, including depression. We expand on prior work by exploring cross-sectional attachment by gene effects on both attachment insecurity and downstream physiological and behavioral measures in a diverse community sample of school-aged children (N=99, 49% girls, M age =10.29years, 66.6% non-White) and their mothers. Specifically, we examined moderating effects of FKBP5 rs3800373 genotype on the links between parenting insensitivity (overcontrol) and child attachment. Further, we assessed whether FKBP5 moderates the links between maternal and child attachment and children's emotion regulation self-report, respiratory sinus arrhythmia (RSA) in response to a standardized laboratory stressor, and depressive symptoms. Higher levels of overcontrol predicted lower child attachment security only in FKBP5 minor allele carriers. Among children with two minor alleles (CC), attachment security was negatively associated with emotion suppression, rumination, depressive symptoms, and RSA reactivity; similarly, for these children, maternal attachment anxiety was positively associated with depressive symptoms. The findings can be conceptualized in a differential susceptibility framework, where the FKBP5 minor allele confers either risk or resilience, depending on the parenting environment. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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    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.

  19. 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.

  20. TTT and PIKK Complex Genes Reverted to Single Copy Following Polyploidization and Retain Function Despite Massive Retrotransposition in Maize

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    Nelson Garcia

    2017-11-01

    Full Text Available The TEL2, TTI1, and TTI2 proteins are co-chaperones for heat shock protein 90 (HSP90 to regulate the protein folding and maturation of phosphatidylinositol 3-kinase-related kinases (PIKKs. Referred to as the TTT complex, the genes that encode them are highly conserved from man to maize. TTT complex and PIKK genes exist mostly as single copy genes in organisms where they have been characterized. Members of this interacting protein network in maize were identified and synteny analyses were performed to study their evolution. Similar to other species, there is only one copy of each of these genes in maize which was due to a loss of the duplicated copy created by ancient allotetraploidy. Moreover, the retained copies of the TTT complex and the PIKK genes tolerated extensive retrotransposon insertion in their introns that resulted in increased gene lengths and gene body methylation, without apparent effect in normal gene expression and function. The results raise an interesting question on whether the reversion to single copy was due to selection against deleterious unbalanced gene duplications between members of the complex as predicted by the gene balance hypothesis, or due to neutral loss of extra copies. Uneven alteration of dosage either by adding extra copies or modulating gene expression of complex members is being proposed as a means to investigate whether the data supports the gene balance hypothesis or not.

  1. Primary fibroblasts from CSPα mutation carriers recapitulate hallmarks of the adult onset neuronal ceroid lipofuscinosis.

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    Benitez, Bruno A; Sands, Mark S

    2017-07-24

    Mutations in the co- chaperone protein, CSPα, cause an autosomal dominant, adult-neuronal ceroid lipofuscinosis (AD-ANCL). The current understanding of CSPα function exclusively at the synapse fails to explain the autophagy-lysosome pathway (ALP) dysfunction in cells from AD-ANCL patients. Here, we demonstrate unexpectedly that primary dermal fibroblasts from pre-symptomatic mutation carriers recapitulate in vitro features found in the brains of AD-ANCL patients including auto-fluorescent storage material (AFSM) accumulation, CSPα aggregates, increased levels of lysosomal proteins and lysosome enzyme activities. AFSM accumulation correlates with CSPα aggregation and both are susceptible to pharmacological modulation of ALP function. In addition, we demonstrate that endogenous CSPα is present in the lysosome-enriched fractions and co-localizes with lysosome markers in soma, neurites and synaptic boutons. Overexpression of CSPα wild-type (WT) decreases lysotracker signal, secreted lysosomal enzymes and SNAP23-mediated lysosome exocytosis. CSPα WT, mutant and aggregated CSPα are degraded mainly by the ALP but this disease-causing mutation exhibits a faster rate of degradation. Co-expression of both WT and mutant CSPα cause a block in the fusion of autophagosomes/lysosomes. Our data suggest that aggregation-dependent perturbation of ALP function is a relevant pathogenic mechanism for AD-ANCL and supports the use of AFSM or CSPα aggregation as biomarkers for drug screening purposes.

  2. Effect of ARA9 on dioxin receptor mediated transcription

    International Nuclear Information System (INIS)

    Lees, M.J.; Whitelaw, M.L.

    2002-01-01

    The dioxin (Aryl hydrocarbon) receptor (DR) is a unique bHLH transcription factor which is activated by binding of planar aromatic hydrocarbons typified by dioxin (TCDD). The active receptor is key to metabolism of aryl hydrocarbon xenobiotics by being a potent inducer of CYP1A1 gene activity. Chlorinated dioxins are inert to metabolism and initiate multifarious toxicities, including potent tumour promotion. These ill-effects are mediated by the activated DR and we are studying the mechanisms by which the ligand binding domain of the DR controls activity of the protein. The DR ligand binding domain resides within a PAS (Per/Arnt/Sim homology) region which is contiguous with the bHLH. The latent bHLH/PAS dioxin receptor (DR) is found in the cytoplasm of most mammalian cell types in a complex with heat shock protein 90, a novel immunophilin like protein termed ARA9/XAP2/AIP, and the co-chaperone p23. Here we use antisense ARA9 constructs to reveal that in the absence of ARA9, the DR is unable to form a transcriptionally active complex. Co-expression of antisense ARA9 with a form of the DR which is constitutively targeted to the nucleus leads to dramatically decreased levels of the nuclear DR protein, implying that ARA9 may function beyond its currently proposed role in cytoplasmic retention of the latent DR

  3. Reversible thermal transition in GrpE, the nucleotide exchange factor of the DnaK heat-shock system.

    Science.gov (United States)

    Grimshaw, J P; Jelesarov, I; Schönfeld, H J; Christen, P

    2001-03-02

    DnaK, a Hsp70 acting in concert with its co-chaperones DnaJ and GrpE, is essential for Escherichia coli to survive environmental stress, including exposure to elevated temperatures. Here we explored the influence of temperature on the structure of the individual components and the functional properties of the chaperone system. GrpE undergoes extensive but fully reversible conformational changes in the physiologically relevant temperature range (transition midpoint at approximately 48 degrees C), as observed with both circular dichroism measurements and differential scanning calorimetry, whereas no thermal transitions occur in DnaK and DnaJ between 15 degrees C and 48 degrees C. The conformational changes in GrpE appear to be important in controlling the interconversion of T-state DnaK (ATP-liganded, low affinity for polypeptide substrates) and R-state DnaK (ADP-liganded, high affinity for polypeptide substrates). The rate of the T --> R conversion of DnaK due to DnaJ-triggered ATP hydrolysis follows an Arrhenius temperature dependence. In contrast, the rate of the R --> T conversion due to GrpE-catalyzed ADP/ATP exchange increases progressively less with increasing temperature and even decreases at temperatures above approximately 40 degrees C, indicating a temperature-dependent reversible inactivation of GrpE. At heat-shock temperatures, the reversible structural changes of GrpE thus shift DnaK toward its high-affinity R state.

  4. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling.

    Science.gov (United States)

    Kandasamy, Saveetha; Loganathan, Karthiba; Muthuraj, Raveendran; Duraisamy, Saravanakumar; Seetharaman, Suresh; Thiruvengadam, Raguchander; Ponnusamy, Balasubramanian; Ramasamy, Samiyappan

    2009-12-24

    Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

  5. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

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    Thiruvengadam Raguchander

    2009-12-01

    Full Text Available Abstract Background Plant Growth Promoting Rhizobacteria (PGPR, Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

  6. Suppression of Cpn10 increases mitochondrial fission and dysfunction in neuroblastoma cells.

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    So Jung Park

    Full Text Available To date, several regulatory proteins involved in mitochondrial dynamics have been identified. However, the precise mechanism coordinating these complex processes remains unclear. Mitochondrial chaperones regulate mitochondrial function and structure. Chaperonin 10 (Cpn10 interacts with heat shock protein 60 (HSP60 and functions as a co-chaperone. In this study, we found that down-regulation of Cpn10 highly promoted mitochondrial fragmentation in SK-N-MC and SH-SY5Y neuroblastoma cells. Both genetic and chemical inhibition of Drp1 suppressed the mitochondrial fragmentation induced by Cpn10 reduction. Reactive oxygen species (ROS generation in 3-NP-treated cells was markedly enhanced by Cpn10 knock down. Depletion of Cpn10 synergistically increased cell death in response to 3-NP treatment. Furthermore, inhibition of Drp1 recovered Cpn10-mediated mitochondrial dysfunction in 3-NP-treated cells. Moreover, an ROS scavenger suppressed cell death mediated by Cpn10 knockdown in 3-NP-treated cells. Taken together, these results showed that down-regulation of Cpn10 increased mitochondrial fragmentation and potentiated 3-NP-mediated mitochondrial dysfunction in neuroblastoma cells.

  7. Elevated CO2 increases R gene-dependent resistance of Medicago truncatula against the pea aphid by up-regulating a heat shock gene.

    Science.gov (United States)

    Sun, Yucheng; Guo, Huijuan; Yuan, Erliang; Ge, Feng

    2018-03-01

    Resistance against pathogens and herbivorous insects in many plant results from the expression of resistance (R) genes. Few reports, however, have considered the effects of elevated CO 2 on R gene-based resistance in plants. The current study determined the responses of two near isogenic Medicago truncatula genotypes (Jester has an R gene and A17 does not) to the pea aphid and elevated CO 2 in open-top chambers in the field. Aphid abundance, mean relative growth rate and feeding efficiency were increased by elevated CO 2 on A17 plants but were reduced on Jester plants. According to proteomic and gene expression data, elevated CO 2 enhanced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) but decreased the effector-triggered immunity (ETI) in aphid-infested A17 plants. For aphid-infested Jester plants, by contrast, elevated CO 2 enhanced the ETI-related heat shock protein (HSP) 90 and its co-chaperones, the jasmonic acid (JA) signaling pathway, and ubiquitin-mediated proteolysis. In a loss-of-function experiment, silencing of the HSP90 gene in Jester plants impaired the JA signaling pathway and ubiquitin-mediated proteolysis against the aphid under ambient CO 2 , and negated the increased resistance against the aphid under elevated CO 2 . Our results suggest that increases in expression of HSP90 are responsible for the enhanced resistance against the aphid under elevated CO 2 . © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  8. The evolutionarily conserved E3 ubiquitin ligase AtCHIP contributes to plant immunity

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    Xin eLi

    2016-03-01

    Full Text Available Plants possess a sophisticated immune system to recognize and respond to microbial threats in their environment. The level of immune signaling must be tightly regulated so that immune responses can be quickly activated in the presence of pathogens, while avoiding autoimmunity. HSP90s, along with their diverse array of co-chaperones, forms chaperone complexes that have been shown to play both positive and negative roles in regulating the accumulation of immune receptors and regulators. In this study, we examined the role of AtCHIP, an evolutionarily conserved E3 ligase that was known to interact with chaperones including HSP90s in multicellular organisms including fruit fly, C. elegans, plants and human. Atchip knockout mutants display enhanced disease susceptibility to a virulent oomycete pathogen, and overexpression of AtCHIP causes enhanced disease resistance at low temperature. Although CHIP was reported to target HSP90 for ubiquitination and degradation, accumulation of HSP90.3 was not affected in Atchip plants. In addition, protein accumulation of nucleotide-binding, leucine-rich repeat domain immune receptor (NLR SNC1 is not altered in Atchip mutant. Thus, while AtCHIP plays a role in immunity, it does not seem to regulate the turnover of HSP90 or SNC1. Further investigation is needed in order to determine the exact mechanism behind AtCHIP’s role in regulating plant immune responses.

  9. Applying chaperones to protein-misfolding disorders: molecular chaperones against α-synuclein in Parkinson's disease.

    Science.gov (United States)

    Chaari, Ali; Hoarau-Véchot, Jessica; Ladjimi, Moncef

    2013-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of a protein called α-synuclein (α-syn) into inclusions known as lewy bodies (LB) within neurons. This accumulation is also due to insufficient formation and activity of dopamine produced in certain neurons within the substantia nigra. Lewy bodies are the pathological hallmark of the idiopathic disorder and the cascade that allows α-synuclein to misfold, aggregate and form these inclusions has been the subject of intensive research. Targeting these early steps of oligomerization is one of the main therapeutic approaches in order to develop neurodegenerative-modifying agents. Because the folding and refolding of alpha synuclein is the key point of this cascade, we are interested in this review to summarize the role of some molecular chaperones proteins such as Hsp70, Hsp90 and small heat shock proteins (sHsp) and Hsp 104. Hsp70 and its co-chaperone, Hsp70 and small heat shock proteins can prevent neurodegeneration by preventing α-syn misfolding, oligomerization and aggregation in vitro and in Parkinson disease animal models. Hsp104 is able to resolve disordered protein aggregates and cross beta amyloid conformers. Together, these chaperones have a complementary effect and can be a target for therapeutic intervention in PD. Published by Elsevier B.V.

  10. Pharmacogenetics of steroid-responsive acute graft-versus-host disease.

    Science.gov (United States)

    Arora, Mukta; Weisdorf, Daniel J; Shanley, Ryan M; Thyagarajan, Bharat

    2017-05-01

    Glucocorticoids are central to effective therapy of acute graft-versus-host disease (GVHD). However, only about half of the patients respond to steroids in initial therapy. Based on postulated mechanisms for anti-inflammatory effectiveness, we explored genetic variations in glucocorticoid receptor, co-chaperone proteins, membrane transporters, inflammatory mediators, and variants in the T-cell receptor complex in hematopoietic cell transplant recipients with acute GVHD requiring treatment with steroids and their donors toward response at day 28 after initiation of therapy. A total of 300 recipient and donor samples were analyzed. Twenty-three SNPs in 17 genes affecting glucocorticoid pathways were included in the analysis. In multiple regression analysis, donor SNP rs3192177 in the ZAP70 gene (O.R. 2.8, 95% CI: 1.3-6.0, P=.008) and donor SNP rs34471628 in the DUSPI gene (O.R. 0.3, 95% CI: 0.1-1.0, P=.048) were significantly associated with complete or partial response. However, after adjustment for multiple testing, these SNPs did not remain statistically significant. Our results, on this small, exploratory, hypothesis generating analysis suggest that common genetic variation in glucocorticoid pathways may help identify subjects with differential response to glucocorticoids. This needs further assessment in larger datasets and if validated could help identify subjects for alternative treatments and design targeted treatments to overcome steroid resistance. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1*

    Science.gov (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

    2010-01-01

    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Δhep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer. PMID:20007714

  12. ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1.

    Science.gov (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

    2010-02-12

    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

  13. Heat Shock Protein 90 (Hsp90 Expression and Breast Cancer

    Directory of Open Access Journals (Sweden)

    Christos A. Papadimitriou

    2012-09-01

    Full Text Available Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc. are currently under evaluation.

  14. BAG3 facilitates the clearance of endogenous tau in primary neurons.

    Science.gov (United States)

    Lei, Zhinian; Brizzee, Corey; Johnson, Gail V W

    2015-01-01

    Tau is a microtubule associated protein that is found primarily in neurons, and in pathologic conditions, such as Alzheimer's disease (AD) it accumulates and contributes to the disease process. Because tau plays a fundamental role in the pathogenesis of AD and other tauopathies, and in AD mouse models reducing tau levels improves outcomes, approaches that facilitate tau clearance are being considered as therapeutic strategies. However, fundamental to the development of such interventions is a clearer understanding of the mechanisms that regulate tau clearance. Here, we report a novel mechanism of tau degradation mediated by the co-chaperone BAG3. BAG3 has been shown to be an essential component of a complex that targets substrates to the autophagy pathway for degradation. In rat primary neurons, activation of autophagy by inhibition of proteasome activity or treatment with trehalose resulted in significant decreases in tau and phospho-tau levels. These treatments also induced an upregulation of BAG3. Proteasome inhibition activated JNK, which was responsible for the upregulation of BAG3 and increased tau clearance. Inhibiting JNK or knocking down BAG3 blocked the proteasome inhibition-induced decreases in tau. Further, BAG3 overexpression alone resulted in significant decreases in tau and phospho-tau levels in neurons. These results indicate that BAG3 plays a critical role in regulating the levels of tau in neurons, and interventions that increase BAG3 levels could provide a therapeutic approach in the treatment of AD. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. BAG3 controls angiogenesis through regulation of ERK phosphorylation.

    Science.gov (United States)

    Falco, A; Festa, M; Basile, A; Rosati, A; Pascale, M; Florenzano, F; Nori, S L; Nicolin, V; Di Benedetto, M; Vecchione, M L; Arra, C; Barbieri, A; De Laurenzi, V; Turco, M C

    2012-12-13

    BAG3 is a co-chaperone of the heat shock protein (Hsp) 70, is expressed in many cell types upon cell stress, however, its expression is constitutive in many tumours. We and others have previously shown that in neoplastic cells BAG3 exerts an anti-apoptotic function thus favoring tumour progression. As a consequence we have proposed BAG3 as a target of antineoplastic therapies. Here we identify a novel role for BAG3 in regulation of neo-angiogenesis and show that its downregulation results in reduced angiogenesis therefore expanding the role of BAG3 as a therapeutical target. In brief we show that BAG3 is expressed in endothelial cells and is essential for the interaction between ERK and its phosphatase DUSP6, as a consequence its removal results in reduced binding of DUSP6 to ERK and sustained ERK phosphorylation that in turn determines increased levels of p21 and p15 and cell-cycle arrest in the G1 phase.

  16. BAG3 affects the nucleocytoplasmic shuttling of HSF1 upon heat stress.

    Science.gov (United States)

    Jin, Young-Hee; Ahn, Sang-Gun; Kim, Soo-A

    2015-08-21

    Bcl2-associated athoanogene (BAG) 3 is a member of the co-chaperone BAG family. It is induced by stressful stimuli such as heat shock and heavy metals, and it regulates cellular adaptive responses against stressful conditions. In this study, we identified a novel role for BAG3 in regulating the nuclear shuttling of HSF1 during heat stress. The expression level of BAG3 was induced by heat stress in HeLa cells. Interestingly, BAG3 rapidly translocalized to the nucleus upon heat stress. Immunoprecipitation assay showed that BAG3 interacts with HSF1 under normal and stressed conditions and co-translocalizes to the nucleus upon heat stress. We also demonstrated that BAG3 interacts with HSF1 via its BAG domain. Over-expression of BAG3 down-regulates the level of nuclear HSF1 by exporting it to the cytoplasm during the recovery period. Depletion of BAG3 using siRNA results in reduced nuclear HSF1 and decreased Hsp70 promoter activity. BAG3 in MEF(hsf1(-/-)) cells actively translocalizes to the nucleus upon heat stress suggesting that BAG3 plays a key role in the processing of the nucleocytoplasmic shuttling of HSF1 upon heat stress. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. WW domain of BAG3 is required for the induction of autophagy in glioma cells.

    Science.gov (United States)

    Merabova, Nana; Sariyer, Ilker Kudret; Saribas, A Sami; Knezevic, Tijana; Gordon, Jennifer; Turco, M Caterina; Rosati, Alessandra; Weaver, Michael; Landry, Jacques; Khalili, Kamel

    2015-04-01

    Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co-chaperone family of proteins that associates with Hsp70 through a conserved BAG domain positioned near the C-terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes that cause stress to cells. Our results show that BAG3 overexpression induces autophagy in glioma cells. Interestingly, inhibition of the proteasome caused an increase in BAG3 levels and induced autophagy. Further analysis using specific siRNA against BAG3 suggests that autophagic activation due to proteosomal inhibition is mediated by BAG3. Analyses of BAG3 domain mutants suggest that the WW domain of BAG3 is crucial for the induction of autophagy. BAG3 overexpression also increased the interaction between Bcl2 and Beclin-1, instead of disrupting them, suggesting that BAG3 induced autophagy is Beclin-1 independent. These observations reveal a novel role for the WW domain of BAG3 in the regulation of autophagy. © 2014 Wiley Periodicals, Inc.

  18. The anti-apoptotic BAG3 protein is involved in BRAF inhibitor resistance in melanoma cells.

    Science.gov (United States)

    Guerriero, Luana; Palmieri, Giuseppe; De Marco, Margot; Cossu, Antonio; Remondelli, Paolo; Capunzo, Mario; Turco, Maria Caterina; Rosati, Alessandra

    2017-10-06

    BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumour types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. In human melanomas, BAG3 positivity is correlated with the aggressiveness of the tumour cells and can sustain IKK-γ levels, allowing a sustained activation of NF-κB. Furthermore, BAG3 is able to modulate BRAFV600E levels and activity in thyroid carcinomas. BRAFV600E is the most frequent mutation detected in malignant melanomas and is targeted by Vemurafenib, a specific inhibitor found to be effective in the treatment of advanced melanoma. However, patients with BRAF-mutated melanoma may result insensitive ab initio or, mostly, develop acquired resistance to the treatment with this molecule. Here we show that BAG3 down-modulation interferes with BRAF levels in melanoma cells and sensitizes them to Vemurafenib treatment. Furthermore, the down-modulation of BAG3 protein in an in vitro model of acquired resistance to Vemurafenib can induce sensitization to the BRAFV600E specific inhibition by interfering with BRAF pathway through reduction of ERK phosphorylation, but also on parallel survival pathways. Future studies on BAG3 molecular interactions with key proteins responsible of acquired BRAF inhibitor resistance may represent a promising field for novel multi-drugs treatment design.

  19. BAG3 regulates epithelial-mesenchymal transition and angiogenesis in human hepatocellular carcinoma.

    Science.gov (United States)

    Xiao, Heng; Cheng, Shaobing; Tong, Rongliang; Lv, Zheng; Ding, Chaofeng; Du, Chengli; Xie, Haiyang; Zhou, Lin; Wu, Jian; Zheng, Shusen

    2014-03-01

    Bcl2-associated athanogene 3 (BAG3) protein is a co-chaperone of heat-shock protein (Hsp) 70 and may regulate major physiological and pathophysiological processes. However, few reports have examined the role of BAG3 in human hepatocellular carcinoma (HCC). In this study, we show that BAG3 regulates epithelial-mesenchymal transition (EMT) and angiogenesis in HCC. BAG3 was overexpressed in HCC tissues and cell lines. BAG3 knockdown resulted in reduction in migration and invasion of HCC cells, which was linked to reversion of EMT by increasing E-cadherin expression and decreasing N-cadherin, vimentin and slug expression, as well as suppressing matrix metalloproteinase 2 (MMP-2) expression. In a xenograft tumorigenicity model, BAG3 knockdown effectively inhibited tumor growth and metastasis through reduction in CD34 and VEGF expression and reversal of the EMT pathway. In conclusion, BAG3 is associated with the invasiveness and angiogenesis in HCC, and the BAG3 gene may be a novel therapeutic approach against HCC.

  20. BAG3: a multifaceted protein that regulates major cell pathways

    Science.gov (United States)

    Rosati, A; Graziano, V; De Laurenzi, V; Pascale, M; Turco, M C

    2011-01-01

    Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression. PMID:21472004

  1. The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease.

    Science.gov (United States)

    Stürner, Elisabeth; Behl, Christian

    2017-01-01

    In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC) system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy). One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 ( BCL-2-associated athanogene 3 ). Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer's disease (tau-protein), Huntington's disease (mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate.

  2. BAG3 promotes stem cell-like phenotype in breast cancer by upregulation of CXCR4 via interaction with its transcript.

    Science.gov (United States)

    Liu, Bao-Qin; Zhang, Song; Li, Si; An, Ming-Xin; Li, Chao; Yan, Jing; Wang, Jia-Mei; Wang, Hua-Qin

    2017-07-13

    BAG3 is an evolutionarily conserved co-chaperone expressed at high levels and has a prosurvival role in many tumor types. The current study reported that BAG3 was induced under specific floating culture conditions that enrich breast cancer stem cell (BCSC)-like cells in spheres. Ectopic BAG3 overexpression increased CD44 + /CD24 - CSC subpopulations, first-generation and second-generation mammosphere formation, indicating that BAG3 promotes CSC self-renewal and maintenance in breast cancer. We further demonstrated that mechanically, BAG3 upregulated CXCR4 expression at the post-transcriptional level. Further studies showed that BAG3 interacted with CXCR4 mRNA and promoted its expression via its coding and 3'-untranslational regions. BAG3 was also found to be positively correlated with CXCR4 expression and unfavorable prognosis in patients with breast cancer. Taken together, our data demonstrate that BAG3 promotes BCSC-like phenotype through CXCR4 via interaction with its transcript. Therefore, this study establishes BAG3 as a potential adverse prognostic factor and a therapeutic target of breast cancer.

  3. A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics, Spindle Orientation and Proper Chromosome Segregation during Mitosis.

    Science.gov (United States)

    Fuchs, Margit; Luthold, Carole; Guilbert, Solenn M; Varlet, Alice Anaïs; Lambert, Herman; Jetté, Alexandra; Elowe, Sabine; Landry, Jacques; Lavoie, Josée N

    2015-10-01

    The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.

  4. 2'-Hydroxycinnamaldehyde induces apoptosis through HSF1-mediated BAG3 expression.

    Science.gov (United States)

    Nguyen, Hai-Anh; Kim, Soo-A

    2017-01-01

    BAG3, a member of BAG co-chaperone family, is induced by stressful stimuli such as heat shock and heavy metals. Through interaction with various binding partners, BAG3 is thought to play a role in cellular adaptive responses against stressful conditions in normal and neoplastic cells. 2'-Hydroxycinnamaldehyde (HCA) is a natural derivative of cinnamaldehyde and has antitumor activity in various cancer cells. In the present study, for the first time, we identified that HCA induced BAG3 expression and BAG3-mediated apoptosis in cancer cells. The apoptotic cell death induced by HCA was demonstrated by caspase-7, -9 and PARP activation, and confirmed by Annexin V staining in both SW480 and SW620 colon cancer cells. Notably, both the mRNA and protein levels of BAG3 were largely induced by HCA in a dose- and time-dependent manner. By showing transcription factor HSF1 activation, we demonstrated that HCA induces the expression of BAG3 through HSF1 activation. More importantly, knockdown of BAG3 expression using siRNA largely inhibited HCA-induced apoptosis, suggesting that BAG3 is actively involved in HCA-induced cancer cell death. Considering the importance of the stress response mechanism in cancer progression, our results strongly suggest that BAG3 could be a potential target for anticancer therapy.

  5. BAG3 is involved in neuronal differentiation and migration.

    Science.gov (United States)

    Santoro, Antonietta; Nicolin, Vanessa; Florenzano, Fulvio; Rosati, Alessandra; Capunzo, Mario; Nori, Stefania L

    2017-05-01

    Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

  6. BAG3-dependent noncanonical autophagy induced by proteasome inhibition in HepG2 cells.

    Science.gov (United States)

    Liu, Bao-Qin; Du, Zhen-Xian; Zong, Zhi-Hong; Li, Chao; Li, Ning; Zhang, Qiang; Kong, De-Hui; Wang, Hua-Qin

    2013-06-01

    Emerging lines of evidence have shown that blockade of ubiquitin-proteasome system (UPS) activates autophagy. The molecular players that regulate the relationship between them remain to be elucidated. Bcl-2 associated athanogene 3 (BAG3) is a member of the BAG co-chaperone family that regulates the ATPase activity of heat shock protein 70 (HSP70) chaperone family. Studies on BAG3 have demonstrated that it plays multiple roles in physiological and pathological processes, including antiapoptotic activity, signal transduction, regulatory role in virus infection, cell adhesion and migration. Recent studies have attracted much attention on its role in initiation of autophagy. The current study, for the first time, demonstrates that proteasome inhibitors elicit noncanonical autophagy, which was not suppressed by inhibitors of class III phosphatidylinositol 3-kinase (PtdIns3K) or shRNA against Beclin 1 (BECN1). In addition, we demonstrate that BAG3 is ascribed to activation of autophagy elicited by proteasome inhibitors and MAPK8/9/10 (also known as JNK1/2/3 respectively) activation is also implicated via upregulation of BAG3. Moreover, we found that noncanonical autophagy mediated by BAG3 suppresses responsiveness of HepG2 cells to proteasome inhibitors.

  7. A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

    Science.gov (United States)

    Judge, Luke M; Perez-Bermejo, Juan A; Truong, Annie; Ribeiro, Alexandre Js; Yoo, Jennie C; Jensen, Christina L; Mandegar, Mohammad A; Huebsch, Nathaniel; Kaake, Robyn M; So, Po-Lin; Srivastava, Deepak; Pruitt, Beth L; Krogan, Nevan J; Conklin, Bruce R

    2017-07-20

    Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

  8. BAG3 Protein Is Over-Expressed in Endometrioid Endometrial Adenocarcinomas.

    Science.gov (United States)

    Esposito, Veronica; Baldi, Carlo; Zeppa, Pio; Festa, Michelina; Guerriero, Luana; d'Avenia, Morena; Chetta, Massimiliano; Zullo, Fulvio; De Laurenzi, Vincenzo; Turco, Maria Caterina; Rosati, Alessandra; Guida, Maurizio

    2017-02-01

    Endometrioid endometrial cancer is the most common gynaecological tumor in developed countries, and its incidence is increasing. The definition of subtypes, based on clinical and endocrine features or on histopathological characteristics, correlate to some extent with patient's prognosis, but there is substantial heterogeneity within tumor types. The search for molecules and mechanisms implied in determining the progression and the response to therapy for this cancer is still ongoing. BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumor types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby, modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. BAG3 expression in human endometrial cancer tissues was not investigated so far. Here, we show that BAG3 protein levels are elevated in tumoral and hyperplastic cells in respect to normal glands. Furthermore, BAG3 subcellular localization appears to be changed in tumoral compared to normal cells. Our results indicate a possible role for BAG3 protein in the maintenance of cell survival in endometrioid endometrial cancer and suggest that this field of studies is worthy of further investigations. J. Cell. Physiol. 232: 309-311, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Overexpression of the anti-apoptotic protein BAG3 in human choroidal melanoma: A case report.

    Science.gov (United States)

    Yunoki, Tatsuya; Tabuchi, Yoshiaki; Kondo, Takashi; Ishii, Yoko; Hayashi, Atsushi

    2017-06-01

    Bcl-2-associated athanogene 3 (BAG3), a co-chaperone of heat shock protein 70 (HSP70), exerts anti-apoptotic effects in various malignant tumors. However, relationships between choroidal melanoma and BAG3 are poorly studied. This study investigated the expression of BAG3 in a case of human choroidal melanoma. Funduscopy, computed tomography, and single-photon emission computed tomography with the intravenous injection of N-isopropyl-p-[ 123 I] iodoamphetamine strongly indicated choroidal melanoma in a 68-year-old woman. Accordingly, we carried out an enucleation and pathological diagnosis. Proteins and total RNA were extracted from normal retinochoroidal and tumor tissues. Proteins were also extracted from ocular nevus tissues of other patients. We examined the expression of BAG3 protein and mRNA using Western blotting and the real-time quantitative polymerase chain reaction, respectively. Immunohistochemical stains were positive for melan-A, HMB-45, and S-100. Histopathology confirmed a choroidal melanoma. The expression of BAG3 protein and mRNA in the choroidal melanoma tissue was upregulated with respect to both normal retinochoroidal tissue and ocular nevus tissues from other patients. Because BAG3 may inhibit apoptosis of choroidal melanoma and facilitate its survival, overexpression of this gene product may be a prognostic marker and therapeutic target.

  10. The anti-apoptotic BAG3 protein is expressed in lung carcinomas and regulates small cell lung carcinoma (SCLC) tumor growth.

    Science.gov (United States)

    Chiappetta, Gennaro; Basile, Anna; Barbieri, Antonio; Falco, Antonia; Rosati, Alessandra; Festa, Michelina; Pasquinelli, Rosa; Califano, Daniela; Palma, Giuseppe; Costanzo, Raffaele; Barcaroli, Daniela; Capunzo, Mario; Franco, Renato; Rocco, Gaetano; Pascale, Maria; Turco, Maria Caterina; De Laurenzi, Vincenzo; Arra, Claudio

    2014-08-30

    BAG3, member the HSP70 co-chaperones family, has been shown to play a relevant role in the survival, growth and invasiveness of different tumor types. In this study, we investigate the expression of BAG3 in 66 specimens from different lung tumors and the role of this protein in small cell lung cancer (SCLC) tumor growth. Normal lung tissue did not express BAG3 while we detected the expression of BAG3 by immunohistochemistry in all the 13 squamous cell carcinomas, 13 adenocarcinomas and 4 large cell carcinomas. Furthermore, we detected BAG3 expression in 22 of the 36 SCLCs analyzed. The role on SCLC cell survival was determined by down-regulating BAG3 levels in two human SCLC cell lines, i.e. H69 and H446, in vitro and measuring cisplatin induced apoptosis. Indeed down-regulation of BAG3 determines increased cell death and sensitizes cells to cisplatin treatment. The effect of BAG3 down-regulation on tumor growth was also investigated in an in vivo xenograft model by treating mice with an adenovirus expressing a specific bag3 siRNA. Treatment with bag3 siRNA-Ad significantly reduced tumor growth and improved animal survival. In conclusion we show that a subset of SCLCs over express BAG3 that exerts an anti-apoptotic effect resulting in resistance to chemotherapy.

  11. The Multifunctional Protein BAG3

    Directory of Open Access Journals (Sweden)

    Valerie D. Myers, MS

    2018-02-01

    Full Text Available The B-cell lymphoma 2–associated anthanogene (BAG3 protein is expressed most prominently in the heart, the skeletal muscle, and in many forms of cancer. In the heart, it serves as a co-chaperone with heat shock proteins in facilitating autophagy; binds to B-cell lymphoma 2, resulting in inhibition of apoptosis; attaches actin to the Z disk, providing structural support for the sarcomere; and links the α-adrenergic receptor with the L-type Ca2+ channel. When BAG3 is overexpressed in cancer cells, it facilitates prosurvival pathways that lead to insensitivity to chemotherapy, metastasis, cell migration, and invasiveness. In contrast, in the heart, mutations in BAG3 have been associated with a variety of phenotypes, including both hypertrophic/restrictive and dilated cardiomyopathy. In murine skeletal muscle and vasculature, a mutation in BAG3 leads to critical limb ischemia after femoral artery ligation. An understanding of the biology of BAG3 is relevant because it may provide a therapeutic target in patients with both cardiac and skeletal muscle disease.

  12. BAG3: a new player in the heart failure paradigm.

    Science.gov (United States)

    Knezevic, Tijana; Myers, Valerie D; Gordon, Jennifer; Tilley, Douglas G; Sharp, Thomas E; Wang, JuFang; Khalili, Kamel; Cheung, Joseph Y; Feldman, Arthur M

    2015-07-01

    BAG3 is a cellular protein that is expressed predominantly in skeletal and cardiac muscle but can also be found in the brain and in the peripheral nervous system. BAG3 functions in the cell include: serving as a co-chaperone with members of the heat-shock protein family of proteins to facilitate the removal of misfolded and degraded proteins, inhibiting apoptosis by interacting with Bcl2 and maintaining the structural integrity of the Z-disk in muscle by binding with CapZ. The importance of BAG3 in the homeostasis of myocytes and its role in the development of heart failure was evidenced by the finding that single allelic mutations in BAG3 were associated with familial dilated cardiomyopathy. Furthermore, significant decreases in the level of BAG3 have been found in end-stage failing human heart and in animal models of heart failure including mice with heart failure secondary to trans-aortic banding and in pigs after myocardial infarction. Thus, it becomes relevant to understand the cellular biology and molecular regulation of BAG3 expression in order to design new therapies for the treatment of patients with both hereditary and non-hereditary forms of dilated cardiomyopathy.

  13. High expression of BAG3 predicts a poor prognosis in human medulloblastoma.

    Science.gov (United States)

    Yang, Dong; Zhou, Ji; Wang, Hao; Wang, Yutao; Yang, Ge; Zhang, Yundong

    2016-10-01

    Bcl2-associated athanogene 3 (BAG3), a co-chaperone of the heat shock protein (Hsp) 70, regulates various physiological and pathological processes. However, its role in human medulloblastoma has not been clarified. First of all, the expression of BAG3 was examined in formalin-fixed, paraffin-embedded specimens by immunohistochemical staining. And then, the prognostic role of BAG3 was analyzed in 51 medulloblastoma samples. Finally, the roles of BAG3 in the proliferation, migration, and invasion of Daoy medulloblastoma cell were investigated using a specific short hairpin RNA (shRNA). The expression of BAG3 in medulloblastoma tissues was higher than nontumorous samples. Furthermore, BAG3 overexpression significantly correlated with poor prognosis of patients with medulloblastoma. The overall survival and tumor-free survival in patients with BAG3 low expression were higher than high expression. Univariate and multivariate analysis showed that BAG3 overexpression was an independent prognostic marker for medulloblastoma. After the BAG3 knockdown, the Daoy cells exhibited decreased the ability to proliferate and form neurosphere. The preliminary mechanism study showed that overexpression of BAG3 might facilitate the cell cycle transition from G1 to S phase by modulating the cyclin-dependent kinase 2 (CDK2) and cyclin E expression. Additionally, we found that BAG3 might enhance the medulloblastoma cell migratory and invasive ability. In summary, BAG3 overexpression may regulate the survival and invasive properties of medulloblastoma and may serve as a potential therapy target for medulloblastoma.

  14. A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics, Spindle Orientation and Proper Chromosome Segregation during Mitosis.

    Directory of Open Access Journals (Sweden)

    Margit Fuchs

    2015-10-01

    Full Text Available The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.

  15. Evidence for the Role of BAG3 in Mitochondrial Quality Control in Cardiomyocytes.

    Science.gov (United States)

    Tahrir, Farzaneh G; Knezevic, Tijana; Gupta, Manish K; Gordon, Jennifer; Cheung, Joseph Y; Feldman, Arthur M; Khalili, Kamel

    2017-04-01

    Mitochondrial abnormalities impact the development of myofibrillar myopathies. Therefore, understanding the mechanisms underlying the removal of dysfunctional mitochondria from cells is of great importance toward understanding the molecular events involved in the genesis of cardiomyopathy. Earlier studies have ascribed a role for BAG3 in the development of cardiomyopathy in experimental animals leading to the identification of BAG3 mutations in patients with heart failure which may play a part in the onset of disease development and progression. BAG3 is co-chaperone of heat shock protein 70 (HSP70), which has been shown to modulate apoptosis and autophagy, in several cell models. In this study, we explore the potential role of BAG3 in mitochondrial quality control. We demonstrate that siRNA mediated suppression of BAG3 production in neonatal rat ventricular cardiomyocytes (NRVCs) significantly elevates the level of Parkin, a key component of mitophagy. We found that both BAG3 and Parkin are recruited to depolarized mitochondria and promote mitophagy. Suppression of BAG3 in NRVCs significantly reduces autophagy flux and eliminates clearance of Tom20, an essential import receptor for mitochondria proteins, after induction of mitophagy. These observations suggest that BAG3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in BAG3 expression impact cardiomyocyte function. J. Cell. Physiol. 232: 797-805, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. An interaction study in mammalian cells demonstrates weak binding of HSPB2 to BAG3, which is regulated by HSPB3 and abrogated by HSPB8.

    Science.gov (United States)

    Morelli, Federica F; Mediani, Laura; Heldens, Lonneke; Bertacchini, Jessika; Bigi, Ilaria; Carrà, Arianna Dorotea; Vinet, Jonathan; Carra, Serena

    2017-07-01

    The ten mammalian small heat shock proteins (sHSPs/HSPBs) show a different expression profile, although the majority of them are abundant in skeletal and cardiac muscles. HSPBs form hetero-oligomers and homo-oligomers by interacting together and complexes containing, e.g., HSPB2/HSPB3 or HSPB1/HSPB5 have been documented in mammalian cells and muscles. Moreover, HSPB8 associates with the Hsc70/Hsp70 co-chaperone BAG3, in mammalian, skeletal, and cardiac muscle cells. Interaction of HSPB8 with BAG3 regulates its stability and function. Weak association of HSPB5 and HSPB6 with BAG3 has been also reported upon overexpression in cells, supporting the idea that BAG3 might indirectly modulate the function of several HSPBs. However, it is yet unknown whether other HSPBs highly expressed in muscles such as HSPB2 and HSPB3 also bind to BAG3. Here, we report that in mammalian cells, upon overexpression, HSPB2 binds to BAG3 with an affinity weaker than HSPB8. HSPB2 competes with HSPB8 for binding to BAG3. In contrast, HSPB3 negatively regulates HSPB2 association with BAG3. In human myoblasts that express HSPB2, HSPB3, HSPB8, and BAG3, the latter interacts selectively with HSPB8. Combining these data, it supports the interpretation that HSPB8-BAG3 is the preferred interaction.

  17. BAG3 Is a Modular, Scaffolding Protein that physically Links Heat Shock Protein 70 (Hsp70) to the Small Heat Shock Proteins.

    Science.gov (United States)

    Rauch, Jennifer N; Tse, Eric; Freilich, Rebecca; Mok, Sue-Ann; Makley, Leah N; Southworth, Daniel R; Gestwicki, Jason E

    2017-01-06

    Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that are important for binding and stabilizing unfolded proteins. In this task, the sHsps have been proposed to coordinate with ATP-dependent chaperones, including heat shock protein 70 (Hsp70). However, it is not yet clear how these two important components of the chaperone network are linked. We report that the Hsp70 co-chaperone, BAG3, is a modular, scaffolding factor to bring together sHsps and Hsp70s. Using domain deletions and point mutations, we found that BAG3 uses both of its IPV motifs to interact with sHsps, including Hsp27 (HspB1), αB-crystallin (HspB5), Hsp22 (HspB8), and Hsp20 (HspB6). BAG3 does not appear to be a passive scaffolding factor; rather, its binding promoted de-oligomerization of Hsp27, likely by competing for the self-interactions that normally stabilize large oligomers. BAG3 bound to Hsp70 at the same time as Hsp22, Hsp27, or αB-crystallin, suggesting that it might physically bring the chaperone families together into a complex. Indeed, addition of BAG3 coordinated the ability of Hsp22 and Hsp70 to refold denatured luciferase in vitro. Together, these results suggest that BAG3 physically and functionally links Hsp70 and sHsps. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Jasmonate signalling in Arabidopsis involves SGT1b-HSP70-HSP90 chaperone complexes.

    Science.gov (United States)

    Zhang, Xue-Cheng; Millet, Yves A; Cheng, Zhenyu; Bush, Jenifer; Ausubel, Frederick M

    Plant hormones play pivotal roles in growth, development and stress responses. Although it is essential to our understanding of hormone signalling, how plants maintain a steady state level of hormone receptors is poorly understood. We show that mutation of the Arabidopsis thaliana co-chaperone SGT1b impairs responses to the plant hormones jasmonate, auxin and gibberellic acid, but not brassinolide and abscisic acid, and that SGT1b and its homologue SGT1a are involved in maintaining the steady state levels of the F-box proteins COI1 and TIR1, receptors for jasmonate and auxin, respectively. The association of SGT1b with COI1 is direct and is independent of the Arabidopsis SKP1 protein, ASK1. We further show that COI1 is a client protein of SGT1b-HSP70-HSP90 chaperone complexes and that the complexes function in hormone signalling by stabilizing the COI1 protein. This study extends the SGT1b-HSP90 client protein list and broadens the functional scope of SGT1b-HSP70-HSP90 chaperone complexes.

  19. AHM1, a Novel Type of Nuclear Matrix–Localized, MAR Binding Protein with a Single AT Hook and a J Domain–Homologous Region

    Science.gov (United States)

    Morisawa, Gaku; Han-yama, Atsushi; Moda, Ichiro; Tamai, Atsushi; Iwabuchi, Masaki; Meshi, Tetsuo

    2000-01-01

    Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook–containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain–homologous region and a Zn finger–like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger–like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones. PMID:11041885

  20. 78 FR 16806 - The Commercial Mobile Alert System

    Science.gov (United States)

    2013-03-19

    ... performs the various functions associated with the authentication, management and dissemination of WEA... transceivers and perform functions associated with authentication of interactions with the Mobile Device. (j... Type; Area Affected; Recommended Action; Expiration Time (with time zone); and Sending Agency. This...

  1. On orbits and admissible subalgebras for the PR groups

    International Nuclear Information System (INIS)

    Lebedenko, V.M.

    1977-01-01

    The PR-groups (Lie groups with commutation relations of [Hsub(i),Hsub(0)] = rsub(ij)Hsub(i) (i< j)-type) are investigated within the Kirillov orbit method. Orbits for all the PR-groups are constructed in a general form. For some classes of PR-groups orbits and admissible subalgebras are obtained in an explicit form

  2. 78 FR 60321 - Biweekly Notice; Applications and Amendments to Facility Operating Licenses and Combined Licenses...

    Science.gov (United States)

    2013-10-01

    .... Nebraska Public Power District, Docket No. 50-298, Cooper Nuclear Station, Nemaha County, Nebraska Date of amendment request: February 12, 2013. Brief description of amendment: The amendment modified the Cooper... licensee from the requirement to perform an appendix J Type A test, containment integrated leakage rate...

  3. 32 CFR 842.128 - Delegations of authority.

    Science.gov (United States)

    2010-07-01

    ... General is the final appellate authority on subpart F type claims without right of further appeal to the... may be liable as that specified for a similar type claim in each subpart of this part. The decision of... delegated authority to settle subparts F, G, and J type claims in any amount without referral to the...

  4. Photoswitchable Intramolecular H-Stacking of Perylenebisimide

    NARCIS (Netherlands)

    Wang, Jiaobing; Kulago, Artem; Browne, Wesley R.; Feringa, Ben L.

    2010-01-01

    Dynamic control over the formation of H- or J-type aggregates of chromophores is of fundamental importance for developing responsive organic optoelectronic materials. In this study, the first example of photoswitching between a nonstacked and an intramolecularly H-stacked arrangement of

  5. Infrared spectroscopy of four carbon stars with 9.8 micron emission from silicate grains

    International Nuclear Information System (INIS)

    Lambert, D.L.; Smith, V.V.; Hinkle, K.H.

    1990-01-01

    High-resolution K band and low resolution 4 micron spectra were obtained for four carbon stars showing IR emission by silicate grains. The results of the analysis of the K band spectra show that they are J-type stars. These results, together with published spectral classifications, show that all known carbon stars with a silicate emission feature are J-type stars. The 4 micron spectra are very similar to the spectra of classical J-type carbon stars, and do not show SiO bands that might come from a M giant companion. A binary model with a luminous M giant companion as a source of the silicate grain is rejected. It is proposed that the silicate grains formed from gas ejecta at or before the He-core flash, and that the flash initiates severe mixing, leading to the star's conversion to a J-type carbon star. The ejecta are stored in an accretion disk around a low mass unevolved companion. If it can be shown that the hypothesized accretion disk is stable and may be heated adequately, this binary model appears to account for these peculiar carbon stars. 41 refs

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

    Science.gov (United States)

    Kaplan, Tommy; Liu, Chih Long; Erkmann, Judith A; Holik, John; Grunstein, Michael; Kaufman, Paul D; Friedman, Nir; Rando, Oliver J

    2008-11-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. DNAJB6 myopathies: Focused review on an emerging and expanding group of myopathies

    Directory of Open Access Journals (Sweden)

    Alessandra Ruggieri

    2016-09-01

    Full Text Available Mutations in the DNAJB6 gene have been associated with the autosomal dominant limb girdle muscular dystrophy type 1D (LGMD1D, a disorder characterized by abnormal protein aggregates and rimmed vacuoles in muscle fibers. DNAJB6 is a ubiquitously expressed Hsp40 co-chaperone characterized by a J domain that specifies Hsp70 functions in the cellular environment. DNAJB6 is also a potent inhibitor of expanded polyglutamine (polyQ aggregation preventing aggregate toxicity in cells. In DNAJB6-mutated patients this anti-aggregation property is significantly reduced, albeit not completely lost. To elucidate the pathogenetic mechanisms underlying the DNAJB6-related myopathy, animal models have been created showing that, indeed, conditional muscular expression of a DNAJB6 mutant in the mouse causes a LGMD1D myofibrillary muscle tissue phenotype. Both mutations and phenotypes reported until recently were rather homogeneous, being exclusively missense mutations of a few amino acids of the protein G/F domain, and with a phenotype characterized by adult-onset slowly progressive muscular dystrophy predominantly affecting proximal muscles. Lately, several novel mutations and new phenotypes of DNAJB6 have been described. These mutations once more affect the G/F domain of DNAJB6 with missense changes and a splice site mutation; and the phenotypes include childhood onset and distal involvement of muscles, or childhood-onset LGMD1D with loss of ambulation in early adulthood and respiratory involvement. Thus, the spectrum of DNAJB6-related phenotypes is widening. Although our knowledge about the role of DNAJB6 in the pathogenesis of muscle diseases has made great progression, several questions remain unsolved, including why a ubiquitous protein affects only, or predominantly, skeletal muscle; why only the G/F domain is involved; and what is the possible role of the DNAJB6a isoform. Clarification of these issues will provide clues to implement possible therapeutic

  9. Hypothalamic-Pituitary-Adrenal Axis Dysfunction and Illness Progression in Bipolar Disorder

    Science.gov (United States)

    Vasconcelos-Moreno, Mirela Paiva; Gubert, Carolina; dos Santos, Bárbara Tietböhl Martins Quadros; Sartori, Juliana; Eisele, Bárbara; Ferrari, Pamela; Fijtman, Adam; Rüegg, Joëlle; Gassen, Nils Christian; Kapczinski, Flávio; Rein, Theo; Kauer-Sant’Anna, Márcia

    2015-01-01

    Background: Impaired stress resilience and a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis are suggested to play key roles in the pathophysiology of illness progression in bipolar disorder (BD), but the mechanisms leading to this dysfunction have never been elucidated. This study aimed to examine HPA axis activity and underlying molecular mechanisms in patients with BD and unaffected siblings of BD patients. Methods: Twenty-four euthymic patients with BD, 18 siblings of BD patients, and 26 healthy controls were recruited for this study. All subjects underwent a dexamethasone suppression test followed by analyses associated with the HPA axis and the glucocorticoid receptor (GR). Results: Patients with BD, particularly those at a late stage of illness, presented increased salivary post-dexamethasone cortisol levels when compared to controls (p = 0.015). Accordingly, these patients presented reduced ex vivo GR responsiveness (p = 0.008) and increased basal protein levels of FK506-binding protein 51 (FKBP51, p = 0.012), a co-chaperone known to desensitize GR, in peripheral blood mononuclear cells. Moreover, BD patients presented increased methylation at the FK506-binding protein 5 (FKBP5) gene. BD siblings presented significantly lower FKBP51 protein levels than BD patients, even though no differences were found in FKBP5 basal mRNA levels. Conclusions: Our data suggest that the epigenetic modulation of the FKBP5 gene, along with increased FKBP51 levels, is associated with the GR hyporesponsiveness seen in BD patients. Our findings are consistent with the notion that unaffected first-degree relatives of BD patients share biological factors that influence the disorder, and that such changes are more pronounced in the late stages of the illness. PMID:25522387

  10. Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

    Science.gov (United States)

    Schilke, Brenda A; Ciesielski, Szymon J; Ziegelhoffer, Thomas; Kamiya, Erina; Tonelli, Marco; Lee, Woonghee; Cornilescu, Gabriel; Hines, Justin K; Markley, John L; Craig, Elizabeth A

    2017-10-01

    By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

  11. Human SGT interacts with Bag-6/Bat-3/Scythe and cells with reduced levels of either protein display persistence of few misaligned chromosomes and mitotic arrest

    International Nuclear Information System (INIS)

    Winnefeld, Marc; Grewenig, Annabel; Schnoelzer, Martina; Spring, Herbert; Knoch, Tobias A.; Gan, Eugene C.; Rommelaere, Jean; Cziepluch, Celina

    2006-01-01

    The human small glutamine-rich TPR-containing protein (hSGT) is essential for cell division since RNA-interference-mediated strong reduction of hSGT protein levels causes mitotic arrest (M. Winnefeld, J. Rommelaere, and C. Cziepluch, The human small glutamine-rich TPR-containing protein is required for progress through cell division, Exp. Cell Res. 293 (2004), 43-57). Analysis of HeLa cells expressing a histone 2A-YFP fusion protein revealed the continuous presence of few mislocalized chromosomes close to the spindle poles as possible cause for hSGT depletion-dependent prometaphase arrest. Cells unable to rescue these mislocalized chromosomes into the metaphase plate died at this stage through apoptosis. In order to address hSGT function at the molecular level, mass spectrometry analysis of proteins which co-immunoprecipitated with Flag-tagged hSGT was performed. Thereby, Hsp70 and Bag-6/Bat-3/Scythe were identified as novel hSGT interaction partners while interaction with Hsc70 was confirmed. Results obtained with truncated versions of the hSGT protein revealed that Bag-6/Bat-3/Scythe and Hsp70 or Hsc70 were independently able to form complexes with hSGT. Interaction of hSGT with Hsc70, Hsp70 or Bag-6/Bat-3/Scythe was demonstrated in prometaphase, thereby suggesting a possible role for complexes containing hSGT and distinct (co)-chaperones during mitosis. Finally, cells from populations with reduced levels of Bag-6/Bat-3/Scythe also displayed persistence of mislocalized chromosomes and mitotic arrest, which strongly indicated that hSGT-Bag-6/Bat-3/Scythe complexes could be directly or indirectly required for complete chromosome congression

  12. Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils.

    Science.gov (United States)

    Lian, Hui-Yong; Zhang, Hong; Zhang, Zai-Rong; Loovers, Harriët M; Jones, Gary W; Rowling, Pamela J E; Itzhaki, Laura S; Zhou, Jun-Mei; Perrett, Sarah

    2007-04-20

    Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.

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

    Directory of Open Access Journals (Sweden)

    Anshuman Dixit

    residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients.

  14. Molecular mechanism of 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL receptor tyrosine kinase degradation.

    Science.gov (United States)

    Krishnamoorthy, Gnana Prakasam; Guida, Teresa; Alfano, Luigi; Avilla, Elvira; Santoro, Massimo; Carlomagno, Francesca; Melillo, Rosa Marina

    2013-06-14

    The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.

  15. Molecular Mechanism of 17-Allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL Receptor Tyrosine Kinase Degradation*

    Science.gov (United States)

    Krishnamoorthy, Gnana Prakasam; Guida, Teresa; Alfano, Luigi; Avilla, Elvira; Santoro, Massimo; Carlomagno, Francesca; Melillo, Rosa Marina

    2013-01-01

    The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [35S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL. PMID:23629654

  16. Hormonal status and age differentially affect tolerance to the disruptive effects of delta-9-tetrahydrocannabinol (Δ9-THC on learning in female rats

    Directory of Open Access Journals (Sweden)

    Peter J Winsauer

    2015-07-01

    Full Text Available The effects of hormone status and age on the development of tolerance to D9-THC were assessed in sham-operated (intact or ovariectomized (OVX female rats that received either intraperitoneal saline or 5.6 mg/kg of D9-THC daily from postnatal day (PD 75 to 180 (early adulthood onward or PD 35 to 140 (adolescence onward. During this time, the 4 groups for each age (i.e., intact/saline, intact/THC, OVX/saline, and OVX/THC were trained in a learning and performance procedure and dose-effect curves were established for D9-THC (0.56-56 mg/kg and the cannabinoid type-1 receptor (CB1R antagonist rimonabant (0.32-10 mg/kg. Despite the persistence of small rate-decreasing and error-increasing effects in intact and OVX females from both ages during chronic D9-THC, all of the D9-THC groups developed tolerance. However, the magnitude of tolerance, as well as the effect of hormone status, varied with the age at which chronic D9-THC was initiated. There was no evidence of dependence in any of the groups. Hippocampal protein expression of CB1R, AHA1 (a co-chaperone of CB1R and HSP90β (a molecular chaperone modulated by AHA-1 was affected more by OVX than chronic D9-THC; striatal protein expression was not consistently affected by either manipulation. Hippocampal BDNF expression varied with age, hormone status, and chronic treatment. Thus, hormonal status differentially affects the development of tolerance to the disruptive effects of delta-9-tetrahydrocannabinol (D9-THC on learning and performance behavior in adolescent, but not adult, female rats. These factors and their interactions also differentially affect cannabinoid signaling proteins in the hippocampus and striatum, and ultimately, neural plasticity.

  17. Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

    Directory of Open Access Journals (Sweden)

    Coppée Jean-Yves

    2010-02-01

    Full Text Available Abstract Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III to As(V as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III. To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54 of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III in this microorganism.

  18. Function of SSA subfamily of Hsp70 within and across species varies widely in complementing Saccharomyces cerevisiae cell growth and prion propagation.

    Directory of Open Access Journals (Sweden)

    Deepak Sharma

    2009-08-01

    Full Text Available The cytosol of most eukaryotic cells contains multiple highly conserved Hsp70 orthologs that differ mainly by their spatio-temporal expression patterns. Hsp70s play essential roles in protein folding, transport or degradation, and are major players of cellular quality control processes. However, while several reports suggest that specialized functions of Hsp70 orthologs were selected through evolution, few studies addressed systematically this issue.We compared the ability of Ssa1p-Ssa4p from Saccharomyces cerevisiae and Ssa5p-Ssa8p from the evolutionary distant yeast Yarrowia lipolytica to perform Hsp70-dependent tasks when expressed as the sole Hsp70 for S. cerevisiae in vivo. We show that Hsp70 isoforms (i supported yeast viability yet with markedly different growth rates, (ii influenced the propagation and stability of the [PSI(+] and [URE3] prions, but iii did not significantly affect the proteasomal degradation rate of CFTR. Additionally, we show that individual Hsp70 orthologs did not induce the formation of different prion strains, but rather influenced the aggregation properties of Sup35 in vivo. Finally, we show that [URE3] curing by the overexpression of Ydj1p is Hsp70-isoform dependent.Despite very high homology and overlapping functions, the different Hsp70 orthologs have evolved to possess distinct activities that are required to cope with different types of substrates or stress situations. Yeast prions provide a very sensitive model to uncover this functional specialization and to explore the intricate network of chaperone/co-chaperone/substrates interactions.

  19. Hsp70/J-protein machinery from Glossina morsitans morsitans, vector of African trypanosomiasis.

    Directory of Open Access Journals (Sweden)

    Stephen J Bentley

    Full Text Available Tsetse flies (Glossina spp. are the sole vectors of the protozoan parasites of the genus Trypanosoma, the causative agents of African Trypanosomiasis. Species of Glossina differ in vector competence and Glossina morsitans morsitans is associated with transmission of Trypanosoma brucei rhodesiense, which causes an acute and often fatal form of African Trypanosomiasis. Heat shock proteins are evolutionarily conserved proteins that play critical roles in proteostasis. The activity of heat shock protein 70 (Hsp70 is regulated by interactions with its J-protein (Hsp40 co-chaperones. Inhibition of these interactions are emerging as potential therapeutic targets. The assembly and annotation of the G. m. morsitans genome provided a platform to identify and characterize the Hsp70s and J-proteins, and carry out an evolutionary comparison to its well-studied eukaryotic counterparts, Drosophila melanogaster and Homo sapiens, as well as Stomoxys calcitrans, a comparator species. In our study, we identified 9 putative Hsp70 proteins and 37 putative J-proteins in G. m. morsitans. Phylogenetic analyses revealed three evolutionarily distinct groups of Hsp70s, with a closer relationship to orthologues from its blood-feeding dipteran relative Stomoxys calcitrans. G. m. morsitans also lacked the high number of heat inducible Hsp70s found in D. melanogaster. The potential localisations, functions, domain organisations and Hsp70/J-protein partnerships were also identified. A greater understanding of the heat shock 70 (Hsp70 and J-protein (Hsp40 families in G. m. morsitans could enhance our understanding of the cell biology of the tsetse fly.

  20. Hsp90: A New Player in DNA Repair?

    Directory of Open Access Journals (Sweden)

    Rosa Pennisi

    2015-10-01

    Full Text Available Heat shock protein 90 (Hsp90 is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis, transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR pathways that include: (i cell cycle arrest; (ii transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted.

  1. Chaperonopathies: spotlight on hereditary motor neuropathies

    Directory of Open Access Journals (Sweden)

    Vincenzo Lupo

    2016-12-01

    Full Text Available Distal hereditary motor neuropathies (dHMN comprise a group of rare hereditary neuromuscular disorders characterized by a peroneal muscular atrophy without sensory symptoms. To date twenty-three genes for dHMN have been reported and four of them encode for chaperones: DNAJB2, which encodes a member of the HSP40/DNAJ co-chaperone family, and HSPB1, HSPB3 and HSPB8, which encode three members of the family of small heat shock proteins. Except for HSPB1, with around thirty different mutations, the remaining three genes comprise a much low number of cases. Thus, only one case has been described caused by an HSPB3 mutation, whereas few DNAJB2 and HSPB8 cases are known, most of them caused by a founder c.352+1G>A mutation in DNAJB2 and by mutations affecting the hot spot K141 residue of the HSPB8 chaperone. This low number of cases makes it difficult to understand the pathomechanism underlying the neuropathy. Chaperones can assemble in multi-chaperone complexes forming an integrative chaperone network in the cell, which plays relevant cellular roles in a variety of processes such as the correct folding of newly synthesized proteins, their escort to their precise cellular locations to form functional proteins and complexes and the response to protein misfolding, including the degradation of proteins that fail to refold properly. Despite of this variety of functions, mutations in some of them lead to diseases with a similar clinical picture, suggesting common pathways. This review gives an overview of the genetics of dHMNs caused by mutations in four genes, DNAJB2, HSPB1, HSPB3 and HSPB8, which encode chaperones and show a common disease mechanism.

  2. Proteomic analysis reveals a role for Bcl2-associated athanogene 3 and major vault protein in resistance to apoptosis in senescent cells by regulating ERK1/2 activation.

    Science.gov (United States)

    Pasillas, Martina P; Shields, Sarah; Reilly, Rebecca; Strnadel, Jan; Behl, Christian; Park, Robin; Yates, John R; Klemke, Richard; Gonias, Steven L; Coppinger, Judith A

    2015-01-01

    Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence and discovered that Bcl2-associated athanogene 3 (Bag3) is up-regulated after adriamycin treatment in MCF7 cells. Bag3 is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major cell-signaling pathways. Mass spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin to favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in therapy-induced senescence by increasing the level of activation of extracellular signal-regulated kinase1/2 (ERK1/2). Silencing of either Bag3 or MVP decreased ERK1/2 activation and promoted apoptosis in adriamycin-treated cells. An increase in nuclear accumulation of MVP is observed during therapy-induced senescence and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We confirmed that silencing of Bag3 or MVP shifts the response toward apoptosis and regulates ERK1/2 activation in a panel of diverse breast cancer cell lines. This study highlights Bag3-MVP as an important complex that regulates a potent prosurvival signaling pathway and contributes to chemotherapy resistance in breast cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea.

    Science.gov (United States)

    Petitjean, Céline; Moreira, David; López-García, Purificación; Brochier-Armanet, Céline

    2012-11-26

    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. 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. 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.

  4. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea

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    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.

  5. Functional mapping of PilF and PilQ in the Pseudomonas aeruginosa type IV pilus system.

    Science.gov (United States)

    Koo, Jason; Tang, Tim; Harvey, Hanjeong; Tammam, Stephanie; Sampaleanu, Liliana; Burrows, Lori L; Howell, P Lynne

    2013-04-30

    Pseudomonas aeruginosa uses type IV pili (T4P) to interact with the environment and as key virulence factors when acting as an opportunistic pathogen. Assembly of the outer membrane PilQ secretin channel through which the pili are extruded is essential for pilus biogenesis. The P. aeruginosa T4P pilotin, PilF, is required for PilQ outer membrane localization and assembly into secretins and contains six tetratricopeptide (TPR) protein-protein interaction motifs, suggesting that the two proteins interact. In this study, we found that the first four TPR motifs of PilF are sufficient for PilQ outer membrane targeting, oligomerization, and function. Guided by our structure of PilF, site-directed mutagenesis of the protein surface revealed that a hydrophobic groove on the first TPR is required for PilF-mediated PilQ assembly. Deletion of individual domains within PilQ suggests that the N0, KH-like, or secretin domain, but not the C-terminus, interacts with PilF. Purified PilQ was found to pull down PilF from Pseudomonas cell lysates. Together, these data allow us to propose a model for PilF function in the T4P system. PilF interacts directly or indirectly with the PilQ monomer after translocation of both proteins through the inner membrane and acts as a co-chaperone with the Lol system to facilitate transit across the periplasm to the outer membrane. The mechanism of PilQ insertion and assembly, which appears to be independent of the Bam system, remains to be determined.

  6. FKBP5 polymorphisms influence pre-learning stress-induced alterations of learning and memory.

    Science.gov (United States)

    Zoladz, Phillip R; Dailey, Alison M; Nagle, Hannah E; Fiely, Miranda K; Mosley, Brianne E; Brown, Callie M; Duffy, Tessa J; Scharf, Amanda R; Earley, McKenna B; Rorabaugh, Boyd R

    2017-03-01

    FK506 binding protein 51 (FKBP5) is a co-chaperone of heat shock protein 90 and significantly influences glucocorticoid receptor sensitivity. Single nucleotide polymorphisms (SNPs) in the FKBP5 gene are associated with altered hypothalamus-pituitary-adrenal (HPA) axis function, changes in the structure and function of several cognitive brain areas, and increased susceptibility to post-traumatic stress disorder, major depression, bipolar disorder and suicidal events. The mechanisms underlying these associations are largely unknown, but it has been speculated that the influence of these SNPs on emotional memory systems may play a role. In the present study, 112 participants were exposed to the socially evaluated cold pressor test (stress) or control (no stress) conditions immediately prior to learning a list of 42 words. Participant memory was assessed immediately after learning (free recall) and 24 h later (free recall and recognition). Participants provided a saliva sample that enabled the genotyping of three FKBP5 polymorphisms: rs1360780, rs3800373 and rs9296158. Results showed that stress impaired immediate recall in risk allele carriers. More importantly, stress enhanced long-term recall and recognition memory in non-carriers of the risk alleles, effects that were completely absent in risk allele carriers. Follow-up analyses revealed that memory performance was correlated with salivary cortisol levels in non-carriers, but not in carriers. These findings suggest that FKBP5 risk allele carriers may possess a sensitized stress response system, perhaps specifically for stress-induced changes in corticosteroid levels, which might aid our understanding of how SNPs in the FKBP5 gene confer increased risk for stress-related psychological disorders and their related phenotypes. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  7. The Not5 subunit of the ccr4-not complex connects transcription and translation.

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    Zoltan Villanyi

    2014-10-01

    Full Text Available Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.

  8. Oral administration of an HSP90 inhibitor, 17-DMAG, intervenes tumor-cell infiltration into multiple organs and improves survival period for ATL model mice

    International Nuclear Information System (INIS)

    Ikebe, E; Kawaguchi, A; Tezuka, K; Taguchi, S; Hirose, S; Matsumoto, T; Mitsui, T; Senba, K; Nishizono, A; Hori, M; Hasegawa, H; Yamada, Y; Ueno, T; Tanaka, Y; Sawa, H; Hall, W; Minami, Y; Jeang, K T; Ogata, M; Morishita, K; Hasegawa, H; Fujisawa, J; Iha, H

    2013-01-01

    In the peripheral blood leukocytes (PBLs) from the carriers of the human T-lymphotropic virus type-1 (HTLV-1) or the patients with adult T-cell leukemia (ATL), nuclear factor kappaB (NF-κB)-mediated antiapoptotic signals are constitutively activated primarily by the HTLV-1-encoded oncoprotein Tax. Tax interacts with the I κB kinase regulatory subunit NEMO (NF-κB essential modulator) to activate NF-κB, and this interaction is maintained in part by a molecular chaperone, heat-shock protein 90 (HSP90), and its co-chaperone cell division cycle 37 (CDC37). The antibiotic geldanamycin (GA) inhibits HSP90's ATP binding for its proper interaction with client proteins. Administration of a novel water-soluble and less toxic GA derivative, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), to Tax-expressing ATL-transformed cell lines, C8166 and MT4, induced significant degradation of Tax. 17-DMAG also facilitated growth arrest and cellular apoptosis to C8166 and MT4 and other ATL cell lines, although this treatment has no apparent effects on normal PBLs. 17-DMAG also downregulated Tax-mediated intracellular signals including the activation of NF-κB, activator protein 1 or HTLV-1 long terminal repeat in Tax-transfected HEK293 cells. Oral administration of 17-DMAG to ATL model mice xenografted with lymphomatous transgenic Lck-Tax (Lck proximal promoter-driven Tax transgene) cells or HTLV-1-producing tumor cells dramatically attenuated aggressive infiltration into multiple organs, inhibited de novo viral production and improved survival period. These observations identified 17-DMAG as a promising candidate for the prevention of ATL progression

  9. Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy.

    Science.gov (United States)

    Flex, Elisabetta; Niceta, Marcello; Cecchetti, Serena; Thiffault, Isabelle; Au, Margaret G; Capuano, Alessandro; Piermarini, Emanuela; Ivanova, Anna A; Francis, Joshua W; Chillemi, Giovanni; Chandramouli, Balasubramanian; Carpentieri, Giovanna; Haaxma, Charlotte A; Ciolfi, Andrea; Pizzi, Simone; Douglas, Ganka V; Levine, Kara; Sferra, Antonella; Dentici, Maria Lisa; Pfundt, Rolph R; Le Pichon, Jean-Baptiste; Farrow, Emily; Baas, Frank; Piemonte, Fiorella; Dallapiccola, Bruno; Graham, John M; Saunders, Carol J; Bertini, Enrico; Kahn, Richard A; Koolen, David A; Tartaglia, Marco

    2016-10-06

    Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αβ-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective β-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/β-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with

  10. FKBP5 and specific microRNAs via glucocorticoid receptor in the basolateral amygdala involved in the susceptibility to depressive disorder in early adolescent stressed rats.

    Science.gov (United States)

    Xu, Jingjing; Wang, Rui; Liu, Yuan; Liu, Dexiang; Jiang, Hong; Pan, Fang

    2017-12-01

    Exposure to stressful events induces depressive-like symptoms and increases susceptibility to depression. However, the molecular mechanisms are not fully understood. Studies reported that FK506 binding protein51 (FKBP5), the co-chaperone protein of glucocorticoid receptors (GR), plays a crucial role. Further, miR-124a and miR-18a are involved in the regulation of FKBP5/GR function. However, few studies have referred to effects of early life stress on depressive-like behaviours, GR and FKBP5, as well as miR-124a and miR-18a in the basolateral amygdala (BLA) from adolescence to adulthood. This study aimed to examine the dynamic alternations of depressive-like behaviours, GR and FKBP5, as well as miR-124a and miR-18a expressions in the BLA of chronic unpredictable mild stress (CUMS) rats and dexamethasone administration rats during the adolescent period. Meanwhile, the GR antagonist, RU486, was used as a means of intervention. We found that CUMS and dexamethasone administration in the adolescent period induced permanent depressive-like behaviours and memory impairment, decreased GR expression, and increased FKBP5 and miR-124a expression in the BLA of both adolescent and adult rats. However, increased miR-18a expression in the BLA was found only in adolescent rats. Depressive-like behaviours were positively correlated with the level of miR-124a, whereas GR levels were negatively correlated with those in both adolescent and adult rats. Our results suggested FKBP5/GR and miR-124a in the BLA were associated with susceptibility to depressive disorder in the presence of stressful experiences in early life. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

    Science.gov (United States)

    Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

    2015-01-01

    ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

  12. BAG3 affects the nucleocytoplasmic shuttling of HSF1 upon heat stress

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Young-Hee [Department of Biochemistry, Dongguk University College of Oriental Medicine, Gyeongju 780-714 (Korea, Republic of); Ahn, Sang-Gun [Department of Pathology, Chosun University College of Dentistry, Gwangju 501-759 (Korea, Republic of); Kim, Soo-A., E-mail: ksooa@dongguk.ac.kr [Department of Biochemistry, Dongguk University College of Oriental Medicine, Gyeongju 780-714 (Korea, Republic of)

    2015-08-21

    Bcl2-associated athoanogene (BAG) 3 is a member of the co-chaperone BAG family. It is induced by stressful stimuli such as heat shock and heavy metals, and it regulates cellular adaptive responses against stressful conditions. In this study, we identified a novel role for BAG3 in regulating the nuclear shuttling of HSF1 during heat stress. The expression level of BAG3 was induced by heat stress in HeLa cells. Interestingly, BAG3 rapidly translocalized to the nucleus upon heat stress. Immunoprecipitation assay showed that BAG3 interacts with HSF1 under normal and stressed conditions and co-translocalizes to the nucleus upon heat stress. We also demonstrated that BAG3 interacts with HSF1 via its BAG domain. Over-expression of BAG3 down-regulates the level of nuclear HSF1 by exporting it to the cytoplasm during the recovery period. Depletion of BAG3 using siRNA results in reduced nuclear HSF1 and decreased Hsp70 promoter activity. BAG3 in MEF(hsf1{sup −/−}) cells actively translocalizes to the nucleus upon heat stress suggesting that BAG3 plays a key role in the processing of the nucleocytoplasmic shuttling of HSF1 upon heat stress. - Highlights: • The expression level of BAG3 is induced by heat stress. • BAG3 translocates to the nucleus upon heat stress. • BAG3 interacts with HSF1 and co-localizes to the nucleus. • BAG3 is a key regulator for HSF1 nuclear shuttling.

  13. Glutathionylation of the Bacterial Hsp70 Chaperone DnaK Provides a Link between Oxidative Stress and the Heat Shock Response.

    Science.gov (United States)

    Zhang, Hong; Yang, Jie; Wu, Si; Gong, Weibin; Chen, Chang; Perrett, Sarah

    2016-03-25

    DnaK is the major bacterial Hsp70, participating in DNA replication, protein folding, and the stress response. DnaK cooperates with the Hsp40 co-chaperone DnaJ and the nucleotide exchange factor GrpE. Under non-stress conditions, DnaK binds to the heat shock transcription factor σ(32)and facilitates its degradation. Oxidative stress results in temporary inactivation of DnaK due to depletion of cellular ATP and thiol modifications such as glutathionylation until normal cellular ATP levels and a reducing environment are restored. However, the biological significance of DnaK glutathionylation remains unknown, and the mechanisms by which glutathionylation may regulate the activity of DnaK are also unclear. We investigated the conditions under which Escherichia coli DnaK undergoesS-glutathionylation. We observed glutathionylation of DnaK in lysates of E. coli cells that had been subjected to oxidative stress. We also obtained homogeneously glutathionylated DnaK using purified DnaK in the apo state. We found that glutathionylation of DnaK reversibly changes the secondary structure and tertiary conformation, leading to reduced nucleotide and peptide binding ability. The chaperone activity of DnaK was reversibly down-regulated by glutathionylation, accompanying the structural changes. We found that interaction of DnaK with DnaJ, GrpE, or σ(32)becomes weaker when DnaK is glutathionylated, and the interaction is restored upon deglutathionylation. This study confirms that glutathionylation down-regulates the functions of DnaK under oxidizing conditions, and this down-regulation may facilitate release of σ(32)from its interaction with DnaK, thus triggering the heat shock response. Such a mechanism provides a link between oxidative stress and the heat shock response in bacteria. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Deficiency of FK506-binding protein (FKBP) 51 alters sleep architecture and recovery sleep responses to stress in mice.

    Science.gov (United States)

    Albu, Stefana; Romanowski, Christoph P N; Letizia Curzi, M; Jakubcakova, Vladimira; Flachskamm, Cornelia; Gassen, Nils C; Hartmann, Jakob; Schmidt, Mathias V; Schmidt, Ulrike; Rein, Theo; Holsboer, Florian; Hausch, Felix; Paez-Pereda, Marcelo; Kimura, Mayumi

    2014-04-01

    FK506-binding protein 51 (FKBP51) is a co-chaperone of the glucocorticoid receptor, functionally linked to its activity via an ultra-short negative feedback loop. Thus, FKBP51 plays an important regulatory role in the hypothalamic-pituitary-adrenocortical (HPA) axis necessary for stress adaptation and recovery. Previous investigations illustrated that HPA functionality is influenced by polymorphisms in the gene encoding FKBP51, which are associated with both increased protein levels and depressive episodes. Because FKBP51 is a key molecule in stress responses, we hypothesized that its deletion impacts sleep. To study FKBP51-involved changes in sleep, polysomnograms of FKBP51 knockout (KO) mice and wild-type (WT) littermates were compared at baseline and in the recovery phase after 6-h sleep deprivation (SD) and 1-h restraint stress (RS). Using another set of animals, the 24-h profiles of hippocampal free corticosterone levels were also determined. The most dominant effect of FKBP51 deletion appeared as increased nocturnal wake, where the bout length was significantly extended while non-rapid eye movement sleep (NREMS) and rapid eye movement sleep were rather suppressed. After both SD and RS, FKBP51KO mice exhibited less recovery or rebound sleep than WTs, although slow-wave activity during NREMS was higher in KOs, particularly after SD. Sleep compositions of KOs were nearly opposite to sleep profiles observed in human depression. This might result from lower levels of free corticosterone in FKBP51KO mice, confirming reduced HPA reactivity. The results indicate that an FKBP51 deletion yields a pro-resilience sleep phenotype. FKBP51 could therefore be a therapeutic target for stress-induced mood and sleep disorders. © 2013 European Sleep Research Society.

  15. The stress protein BAG3 stabilizes Mcl-1 protein and promotes survival of cancer cells and resistance to antagonist ABT-737.

    Science.gov (United States)

    Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D; Marnett, Lawrence J

    2013-03-08

    Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.

  16. The Role of the Heat Shock Protein B8 (HSPB8 in Motoneuron Diseases

    Directory of Open Access Journals (Sweden)

    Paola Rusmini

    2017-06-01

    Full Text Available Amyotrophic lateral sclerosis (ALS and spinal and bulbar muscular atrophy (SBMA are two motoneuron diseases (MNDs characterized by aberrant protein behavior in affected cells. In familial ALS (fALS and in SBMA specific gene mutations lead to the production of neurotoxic proteins or peptides prone to misfold, which then accumulate in form of aggregates. Notably, some of these proteins accumulate into aggregates also in sporadic ALS (sALS even if not mutated. To prevent proteotoxic stresses detrimental to cells, misfolded and/or aggregated proteins must be rapidly removed by the protein quality control (PQC system. The small heat shock protein B8 (HSPB8 is a chaperone induced by harmful events, like proteasome inhibition. HSPB8 is expressed both in motoneuron and muscle cells, which are both targets of misfolded protein toxicity in MNDs. In ALS mice models, in presence of the mutant proteins, HSPB8 is upregulated both in spinal cord and muscle. HSPB8 interacts with the HSP70 co-chaperone BAG3 and enhances the degradation of misfolded proteins linked to sALS, or causative of fALS and of SBMA. HSPB8 acts by facilitating autophagy, thereby preventing misfolded protein accumulation in affected cells. BAG3 and BAG1 compete for HSP70-bound clients and target them for disposal to the autophagy or proteasome, respectively. Enhancing the selective targeting of misfolded proteins by HSPB8-BAG3-HSP70 to autophagy may also decrease their delivery to the proteasome by the BAG1-HSP70 complex, thereby limiting possible proteasome overwhelming. Thus, approaches aimed at potentiating HSPB8-BAG3 may contribute to the maintenance of proteostasis and may delay MNDs progression.

  17. The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737*

    Science.gov (United States)

    Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D.; Marnett, Lawrence J.

    2013-01-01

    Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery. PMID:23341456

  18. BAG3 protects against hyperthermic stress by modulating NF-κB and ERK activities in human retinoblastoma cells.

    Science.gov (United States)

    Yunoki, Tatsuya; Tabuchi, Yoshiaki; Hayashi, Atsushi; Kondo, Takashi

    2015-03-01

    BCL2-associated athanogene 3 (BAG3), a co-chaperone of HSP70, is a cytoprotective and anti-apoptotic protein that acts against various stresses, including heat stress. Here, we examined the effect of BAG3 on the sensitivity of human retinoblastoma cells to hyperthermia (HT). We examined the effects of BAG3 knockdown on the sensitivity of Y79 and WERI-Rb-1cells to HT (44 °C, 1 h) by evaluating apoptosis and cell proliferation using western blotting, real-time quantitative PCR (qPCR), flow cytometry, and a WST-8 assay kit. Furthermore, we examined the effects of activating nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK) using western blotting and real time qPCR. HT induced considerable apoptosis along with the activation of caspase-3 and chromatin condensation. The sensitivity of Y79 and WERI-Rb-1 cells to HT was significantly enhanced by BAG3 knockdown. Compared to HT alone, the combination of BAG3 knockdown and HT reduced phosphorylation of the inhibitors of kappa B α (IκBα) and p65, a subunit of NF-κB, and degraded IκB kinase γ (IKKγ) during the recovery period after HT. Furthermore, BAG3 knockdown increased the HT-induced phosphorylation of ERK after HT treatment, and the ERK inhibitor U0126 significantly improved the viability of the cells treated with a combination of BAG3 knockdown and HT. The silencing of BAG3 seems to enhance the effects of HT, at least in part, by maintaining HT-induced inactivity of NF-κB and the phosphorylation of ERK. These findings indicate that BAG3 may be a potential molecular target for modifying the outcomes of HT in retinoblastoma.

  19. NIK is required for NF-κB-mediated induction of BAG3 upon inhibition of constitutive protein degradation pathways.

    Science.gov (United States)

    Rapino, F; Abhari, B A; Jung, M; Fulda, S

    2015-03-12

    Recently, we reported that induction of the co-chaperone Bcl-2-associated athanogene 3 (BAG3) is critical for recovery of rhabdomyosarcoma (RMS) cells after proteotoxic stress upon inhibition of the two constitutive protein degradation pathways, that is, the ubiquitin-proteasome system by Bortezomib and the aggresome-autophagy system by histone deacetylase 6 (HDAC6) inhibitor ST80. In the present study, we investigated the molecular mechanisms mediating BAG3 induction under these conditions. Here, we identify nuclear factor-kappa B (NF-κB)-inducing kinase (NIK) as a key mediator of ST80/Bortezomib-stimulated NF-κB activation and transcriptional upregulation of BAG3. ST80/Bortezomib cotreatment upregulates mRNA and protein expression of NIK, which is accompanied by an initial increase in histone H3 acetylation. Importantly, NIK silencing by siRNA abolishes NF-κB activation and BAG3 induction by ST80/Bortezomib. Furthermore, ST80/Bortezomib cotreatment stimulates NF-κB transcriptional activity and upregulates NF-κB target genes. Genetic inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) or by knockdown of p65 blocks the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein expression. Interestingly, inhibition of lysosomal activity by Bafilomycin A1 inhibits ST80/Bortezomib-stimulated IκBα degradation, NF-κB activation and BAG3 upregulation, indicating that IκBα is degraded via the lysosome in the presence of Bortezomib. Thus, by demonstrating a critical role of NIK in mediating NF-κB activation and BAG3 induction upon ST80/Bortezomib cotreatment, our study provides novel insights into mechanisms of resistance to proteotoxic stress in RMS.

  20. BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Chandan Kanta Das

    2018-03-01

    Full Text Available Target-specific treatment modalities are currently not available for triple-negative breast cancer (TNBC, and acquired chemotherapy resistance is a primary obstacle for the treatment of these tumors. Here we employed derivatives of BT-549 and MDA-MB-468 TNBC cell lines that were adapted to grow in the presence of either 5-Fluorouracil, Doxorubicin or Docetaxel in an aim to identify molecular pathways involved in the adaptation to drug-induced cell killing. All six drug-adapted BT-549 and MDA-MB-468 cell lines displayed cross resistance to chemotherapy and decreased apoptosis sensitivity. Expression of the anti-apoptotic co-chaperone BAG3 was notably enhanced in two thirds (4/6 of the six resistant lines simultaneously with higher expression of HSP70 in comparison to parental controls. Doxorubicin-resistant BT-549 (BT-549rDOX20 and 5-Fluorouracil-resistant MDA-MB-468 (MDA-MB-468r5-FU2000 cells were chosen for further analysis with the autophagy inhibitor Bafilomycin A1 and lentiviral depletion of ATG5, indicating that enhanced cytoprotective autophagy partially contributes to increased drug resistance and cell survival. Stable lentiviral BAG3 depletion was associated with a robust down-regulation of Mcl-1, Bcl-2 and Bcl-xL, restoration of drug-induced apoptosis and reduced cell adhesion in these cells, and these death-sensitizing effects could be mimicked with the BAG3/Hsp70 interaction inhibitor YM-1 and by KRIBB11, a selective transcriptional inhibitor of HSF-1. Furthermore, BAG3 depletion was able to revert the EMT-like transcriptional changes observed in BT-549rDOX20 and MDA-MB-468r5-FU2000 cells. In summary, genetic and pharmacological interference with BAG3 is capable to resensitize TNBC cells to treatment, underscoring its relevance for cell death resistance and as a target to overcome therapy resistance of breast cancer.

  1. Proteomic Analysis Reveals a Role for Bcl2-associated Athanogene 3 and Major Vault Protein in Resistance to Apoptosis in Senescent Cells by Regulating ERK1/2 Activation*

    Science.gov (United States)

    Pasillas, Martina P.; Shields, Sarah; Reilly, Rebecca; Strnadel, Jan; Behl, Christian; Park, Robin; Yates, John R.; Klemke, Richard; Gonias, Steven L.; Coppinger, Judith A.

    2015-01-01

    Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence and discovered that Bcl2-associated athanogene 3 (Bag3) is up-regulated after adriamycin treatment in MCF7 cells. Bag3 is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major cell-signaling pathways. Mass spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin to favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in therapy-induced senescence by increasing the level of activation of extracellular signal-regulated kinase1/2 (ERK1/2). Silencing of either Bag3 or MVP decreased ERK1/2 activation and promoted apoptosis in adriamycin-treated cells. An increase in nuclear accumulation of MVP is observed during therapy-induced senescence and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We confirmed that silencing of Bag3 or MVP shifts the response toward apoptosis and regulates ERK1/2 activation in a panel of diverse breast cancer cell lines. This study highlights Bag3-MVP as an important complex that regulates a potent prosurvival signaling pathway and contributes to chemotherapy resistance in breast cancer. PMID:24997994

  2. Fine-tuning of actin dynamics by the HSPB8-BAG3 chaperone complex facilitates cytokinesis and contributes to its impact on cell division.

    Science.gov (United States)

    Varlet, Alice Anaïs; Fuchs, Margit; Luthold, Carole; Lambert, Herman; Landry, Jacques; Lavoie, Josée N

    2017-07-01

    The small heat shock protein HSPB8 and its co-chaperone BAG3 are proposed to regulate cytoskeletal proteostasis in response to mechanical signaling in muscle cells. Here, we show that in dividing cells, the HSPB8-BAG3 complex is instrumental to the accurate disassembly of the actin-based contractile ring during cytokinesis, a process required to allow abscission of daughter cells. Silencing of HSPB8 markedly decreased the mitotic levels of BAG3 in HeLa cells, supporting its crucial role in BAG3 mitotic functions. Cells depleted of HSPB8 were delayed in cytokinesis, remained connected via a disorganized intercellular bridge, and exhibited increased incidence of nuclear abnormalities that result from failed cytokinesis (i.e., bi- and multi-nucleation). Such phenotypes were associated with abnormal accumulation of F-actin at the intercellular bridge of daughter cells at telophase. Remarkably, the actin sequestering drug latrunculin A, like the inhibitor of branched actin polymerization CK666, normalized F-actin during cytokinesis and restored proper cell division in HSPB8-depleted cells, implicating deregulated actin dynamics as a cause of abscission failure. Moreover, this HSPB8-dependent phenotype could be corrected by rapamycin, an autophagy-promoting drug, whereas it was mimicked by drugs impairing lysosomal function. Together, the results further support a role for the HSPB8-BAG3 chaperone complex in quality control of actin-based structure dynamics that are put under high tension, notably during cell cytokinesis. They expand a so-far under-appreciated connection between selective autophagy and cellular morphodynamics that guide cell division.

  3. Zebrafish models of BAG3 myofibrillar myopathy suggest a toxic gain of function leading to BAG3 insufficiency.

    Science.gov (United States)

    Ruparelia, Avnika A; Oorschot, Viola; Vaz, Raquel; Ramm, Georg; Bryson-Richardson, Robert J

    2014-12-01

    Mutations in the co-chaperone Bcl2-associated athanogene 3 (BAG3) can cause myofibrillar myopathy (MFM), a childhood-onset progressive muscle disease, characterized by the formation of protein aggregates and myofibrillar disintegration. In contrast to other MFM-causing proteins, BAG3 has no direct structural role, but regulates autophagy and the degradation of misfolded proteins. To investigate the mechanism of disease in BAG3-related MFM, we expressed wild-type BAG3 or the dominant MFM-causing BAG3 (BAG3(P209L)) in zebrafish. Expression of the mutant protein results in the formation of aggregates that contain wild-type BAG3. Through the stimulation and inhibition of autophagy, we tested the prevailing hypothesis that impaired autophagic function is responsible for the formation of protein aggregates. Contrary to the existing theory, our studies reveal that inhibition of autophagy is not sufficient to induce protein aggregation. Expression of the mutant protein, however, did not induce myofibrillar disintegration and we therefore examined the effect of knocking down Bag3 function. Loss of Bag3 resulted in myofibrillar disintegration, but not in the formation of protein aggregates. Remarkably, BAG3(P209L) is able to rescue the myofibrillar disintegration phenotype, further demonstrating that its function is not impaired. Together, our knockdown and overexpression experiments identify a mechanism whereby BAG3(P209L) aggregates form, gradually reducing the pool of available BAG3, which eventually results in BAG3 insufficiency and myofibrillar disintegration. This mechanism is consistent with the childhood onset and progressive nature of MFM and suggests that reducing aggregation through enhanced degradation or inhibition of nucleation would be an effective therapy for this disease.

  4. BAG3 Expression in Glioblastoma Cells Promotes Accumulation of Ubiquitinated Clients in an Hsp70-dependent Manner*

    Science.gov (United States)

    Gentilella, Antonio; Khalili, Kamel

    2011-01-01

    Disposal of damaged proteins and protein aggregates is a prerequisite for the maintenance of cellular homeostasis and impairment of this disposal can lead to a broad range of pathological conditions, most notably in brain-associated disorders including Parkinson and Alzheimer diseases, and cancer. In this respect, the Protein Quality Control (PQC) pathway plays a central role in the clearance of damaged proteins. The Hsc/Hsp70-co-chaperone BAG3 has been described as a new and critical component of the PQC in several cellular contexts. For example, the expression of BAG3 in the rodent brain correlates with the engagement of protein degradation machineries in response to proteotoxic stress. Nevertheless, little is known about the molecular events assisted by BAG3. Here we show that ectopic expression of BAG3 in glioblastoma cells leads to the activation of an HSF1-driven stress response, as attested by transcriptional activation of BAG3 and Hsp70. BAG3 overexpression determines an accumulation of ubiquitinated proteins and this event requires the N-terminal region, WW domain of BAG3 and the association of BAG3 with Hsp70. The ubiquitination mainly occurs on BAG3-client proteins and the inhibition of proteasomal activity results in a further accumulation of ubiquitinated clients. At the cellular level, overexpression of BAG3 in glioblastoma cell lines, but not in non-glial cells, results in a remarkable decrease in colony formation capacity and this effect is reverted when the binding of BAG3 to Hsp70 is impaired. These observations provide the first evidence for an involvement of BAG3 in the ubiquitination and turnover of its partners. PMID:21233200

  5. BAG3 affects the nucleocytoplasmic shuttling of HSF1 upon heat stress

    International Nuclear Information System (INIS)

    Jin, Young-Hee; Ahn, Sang-Gun; Kim, Soo-A.

    2015-01-01

    Bcl2-associated athoanogene (BAG) 3 is a member of the co-chaperone BAG family. It is induced by stressful stimuli such as heat shock and heavy metals, and it regulates cellular adaptive responses against stressful conditions. In this study, we identified a novel role for BAG3 in regulating the nuclear shuttling of HSF1 during heat stress. The expression level of BAG3 was induced by heat stress in HeLa cells. Interestingly, BAG3 rapidly translocalized to the nucleus upon heat stress. Immunoprecipitation assay showed that BAG3 interacts with HSF1 under normal and stressed conditions and co-translocalizes to the nucleus upon heat stress. We also demonstrated that BAG3 interacts with HSF1 via its BAG domain. Over-expression of BAG3 down-regulates the level of nuclear HSF1 by exporting it to the cytoplasm during the recovery period. Depletion of BAG3 using siRNA results in reduced nuclear HSF1 and decreased Hsp70 promoter activity. BAG3 in MEF(hsf1 −/− ) cells actively translocalizes to the nucleus upon heat stress suggesting that BAG3 plays a key role in the processing of the nucleocytoplasmic shuttling of HSF1 upon heat stress. - Highlights: • The expression level of BAG3 is induced by heat stress. • BAG3 translocates to the nucleus upon heat stress. • BAG3 interacts with HSF1 and co-localizes to the nucleus. • BAG3 is a key regulator for HSF1 nuclear shuttling

  6. Network analysis of genes involved in the enhancement of hyperthermia sensitivity by the knockdown of BAG3 in human oral squamous cell carcinoma cells.

    Science.gov (United States)

    Yunoki, Tatsuya; Tabuchi, Yoshiaki; Hayashi, Atsushi; Kondo, Takashi

    2016-07-01

    BCL2-associated athanogene 3 (BAG3), a co-chaperone of the heat shock 70 kDa protein (HSPA) family of proteins, is a cytoprotective protein that acts against various stresses, including heat stress. The aim of the present study was to identify gene networks involved in the enhancement of hyperthermia (HT) sensitivity by the knockdown (KD) of BAG3 in human oral squamous cell carcinoma (OSCC) cells. Although a marked elevation in the protein expression of BAG3 was detected in human the OSCC HSC-3 cells exposed to HT at 44˚C for 90 min, its expression was almost completely suppressed in the cells transfected with small interfering RNA against BAG3 (siBAG) under normal and HT conditions. The silencing of BAG3 also enhanced the cell death that was increased in the HSC-3 cells by exposure to HT. Global gene expression analysis revealed many genes that were differentially expressed by >2-fold in the cells exposed to HT and transfected with siBAG. Moreover, Ingenuity® pathways analysis demonstrated two unique gene networks, designated as Pro-cell death and Anti-cell death, which were obtained from upregulated genes and were mainly associated with the biological functions of induction and the prevention of cell death, respectively. Of note, the expression levels of genes in the Pro-cell death and Anti-cell death gene networks were significantly elevated and reduced in the HT + BAG3-KD group compared to those in the HT control group, respectively. These results provide further insight into the molecular mechanisms involved in the enhancement of HT sensitivity by the silencing of BAG3 in human OSCC cells.

  7. Bcl2-associated Athanogene 3 Interactome Analysis Reveals a New Role in Modulating Proteasome Activity*

    Science.gov (United States)

    Chen, Ying; Yang, Li-Na; Cheng, Li; Tu, Shun; Guo, Shu-Juan; Le, Huang-Ying; Xiong, Qian; Mo, Ran; Li, Chong-Yang; Jeong, Jun-Seop; Jiang, Lizhi; Blackshaw, Seth; Bi, Li-Jun; Zhu, Heng; Tao, Sheng-Ce; Ge, Feng

    2013-01-01

    Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach. PMID:23824909

  8. The HSPB8-BAG3 chaperone complex is upregulated in astrocytes in the human brain affected by protein aggregation diseases.

    Science.gov (United States)

    Seidel, K; Vinet, J; Dunnen, W F A den; Brunt, E R; Meister, M; Boncoraglio, A; Zijlstra, M P; Boddeke, H W G M; Rüb, U; Kampinga, H H; Carra, S

    2012-02-01

    HSPB8 is a small heat shock protein that forms a complex with the co-chaperone BAG3. Overexpression of the HSPB8-BAG3 complex in cells stimulates autophagy and facilitates the clearance of mutated aggregation-prone proteins, whose accumulation is a hallmark of many neurodegenerative disorders. HSPB8-BAG3 could thus play a protective role in protein aggregation diseases and might be specifically upregulated in response to aggregate-prone protein-mediated toxicity. Here we analysed HSPB8-BAG3 expression levels in post-mortem human brain tissue from patients suffering of the following protein conformation disorders: Alzheimer's disease, Parkinson's disease, Huntington's disease and spinocerebellar ataxia type 3 (SCA3). Western blotting and immunohistochemistry techniques were used to analyse HSPB8 and BAG3 expression levels in fibroblasts from SCA3 patients and post-mortem brain tissues, respectively. In all diseases investigated, we observed a strong upregulation of HSPB8 and a moderate upregulation of BAG3 specifically in astrocytes in the cerebral areas affected by neuronal damage and degeneration. Intriguingly, no significant change in the HSPB8-BAG3 expression levels was observed within neurones, irrespective of their localization or of the presence of proteinaceous aggregates. We propose that the upregulation of HSPB8 and BAG3 may enhance the ability of astrocytes to clear aggregated proteins released from neurones and cellular debris, maintain the local tissue homeostasis and/or participate in the cytoskeletal remodelling that astrocytes undergo during astrogliosis. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological Society.

  9. Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

    Science.gov (United States)

    Antonietti, Patrick; Linder, Benedikt; Hehlgans, Stephanie; Mildenberger, Iris C; Burger, Michael C; Fulda, Simone; Steinbach, Joachim P; Gessler, Florian; Rödel, Franz; Mittelbronn, Michel; Kögel, Donat

    2017-01-01

    Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR. ©2016 American Association for Cancer Research.

  10. The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease

    Directory of Open Access Journals (Sweden)

    Elisabeth Stürner

    2017-06-01

    Full Text Available In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy. One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 (BCL-2-associated athanogene 3. Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer’s disease (tau-protein, Huntington’s disease (mutated huntingtin/polyQ proteins, and amyotrophic lateral sclerosis (mutated SOD1. In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate.

  11. Mutations of the interferon sensitivity-determining region (ISDR) correlate with the complexity of hypervariable region (HVR)-1 in the Japanese variant of hepatitis C virus (HCV) type 1b.

    Science.gov (United States)

    Nakano, Isao; Fukuda, Yoshihide; Katano, Yoshiaki; Toyoda, Hidenori; Hayashi, Kazuhiko; Kumada, Takashi; Nakano, Satoshi

    2004-09-01

    Hepatitis C virus (HCV) genotype 1b comprises mainly two subtypes in Japan, each named for its geographic prevalence (Japan-specific, J type; worldwide, W type). Because the newly identified subtypes have not been fully characterized, the present study directed this issue from virological viewpoints such as hypervariable region (HVR)-1 as well as interferon (IFN) sensitivity-determining region (ISDR). Fifty chronic hepatitis patients with HCV 1b (31 men and 19 women; mean age 50.5 years) were enrolled, and J/W type was determined according to envelope 1 (E1) sequence as described previously (23 J type and 27 W type). Correlations between age, number of HVR-1 clones, HVR-1 diversity, and ISDR mutations were analyzed in J and W type patients independently. In addition, the sequences of the three HCV regions obtained for the determination of the above genetic factors were studied phylogenetically. The number of HVR-1 clones was significantly higher for J type in comparison with W type (P = 0.044). In the J type-infected patients, the ISDR mutation number was correlated inversely with HVR-1 clone number (P = 0.0001, r = -0.734) and HVR-1 diversity (P = 0.0001, r = -0.722). However, this correlation was not observed in the W type patients. W type patients showed a significant correlation between age and HVR-1 clone number (P = 0.015, r = 0.462). Phylogenetic study revealed that the nonstructural (NS) 5A sequence, which is obtained for ISDR type determination, can distinguish between J and W types. The inverse correlation in J type patients between ISDR mutations and HVR-1 complexity may explain the usefulness of the ISDR for prediction of IFN response only in Japanese patients. This suggests that the ISDR is not directly related to IFN responsiveness, but the degree of HVR-1 complexity may be more important.

  12. Pengaruh Agency Cost of Free Cash Flow Terhadap Tingkat Konservatisme dan Pengujian Efek Moderasi Kebijakan Hutang, Pendistribusian Kas, Persistensi Kas, dan Tata Kelola Perusahaan

    Directory of Open Access Journals (Sweden)

    Hendro Hendro

    2015-01-01

    Full Text Available This research investigates whether the J-type firm (high agency cost of free cash flow provide more conservative financial statements than non J-type firm. Besides, this research also aim to examine the moderating effects of debt, dividend, stock repurchase, cash persistency, and corporate governance on the relationship between the level of agency cost of free cash flow and conservatism level of financial statements. This research uses two measurement of conservatism, namely accrual conservatism and market value conservatism. Research sample includes manufacturing companies listed in the Indonesian Stock Exchange for the year 2007, 2008 and 2010. The result proves that there is a positive and significant relationship between the level of agency cost of free cash flow and the two measurement of conservatism level of financial stataments. However, this research shows that there is no effect of the moderating variables on that relationship.

  13. On PR group classes and PR algebra membership

    International Nuclear Information System (INIS)

    Lebedenko, V.M.

    1978-01-01

    The necessary and sufficient conditions are found for the membership of Lee algebras to PR algebra class, to algebras with commutation relations of [Hsub(i), Hsub(j)]=rsub(ij)Hsub(i) (i< j) type. Due to this, a criterion is obtained for the membership of the Lee froups to PR group classes, connected and simply connected Lee groups, which Lee algebras are PR algebras

  14. On the PR-algebras

    International Nuclear Information System (INIS)

    Lebedenko, V.M.

    1978-01-01

    The PR-algebras, i.e. the Lie algebras with commutation relations of [Hsub(i),Hsub(j)]=rsub(ij)Hsub(i)(i< j) type are investigated. On the basis of former results a criterion for the membership of 2-solvable Lie algebras to the PR-algebra class is given. The conditions imposed by the criterion are formulated in the linear algebra language

  15. SPIRAL COUNTER-CURRENT CHROMATOGRAPHY OF SMALL MOLECULES, PEPTIDES AND PROTEINS USING THE SPIRAL TUBING SUPPORT ROTOR

    OpenAIRE

    Knight, Martha; Finn, Thomas M.; Zehmer, John; Clayton, Adam; Pilon, Aprile

    2011-01-01

    An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid-liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was c...

  16. Supramolecular Assembly of Complementary Cyanine Salt J-Aggregates

    KAUST Repository

    Li, Zhong’ an; Mukhopadhyay, Sukrit; Jang, Sei-Hum; Bredas, Jean-Luc; Jen, Alex K.-Y.

    2015-01-01

    An understanding of structure–property relationships in cyanine dyes is critical for their design and application. Anionic and cationic cyanines can be organized into complementary cyanine salts, offering potential building blocks to modulate their intra/intermolecular interactions in the solid state. Here, we demonstrate how the structures of these complementary salts can be tuned to achieve highly ordered J-type supramolecular aggregate structures of heptamethine dyes in crystalline solids.

  17. Supramolecular Assembly of Complementary Cyanine Salt J-Aggregates

    KAUST Repository

    Li, Zhong’an

    2015-09-09

    An understanding of structure–property relationships in cyanine dyes is critical for their design and application. Anionic and cationic cyanines can be organized into complementary cyanine salts, offering potential building blocks to modulate their intra/intermolecular interactions in the solid state. Here, we demonstrate how the structures of these complementary salts can be tuned to achieve highly ordered J-type supramolecular aggregate structures of heptamethine dyes in crystalline solids.

  18. Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability.

    Directory of Open Access Journals (Sweden)

    Wai-Lok Yau

    Full Text Available BACKGROUND: Cyclosporin A (CsA has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development. METHODOLOGY/PRINCIPAL FINDINGS: Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 microM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 microM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function. CONCLUSIONS/SIGNIFICANCE: The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate

  19. Identification of regions involved in substrate binding and dimer stabilization within the central domains of yeast Hsp40 Sis1.

    Directory of Open Access Journals (Sweden)

    Júlio C Borges

    Full Text Available Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1 Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2 The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3 The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4 Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.

  20. TaHsfA6f is a transcriptional activator that regulates a suite of heat stress protection genes in wheat (Triticum aestivum L.) including previously unknown Hsf targets.

    Science.gov (United States)

    Xue, Gang-Ping; Drenth, Janneke; McIntyre, C Lynne

    2015-02-01

    Heat stress is a significant environmental factor adversely affecting crop yield. Crop adaptation to high-temperature environments requires transcriptional reprogramming of a suite of genes involved in heat stress protection. This study investigated the role of TaHsfA6f, a member of the A6 subclass of heat shock transcription factors, in the regulation of heat stress protection genes in Triticum aestivum (bread wheat), a poorly understood phenomenon in this crop species. Expression analysis showed that TaHsfA6f was expressed constitutively in green organs but was markedly up-regulated during heat stress. Overexpression of TaHsfA6f in transgenic wheat using a drought-inducible promoter resulted in up-regulation of heat shock proteins (HSPs) and a number of other heat stress protection genes that included some previously unknown Hsf target genes such as Golgi anti-apoptotic protein (GAAP) and the large isoform of Rubisco activase. Transgenic wheat plants overexpressing TaHsfA6f showed improved thermotolerance. Transactivation assays showed that TaHsfA6f activated the expression of reporter genes driven by the promoters of several HSP genes (TaHSP16.8, TaHSP17, TaHSP17.3, and TaHSP90.1-A1) as well as TaGAAP and TaRof1 (a co-chaperone) under non-stress conditions. DNA binding analysis revealed the presence of high-affinity TaHsfA6f-binding heat shock element-like motifs in the promoters of these six genes. Promoter truncation and mutagenesis analyses identified TaHsfA6f-binding elements that were responsible for transactivation of TaHSP90.1-A1 and TaGAAP by TaHsfA6f. These data suggest that TaHsfA6f is a transcriptional activator that directly regulates TaHSP, TaGAAP, and TaRof1 genes in wheat and its gene regulatory network has a positive impact on thermotolerance. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  1. Rice sHsp genes: genomic organization and expression profiling under stress and development

    Directory of Open Access Journals (Sweden)

    Grover Anil

    2009-08-01

    Full Text Available Abstract Background Heat shock proteins (Hsps constitute an important component in the heat shock response of all living systems. Among the various plant Hsps (i.e. Hsp100, Hsp90, Hsp70 and Hsp20, Hsp20 or small Hsps (sHsps are expressed in maximal amounts under high temperature stress. The characteristic feature of the sHsps is the presence of α-crystallin domain (ACD at the C-terminus. sHsps cooperate with Hsp100/Hsp70 and co-chaperones in ATP-dependent manner in preventing aggregation of cellular proteins and in their subsequent refolding. Database search was performed to investigate the sHsp gene family across rice genome sequence followed by comprehensive expression analysis of these genes. Results We identified 40 α-crystallin domain containing genes in rice. Phylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. The additional 17 genes containing ACD clustered with Acd proteins of Arabidopsis. Detailed scrutiny of 23 sHsp sequences enabled us to categorize these proteins in a revised scheme of classification constituting of 16 cytoplasmic/nuclear, 2 ER, 3 mitochondrial, 1 plastid and 1 peroxisomal genes. In the new classification proposed herein nucleo-cytoplasmic class of sHsps with 9 subfamilies is more complex in rice than in Arabidopsis. Strikingly, 17 of 23 rice sHsp genes were noted to be intronless. Expression analysis based on microarray and RT-PCR showed that 19 sHsp genes were upregulated by high temperature stress. Besides heat stress, expression of sHsp genes was up or downregulated by other abiotic and biotic stresses. In addition to stress regulation, various sHsp genes were differentially upregulated at different developmental stages of the rice plant. Majority of sHsp genes were expressed in seed. Conclusion We identified twenty three sHsp genes and seventeen Acd genes in rice. Three nucleocytoplasmic sHsp genes were found only in monocots. Analysis of expression profiling of sHsp genes revealed

  2. The phosducin-like protein PhLP1 impacts regulation of glycoside hydrolases and light response in Trichoderma reesei

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    Tisch Doris

    2011-12-01

    Full Text Available Abstract Background In the biotechnological workhorse Trichoderma reesei (Hypocrea jecorina transcription of cellulase genes as well as efficiency of the secreted cellulase mixture are modulated by light. Components of the heterotrimeric G-protein pathway interact with light-dependent signals, rendering this pathway a key regulator of cellulase gene expression. Results As regulators of heterotrimeric G-protein signaling, class I phosducin-like proteins, are assumed to act as co-chaperones for G-protein beta-gamma folding and exert their function in response to light in higher eukaryotes. Our results revealed light responsive transcription of the T. reesei class I phosducin-like protein gene phlp1 and indicate a light dependent function of PhLP1 also in fungi. We showed the functions of PhLP1, GNB1 and GNG1 in the same pathway, with one major output being the regulation of transcription of glycoside hydrolase genes including cellulase genes in T. reesei. We found no direct correlation between the growth rate and global regulation of glycoside hydrolases, which suggests that regulation of growth does not occur only at the level of substrate degradation efficiency. Additionally, PhLP1, GNB1 and GNG1 are all important for proper regulation of light responsiveness during long term exposure. In their absence, the amount of light regulated genes increased from 2.7% in wild type to 14% in Δphlp1. Besides from the regulation of degradative enzymes, PhLP1 was also found to impact on the transcription of genes involved in sexual development, which was in accordance with decreased efficiency of fruiting body formation in Δphlp1. The lack of GNB1 drastically diminished ascospore discharge in T. reesei. Conclusions The heterotrimeric G-protein pathway is crucial for the interconnection of nutrient signaling and light response of T. reesei, with the class I phosducin-like protein PhLP1, GNB1 and GNG1 acting as important nodes, which influence light

  3. Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

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    Gonzalez-Mendez Ricardo

    2011-07-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. Results The presence of RNA interference (RNAi mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1 gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90 as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM, an inhibitor of HSP90. Conclusions Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These

  4. Molecular chaperone complexes with antagonizing activities regulate stability and activity of the tumor suppressor LKB1.

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    Gaude, H; Aznar, N; Delay, A; Bres, A; Buchet-Poyau, K; Caillat, C; Vigouroux, A; Rogon, C; Woods, A; Vanacker, J-M; Höhfeld, J; Perret, C; Meyer, P; Billaud, M; Forcet, C

    2012-03-22

    LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90-Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1-STRAD and LKB1-Hsp90-Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1-Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90-Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing activities, Hsp90-Cdc37 and Hsp/Hsc70-CHIP, finely control the cellular level of LKB1 protein.

  5. SIL1 mutations and clinical spectrum in patients with Marinesco-Sjogren syndrome.

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    Krieger, Michael; Roos, Andreas; Stendel, Claudia; Claeys, Kristl G; Sonmez, Fatma Mujgan; Baudis, Michael; Bauer, Peter; Bornemann, Antje; de Goede, Christian; Dufke, Andreas; Finkel, Richard S; Goebel, Hans H; Häussler, Martin; Kingston, Helen; Kirschner, Janbernd; Medne, Livija; Muschke, Petra; Rivier, François; Rudnik-Schöneborn, Sabine; Spengler, Sabrina; Inzana, Francesca; Stanzial, Franco; Benedicenti, Francesco; Synofzik, Matthis; Lia Taratuto, Ana; Pirra, Laura; Tay, Stacey Kiat-Hong; Topaloglu, Haluk; Uyanik, Gökhan; Wand, Dorothea; Williams, Denise; Zerres, Klaus; Weis, Joachim; Senderek, Jan

    2013-12-01

    Marinesco-Sjögren syndrome is a rare autosomal recessive multisystem disorder featuring cerebellar ataxia, early-onset cataracts, chronic myopathy, variable intellectual disability and delayed motor development. More recently, mutations in the SIL1 gene, which encodes an endoplasmic reticulum resident co-chaperone, were identified as the main cause of Marinesco-Sjögren syndrome. Here we describe the results of SIL1 mutation analysis in 62 patients presenting with early-onset ataxia, cataracts and myopathy or combinations of at least two of these. We obtained a mutation detection rate of 60% (15/25) among patients with the characteristic Marinesco-Sjögren syndrome triad (ataxia, cataracts, myopathy) whereas the detection rate in the group of patients with more variable phenotypic presentation was below 3% (1/37). We report 16 unrelated families with a total of 19 different SIL1 mutations. Among these mutations are 15 previously unreported changes, including single- and multi-exon deletions. Based on data from our screening cohort and data compiled from the literature we found that SIL1 mutations are invariably associated with the combination of a cerebellar syndrome and chronic myopathy. Cataracts were observed in all patients beyond the age of 7 years, but might be missing in infants. Six patients with SIL1 mutations had no intellectual disability, extending the known wide range of cognitive capabilities in Marinesco-Sjögren syndrome to include normal intelligence. Modestly constant features were somatic growth retardation, skeletal abnormalities and pyramidal tract signs. Examination of mutant SIL1 expression in cultured patient lymphoblasts suggested that SIL1 mutations result in severely reduced SIL1 protein levels irrespective of the type and position of mutations. Our data broaden the SIL1 mutation spectrum and confirm that SIL1 is the major Marinesco-Sjögren syndrome gene. SIL1 patients usually present with the characteristic triad but cataracts might be

  6. Reduced DNA methylation of FKBP5 in Cushing's syndrome.

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    Resmini, Eugenia; Santos, Alicia; Aulinas, Anna; Webb, Susan M; Vives-Gilabert, Yolanda; Cox, Olivia; Wand, Gary; Lee, Richard S

    2016-12-01

    FKBP5 encodes a co-chaperone of HSP90 protein that regulates intracellular glucocorticoid receptor sensitivity. When it is bound to the glucocorticoid receptor complex, cortisol binds with lower affinity to glucocorticoid receptor. Cushing's syndrome is associated with memory deficits, smaller hippocampal volumes, and wide range of cognitive impairments. We aimed at evaluating blood DNA methylation of FKBP5 and its relationship with memory and hippocampal volumes in Cushing's syndrome patients. Polymorphism rs1360780 in FKBP5 has also been assessed to determine whether genetic variations can also govern CpG methylation. Thirty-two Cushing's syndrome patients and 32 matched controls underwent memory tests, 3-Tesla MRI of the brain, and DNA extraction from total leukocytes. DNA samples were bisulfite treated, PCR amplified, and pyrosequenced to assess a total of 41CpG-dinucleotides in the introns 1, 2, 5, and 7 of FKBP5. Significantly lower intronic FKBP5 DNA methylation in CS patients compared to controls was observed in ten CpG-dinucleotides. DNA methylation at these CpGs correlated with left and right HV (Intron-2-Region-2-CpG-3: LHV, r = 0.73, p = 0.02; RHV, r = 0.58, p = 0.03). Cured and active CS patients showed both lower methylation of intron 2 (92.37, 91.8, and 93.34 %, respectively, p = 0.03 for both) and of intron 7 (77.08, 73.74, and 79.71 %, respectively, p = 0.02 and p < 0.01) than controls. Twenty-two subjects had the CC genotype, 34 had the TC genotype, and eight had the TT genotype. Lower average DNA methylation in intron 7 was observed in the TT subjects compared to CC (72.5vs. 79.5 %, p = 0.02) and to TC (72.5 vs. 79.0 %, p = 0.03). Our data demonstrate, for the first time, a reduction of intronic DNA methylation of FKBP5 in CS patients.

  7. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli

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    Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

    2016-01-01

    S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

  8. Insight into PreImplantation Factor (PIF* mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP through essential RIKP [corrected] binding site.

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    Eytan R Barnea

    Full Text Available Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control. Murine embryo (d10 lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS. In silico evaluation examined binding of PIF to critical targets, using mutation analysis.PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90, co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a

  9. BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells.

    Science.gov (United States)

    Das, Chandan Kanta; Linder, Benedikt; Bonn, Florian; Rothweiler, Florian; Dikic, Ivan; Michaelis, Martin; Cinatl, Jindrich; Mandal, Mahitosh; Kögel, Donat

    2018-03-01

    Target-specific treatment modalities are currently not available for triple-negative breast cancer (TNBC), and acquired chemotherapy resistance is a primary obstacle for the treatment of these tumors. Here we employed derivatives of BT-549 and MDA-MB-468 TNBC cell lines that were adapted to grow in the presence of either 5-Fluorouracil, Doxorubicin or Docetaxel in an aim to identify molecular pathways involved in the adaptation to drug-induced cell killing. All six drug-adapted BT-549 and MDA-MB-468 cell lines displayed cross resistance to chemotherapy and decreased apoptosis sensitivity. Expression of the anti-apoptotic co-chaperone BAG3 was notably enhanced in two thirds (4/6) of the six resistant lines simultaneously with higher expression of HSP70 in comparison to parental controls. Doxorubicin-resistant BT-549 (BT-549 r DOX 20 ) and 5-Fluorouracil-resistant MDA-MB-468 (MDA-MB-468 r 5-FU 2000 ) cells were chosen for further analysis with the autophagy inhibitor Bafilomycin A1 and lentiviral depletion of ATG5, indicating that enhanced cytoprotective autophagy partially contributes to increased drug resistance and cell survival. Stable lentiviral BAG3 depletion was associated with a robust down-regulation of Mcl-1, Bcl-2 and Bcl-xL, restoration of drug-induced apoptosis and reduced cell adhesion in these cells, and these death-sensitizing effects could be mimicked with the BAG3/Hsp70 interaction inhibitor YM-1 and by KRIBB11, a selective transcriptional inhibitor of HSF-1. Furthermore, BAG3 depletion was able to revert the EMT-like transcriptional changes observed in BT-549 r DOX 20 and MDA-MB-468 r 5-FU 2000 cells. In summary, genetic and pharmacological interference with BAG3 is capable to resensitize TNBC cells to treatment, underscoring its relevance for cell death resistance and as a target to overcome therapy resistance of breast cancer. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Heat Stress and Lipopolysaccharide Stimulation of Chicken Macrophage-Like Cell Line Activates Expression of Distinct Sets of Genes.

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    Anna Slawinska

    Full Text Available Acute heat stress requires immediate adjustment of the stressed individual to sudden changes of ambient temperatures. Chickens are particularly sensitive to heat stress due to development of insufficient physiological mechanisms to mitigate its effects. One of the symptoms of heat stress is endotoxemia that results from release of the lipopolysaccharide (LPS from the guts. Heat-related cytotoxicity is mitigated by the innate immune system, which is comprised mostly of phagocytic cells such as monocytes and macrophages. The objective of this study was to analyze the molecular responses of the chicken macrophage-like HD11 cell line to combined heat stress and lipopolysaccharide treatment in vitro. The cells were heat-stressed and then allowed a temperature-recovery period, during which the gene expression was investigated. LPS was added to the cells to mimic the heat-stress-related endotoxemia. Semi high-throughput gene expression analysis was used to study a gene panel comprised of heat shock proteins, stress-related genes, signaling molecules and immune response genes. HD11 cell line responded to heat stress with increased mRNA abundance of the HSP25, HSPA2 and HSPH1 chaperones as well as DNAJA4 and DNAJB6 co-chaperones. The anti-apoptotic gene BAG3 was also highly up-regulated, providing evidence that the cells expressed pro-survival processes. The immune response of the HD11 cell line to LPS in the heat stress environment (up-regulation of CCL4, CCL5, IL1B, IL8 and iNOS was higher than in thermoneutral conditions. However, the peak in the transcriptional regulation of the immune genes was after two hours of temperature-recovery. Therefore, we propose the potential influence of the extracellular heat shock proteins not only in mitigating effects of abiotic stress but also in triggering the higher level of the immune responses. Finally, use of correlation networks for the data analysis aided in discovering subtle differences in the gene

  11. Age-Related Decrease in Stress Responsiveness and Proactive Coping in Male Mice.

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    Oh, Hee-Jin; Song, Minah; Kim, Young Ki; Bae, Jae Ryong; Cha, Seung-Yun; Bae, Ji Young; Kim, Yeongmin; You, Minsu; Lee, Younpyo; Shim, Jieun; Maeng, Sungho

    2018-01-01

    Coping is a strategic approach to dealing with stressful situations. Those who use proactive coping strategies tend to accept changes and act before changes are expected. In contrast, those who use reactive coping are less flexible and more likely to act in response to changes. However, little research has assessed how coping style changes with age. This study investigated age-related changes in coping strategies and stress responsiveness and the influence of age on the processing of conditioned fear memory in 2-, 12- and 23-month-old male mice. Coping strategy was measured by comparing the escape latency in an active avoidance test and by comparing responses to a shock prod. The results showed that proactivity in coping response gradually decreased with age. Stress responsiveness, measured by stress-induced concentration of corticosterone, was also highest in 2-month-old mice and decreased with age. Consolidation of fear memory was highest in 12-month-old mice and was negatively correlated with the degree of stress responsiveness and proactivity in coping. Fear extinction did not differ among age groups and was not correlated with stress responsiveness or the proactivity of coping. However, the maintenance of extinct fear memory, which was best in 2-month-old mice and worst in 12-month-old mice, was negatively correlated with stress responsiveness but not with coping style. Age-dependent changes in the expression of glucocorticoid receptor (GR) and its regulatory co-chaperones, which are accepted mechanisms for stress hormone stimulation, were measured in the hippocampus. The expression of GR was increased at 12 months compared to other age groups. There were no differences in Hsp70 and BAG1 expression by age. These results can be summarized as follows: (1) stress responsiveness and proactivity in coping decreased with age class; (2) consolidation of fear memory was negatively correlated with both stress responsiveness and proactivity; however, maintenance of

  12. Introduction of a unique tryptophan residue into various positions of Bacillus licheniformis DnaK.

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    Chen, Bo-En; Lin, Min-Guan; Lo, Huei-Fen; Wang, Tzu-Fan; Chi, Meng-Chun; Lin, Long-Liu

    2013-01-01

    Site-directed mutagenesis together with biochemical and biophysical techniques were used to probe effects of single-tryptophan-incorporated mutations on a bacterial molecular chaperone, Bacillus licheniformis DnaK (BlDnaK). Specifically, five phenylalanine residues (Phe(120), Phe(174), Phe(186), Phe(378) and Phe(396)) of BlDnaK were individually replaced by single tryptophans, thus creating site-specific probes for the fluorescence analysis of the protein. The steady-state ATPase activity for BlDnaK, F120W, F174W, F186W, F378W, and F396W was determined to be 76.01, 52.82, 25.32, 53.31, 58.84, and 47.53 nmol Pi/min/mg, respectively. Complementation test revealed that the single mutation at codons 120, 186, and 378 of the dnaK gene still allowed an Escherichia coli dnaK756-Ts strain to grow at a stringent temperature of 44°C. Simultaneous addition of co-chaperones and NR-peptide did not synergistically stimulate the ATPase activity of F174W and F396W, and these two proteins were unable to assist the refolding of GdnHCl-denatured luciferase. The heat-induced denaturation of all variants could be fitted adequately to a three-state model, in agreement with the observation for the wild-type protein. By CD spectral analysis, GdnHCl-induced unfolding transition for BlDnaK was 1.51 M corresponding to ΔG(N-U) of 1.69 kcal/mol; however, the transitions for mutant proteins were 1.07-1.55 M equivalent to ΔG(N-U) of 0.94-2.93 kcal/mol. The emission maximum of single-tryptophan-incorporated variants was in the range of 333.2-335.8 nm. Acrylamide quenching analysis showed that the mutant proteins had a dynamic quenching constant of 3.0-4.2 M(-1). Taken together, these results suggest that the molecular properties of BlDnaK have been significantly changed upon the individual replacement of selected phenylalanine residues by tryptophan. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. A role for synapsin in FKBP51 modulation of stress responsiveness: Convergent evidence from animal and human studies.

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    Schmidt, Ulrike; Buell, Dominik R; Ionescu, Irina A; Gassen, Nils C; Holsboer, Florian; Cox, Marc B; Novak, Bozidar; Huber, Christine; Hartmann, Jakob; Schmidt, Mathias V; Touma, Chadi; Rein, Theo; Herrmann, Leonie

    2015-02-01

    Both the molecular co-chaperone FKBP51 and the presynaptic vesicle protein synapsin (alternatively spliced from SYN1-3) are intensively discussed players in the still insufficiently explored pathobiology of psychiatric disorders such as major depression, schizophrenia and posttraumatic stress disorder (PTSD). To address their still unknown interaction, we compared the expression levels of synapsin and five other neurostructural and HPA axis related marker proteins in the prefrontal cortex (PFC) and the hippocampus of restrained-stressed and unstressed Fkbp5 knockout mice and corresponding wild-type littermates. In addition, we compared and correlated the gene expression levels of SYN1, SYN2 and FKBP5 in three different online datasets comprising expression data of human healthy subjects as well as of predominantly medicated patients with different psychiatric disorders. In summary, we found that Fkbp5 deletion, which we previously demonstrated to improve stress-coping behavior in mice, prevents the stress-induced decline in prefrontal cortical (pc), but not in hippocampal synapsin expression. Accordingly, pc, but not hippocampal, synapsin protein levels correlated positively with a more active mouse stress coping behavior. Searching for an underlying mechanism, we found evidence that deletion of Fkbp5 might prevent stress-induced pc synapsin loss, at least in part, through improvement of pc Akt kinase activity. These results, together with our finding that FKBP5 and SYN1 mRNA levels were regulated in opposite directions in the PFC of schizophrenic patients, who are known for exhibiting an altered stress-coping behavior, provide the first evidence of a role for pc synapsin in FKBP51 modulation of stress responsiveness. This role might extend to other tissues, as we found FKBP5 and SYN1 levels to correlate inversely not only in human PFC samples but also in other expression sites. The main limitation of this study is the small number of individuals included in the

  14. A micellar model system for the role of zeaxanthin in the non-photochemical quenching process of photosynthesis--chlorophyll fluorescence quenching by the xanthophylls.

    Science.gov (United States)

    Avital, Shlomo; Brumfeld, Vlad; Malkin, Shmuel

    2006-07-01

    To get an insight to the mechanism of the zeaxanthin-dependent non-photochemical quenching in photosystem II of photosynthesis, we probed the interaction of some xanthophylls with excited chlorophyll-a by trapping both pigments in micelles of triton X-100. Optimal distribution of pigments among micelles was obtained by proper control of the micelle concentration, using formamide in the reaction mixture, which varies the micellar aggregation number over three orders of magnitude. The optimal reaction mixture was obtained around 40% (v/v) formamide in 0.2-0.4% (v/v) triton X-100 in water. Zeaxanthin in the micellar solution exhibited initially absorption and circular dichroism spectral features corresponding to a J-type aggregate. The spectrum was transformed over time (half-time values vary-an average characteristic figure is roughly 20 min) to give features representing an H-type aggregate. The isosbestic point in the series of spectral curves favors the supposition of a rather simple reaction between two pure J and H-types dimeric species. Violaxanthin exhibited immediately stable spectral features corresponding to a mixture of J-type and more predominately H-type dimers. Lutein, neoxanthin and beta-carotene did not show any aggregated spectral forms in micelles. The spectral features in micelles were compared to spectra in aqueous acetone, where the assignment to various aggregated types was established previously. The specific tendency of zeaxanthin to form the J-type dimer (or aggregate) could be important for its function in photosynthesis. The abilities of five carotenoids (zeaxanthin, violaxanthin, lutein, neoxanthin and beta-carotene) to quench chlorophyll-a fluorescence were compared. Zeaxanthin, in its two micellar dimeric forms, and beta-carotene were comparable good quenchers of chlorophyll-a fluorescence. Violaxanthin was a much weaker quencher, if at all. Lutein and neoxanthin rather enhanced the fluorescence. The implications to non

  15. Comparative Analysis of Putative Orthologues of Mitochondrial Import Motor Subunit: Pam18 and Pam16 in Plants

    OpenAIRE

    Chen, Xuejin; Ghazanfar, Bushra; Khan, Abdul Rehman; Hayat, Sikandar; Cheng, Zhihui

    2013-01-01

    Pam18/Tim14 and Pam16/Tim16, highly conserved proteins among eukaryotes, are two essential subunits of protein import motors localized in the inner mitochondrial membrane. The heterodimer formed by Pam18 and Pam16 via their J-type domains serves a regulatory function in protein translocation. Here, we report that thirty-one Pam18 and twenty-six Pam16 putative orthologues in twelve plant species were identified and analyzed through bioinformatics strategy. Results data revealed that Pam18 and ...

  16. Association of FKBP51 with priming of autophagy pathways and mediation of antidepressant treatment response: evidence in cells, mice, and humans.

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    Nils C Gassen

    2014-11-01

    Full Text Available FK506 binding protein 51 (FKBP51 is an Hsp90 co-chaperone and regulator of the glucocorticoid receptor, and consequently of stress physiology. Clinical studies suggest a genetic link between FKBP51 and antidepressant response in mood disorders; however, the underlying mechanisms remain elusive. The objective of this study was to elucidate the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy.Established cell lines, primary neural cells, human blood cells of healthy individuals and patients with depression, and mice were treated with antidepressants. Mice were tested for several neuroendocrine and behavioral parameters. Protein interactions and autophagic pathway activity were mainly evaluated by co-immunoprecipitation and Western blots. We first show that the effects of acute antidepressant treatment on behavior are abolished in FKBP51 knockout (51KO mice. Autophagic markers, such as the autophagy initiator Beclin1, were increased following acute antidepressant treatment in brains from wild-type, but not 51KO, animals. FKBP51 binds to Beclin1, changes decisive protein interactions and phosphorylation of Beclin1, and triggers autophagic pathways. Antidepressants and FKBP51 exhibited synergistic effects on these pathways. Using chronic social defeat as a depression-relevant stress model in combination with chronic paroxetine (PAR treatment revealed that the stress response, as well as the effects of antidepressants on behavior and autophagic markers, depends on FKBP51. In human blood cells of healthy individuals, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways. Importantly, the clinical antidepressant response of patients with depression (n = 51 could be predicted by the antidepressant response of autophagic markers in patient-derived peripheral blood lymphocytes cultivated and treated ex vivo (Beclin1/amitriptyline: r = 0.572, p = 0.003; Beclin1/PAR: r

  17. Microsecond-scale electric field pulses in cloud lightning discharges

    Science.gov (United States)

    Villanueva, Y.; Rakov, V. A.; Uman, M. A.; Brook, M.

    1994-01-01

    From wideband electric field records acquired using a 12-bit digitizing system with a 500-ns sampling interval, microsecond-scale pulses in different stages of cloud flashes in Florida and New Mexico are analyzed. Pulse occurrence statistics and waveshape characteristics are presented. The larger pulses tend to occur early in the flash, confirming the results of Bils et al. (1988) and in contrast with the three-stage representation of cloud-discharge electric fields suggested by Kitagawa and Brook (1960). Possible explanations for the discrepancy are discussed. The tendency for the larger pulses to occur early in the cloud flash suggests that they are related to the initial in-cloud channel formation processes and contradicts the common view found in the atmospheric radio-noise literature that the main sources of VLF/LF electromagnetic radiation in cloud flashes are the K processes which occur in the final, or J type, part of the cloud discharge.

  18. Abundance analyses of thirty cool carbon stars

    International Nuclear Information System (INIS)

    Utsumi, Kazuhiko

    1985-01-01

    The results were previously obtained by use of the absolute gf-values and the cosmic abundance as a standard. These gf-values were found to contain large systematic errors, and as a result, the solar photospheric abundances were revised. Our previous results, therefore, must be revised by using new gf-values, and abundance analyses are extended for as many carbon stars as possible. In conclusion, in normal cool carbon stars heavy metals are overabundant by factors of 10 - 100 and rare-earth elements are overabundant by a factor of about 10, and in J-type cool carbon stars, C 12 /C 13 ratio is smaller, C 2 and CN bands and Li 6708 are stronger than in normal cool carbon stars, and the abundances of s-process elements with respect to Fe are nearly normal. (Mori, K.)

  19. FLUORINE ABUNDANCES IN GALACTIC ASYMPTOTIC GIANT BRANCH STARS

    International Nuclear Information System (INIS)

    Abia, C.; Cristallo, S.; DomInguez, I.; Cunha, K.; Hinkle, K.; Smith, V. V.; De Laverny, P.; Recio-Blanco, A.; Eriksson, K.; Wahlin, R.; Gialanella, L.; Imbriani, G.; Straniero, O.

    2010-01-01

    An analysis of the fluorine abundance in Galactic asymptotic giant branch (AGB) carbon stars (24 N-type, 5 SC-type, and 5 J-type) is presented. This study uses the state-of-the-art carbon-rich atmosphere models and improved atomic and molecular line lists in the 2.3 μm region. Significantly lower F abundances are obtained in comparison to previous studies in the literature. This difference is mainly due to molecular blends. In the case of carbon stars of SC-type, differences in the model atmospheres are also relevant. The new F enhancements are now in agreement with the most recent theoretical nucleosynthesis models in low-mass AGB stars, solving the long-standing problem of F in Galactic AGB stars. Nevertheless, some SC-type carbon stars still show larger F abundances than predicted by stellar models. The possibility that these stars are of larger mass is briefly discussed.

  20. Aggregation of non-amphiphilic bathophenanthroline in the restricted geometry of Langmuir-Blodgett films with two different matrices

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Ajitesh [Centre of Studies in Surface Science and Technology, School of Chemistry, Sambalpur University, Jyoti Vihar-768019, Orissa (India); Panigrahi, Simanchalo [Department of Physics, National Institute of Technology, Rourkela-788011, Orissa (India); Nath, Ranendu Kumar [Department of Chemistry, Tripura University, Suryamaninagar-799130, Tripura (India); Deb, Subrata [Department of Physics, Iswar Chandra Vidyasagar College, Belonia-799155, Tripura (India); Sinha, Tripurari Prasad [Department of Physics, Bose Institute, Kolkata-700009, West Bengal (India); Mishra, Bijay Kumar, E-mail: bijaym@hotmail.com [Centre of Studies in Surface Science and Technology, School of Chemistry, Sambalpur University, Jyoti Vihar-768019, Orissa (India)

    2011-10-31

    The behavior of binary mixed Langmuir monolayers from the mixture of non-amphiphilic bathophenanthroline (BPH) and behenic acid (BA)/poly(methyl methacrylate) (PMMA) spread on aqueous subphase was investigated on the basis of the analysis of surface pressure-average area per molecule ({pi}-A) isotherms complemented with UV-vis absorption spectroscopy and scanning electron microscopy. In addition, the miscibility of the components in the two investigated mixed systems (BPH/BA and BPH/PMMA) was also tested by using additivity and surface phase rules. The plots of area per molecule versus mole fraction suggest that BPH and BA are immiscible, whereas BPH and PMMA mixtures show non-ideal behavior at low surface pressures and complete miscibility or immiscibility at higher surface pressures. Spectroscopic study reveals that J-type of aggregates is formed in the mixed films. Scanning electron microscopic study supports the presence of aggregates.

  1. Massive boson-fermion degeneracy and the early structure of the universe

    International Nuclear Information System (INIS)

    Kounnas, C.

    2008-01-01

    The existence of a new kind of massive boson-fermion symmetry is shown explicitly in the framework of the heterotic, type II and type II orientifold superstring theories. The target space-time is two-dimensional. Higher dimensional models are defined via large marginal deformations of J anti J-type. The spectrum of the initial undeformed two dimensional vacuum consists of massless boson degrees of freedom, while all massive boson and fermion degrees of freedom exhibit a new Massive Spectrum Degeneracy Symmetry (MSDS). This precise property, distinguishes the MSDS theories from the well known supersymmetric SUSY-theories. Some proposals are stated in the framework of these theories concerning the structure of: (i) The Early Non-singular Phase of the Universe, (ii) The two dimensional boundary theory of AdS 3 Black-Holes, (iii) Plausible applications of the MSDS theories in particle physics, alternative to SUSY. (Abstract Copyright [2008], Wiley Periodicals, Inc.)

  2. Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

    International Nuclear Information System (INIS)

    Kanakura, Y.; Kuriu, A.; Waki, N.; Nakano, T.; Asai, H.; Yonezawa, T.; Kitamura, Y.

    1988-01-01

    Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. Large mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas medium and small mast cell colonies are produced by morphologically identifiable mast cells (M-CFU-Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared, suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU-Mast from the bloodstream and to inhibit the differentiation of L-CFU-Mast to M-CFU-Mast

  3. Dust, ice and gas in time (DIGIT): Herschel and Spitzer spectro-imaging of SMM3 and SMM4 in Serpens

    Science.gov (United States)

    Dionatos, O.; Jørgensen, J. K.; Green, J. D.; Herczeg, G. J.; Evans, N. J.; Kristensen, L. E.; Lindberg, J. E.; van Dishoeck, E. F.

    2013-10-01

    Context. Mid- and far-infrared observations of the environment around embedded protostars reveal a plethora of high excitation molecular and atomic emission lines. Different mechanisms for the origin of these lines have been proposed, including shocks induced by protostellar jets and radiation or heating by the embedded protostar of its immediate surroundings. Aims: By studying of the most important molecular and atomic coolants, we aim at constraining the physical conditions around the embedded protostars SMM3 and SMM4 in the Serpens molecular cloud core and measuring the CO/H2 ratio in warm gas. Methods: Spectro-imaging observations from the Spitzer Infrared Spectrograph (IRS) and the Herschel Photodetector Array Camera and Spectrometer (PACS) provide an almost complete wavelength coverage between 5 and 200 μm. Within this range, emission from all major molecular (H2, CO, H2O and OH) and many atomic ([OI], [CII], [FeII], [SiII] and [SI]) coolants of excited gas are detected. Emission line maps reveal the morphology of the observed emission and indicate associations between the different species. The excitation conditions for molecular species are assessed through rotational diagrams. Emission lines from major coolants are compared to the results of steady-state C- and J-type shock models. Results: Line emission tends to peak at distances of ~10-20″ from the protostellar sources with all but [CII] peaking at the positions of outflow shocks seen in near-IR and sub-millimeter interferometric observations. The [CII] emission pattern suggests that it is most likely excited from energetic UV radiation originating from the nearby flat-spectrum source SMM6. Excitation analysis indicates that H2 and CO originate in gas at two distinct rotational temperatures of ~300 K and 1000 K, while the excitation temperature for H2O and OH is ~100-200 K. The morphological and physical association between CO and H2 suggests a common excitation mechanism, which allows direct

  4. Theoretical studies of superconductivity in doped BaCoSO

    Science.gov (United States)

    Qin, Shengshan; Li, Yinxiang; Zhang, Qiang; Le, Congcong; Hu, Jiangping

    2018-06-01

    We investigate superconductivity that may exist in the doped BaCoSO, a multi-orbital Mott insulator with a strong antiferromagnetic ground state. The superconductivity is studied in both t-J type and Hubbard type multi-orbital models by mean field approach and random phase approximation (RPA) analysis. Even if there is no C 4 rotational symmetry, it is found that the system still carries a d-wave like pairing symmetry state with gapless nodes and sign changed superconducting order parameters on Fermi surfaces. The results are largely doping insensitive. In this superconducting state, the three {t_{{2_g}}} orbitals have very different superconducting form factors in momentum space. In particular, the intra-orbital pairing of the {d_{{x^2} - {y^2}}} orbital has an s-wave like pairing form factor. The two methods also predict very different pairing strength on different parts of Fermi surfaces. These results suggest that BaCoSO and related materials can be a new ground to test and establish fundamental principles for unconventional high temperature superconductivity.

  5. Polyfunctional inorganic-organic hybrid materials: an unusual kind of NLO active layered mixed metal oxalates with tunable magnetic properties and very large second harmonic generation.

    Science.gov (United States)

    Cariati, Elena; Macchi, Roberto; Roberto, Dominique; Ugo, Renato; Galli, Simona; Casati, Nicola; Macchi, Piero; Sironi, Angelo; Bogani, Lapo; Caneschi, Andrea; Gatteschi, Dante

    2007-08-01

    Mixed M(II)/M(III) metal oxalates, as "stripes" connected through strong hydrogen bonding by para-dimethylaminobenzaldeide (DAMBA) and water, form an organic-inorganic 2D network that enables segregation in layers of the cationic organic NLO-phore trans-4-(4-dimethylaminostyryl)-1-methylpyridinium, [DAMS+]. The crystalline hybrid materials obtained have the general formula [DAMS]4[M2M'(C2O4)6].2DAMBA.2H2O (M = Rh, Fe, Cr; M' = Mn, Zn), and their overall three-dimensional packing is non-centrosymmetric and polar, therefore suitable for second harmonic generation (SHG). All the compounds investigated are characterized by an exceptional SHG activity, due both to the large molecular quadratic hyperpolarizability of [DAMS+] and to the efficiency of the crystalline network which organizes [DAMS+] into head-to-tail arranged J-type aggregates. The tunability of the pairs of metal ions allows exploiting also the magnetic functionality of the materials. Examples containing antiferro-, ferro-, and ferri-magnetic interactions (mediated by oxalato bridges) are obtained by coupling proper M(III) ions (Fe, Cr, Rh) with M(II) (Mn, Zn). This shed light on the role of weak next-nearest-neighbor interactions and main nearest-neighbor couplings along "stripes" of mixed M(II)/M(III) metal oxalates of the organic-inorganic 2D network, thus suggesting that these hybrid materials may display isotropic 1D magnetic properties along the mixed M(II)/M(III) metal oxalates "stripes".

  6. Inhomogeneities and the Modeling of Radio Supernovae

    Energy Technology Data Exchange (ETDEWEB)

    Björnsson, C.-I.; Keshavarzi, S. T., E-mail: bjornsson@astro.su.se [Department of Astronomy, AlbaNova University Center, Stockholm University, SE–106 91 Stockholm (Sweden)

    2017-05-20

    Observations of radio supernovae (SNe) often exhibit characteristics not readily accounted for by a homogeneous, spherically symmetric synchrotron model; e.g., flat-topped spectra/light curves. It is shown that many of these deviations from the standard model can be attributed to an inhomogeneous source structure. When inhomogeneities are present, the deduced radius of the source and, hence, the shock velocity, is sensitive to the details of the modeling. As the inhomogeneities are likely to result from the same mechanism that amplify the magnetic field, a comparison between observations and the detailed numerical simulations now under way may prove mutually beneficial. It is argued that the radio emission in Type Ib/c SNe has a small volume filling factor and comes from a narrow region associated with the forward shock, while the radio emission region in SN 1993J (Type IIb) is determined by the extent of the Rayleigh–Taylor instability emanating from the contact discontinuity. Attention is also drawn to the similarities between radio SNe and the structural properties of SN remnants.

  7. The design and fabrication of supramolecular semiconductor nanowires formed by benzothienobenzothiophene (BTBT)-conjugated peptides.

    Science.gov (United States)

    Khalily, Mohammad Aref; Usta, Hakan; Ozdemir, Mehmet; Bakan, Gokhan; Dikecoglu, F Begum; Edwards-Gayle, Charlotte; Hutchinson, Jessica A; Hamley, Ian W; Dana, Aykutlu; Guler, Mustafa O

    2018-05-18

    π-Conjugated small molecules based on a [1]benzothieno[3,2-b]benzothiophene (BTBT) unit are of great research interest in the development of solution-processable semiconducting materials owing to their excellent charge-transport characteristics. However, the BTBT π-core has yet to be demonstrated in the form of electro-active one-dimensional (1D) nanowires that are self-assembled in aqueous media for potential use in bioelectronics and tissue engineering. Here we report the design, synthesis, and self-assembly of benzothienobenzothiophene (BTBT)-peptide conjugates, the BTBT-peptide (BTBT-C3-COHN-Ahx-VVAGKK-Am) and the C8-BTBT-peptide (C8-BTBT-C3-COHN-Ahx-VVAGKK-Am), as β-sheet forming amphiphilic molecules, which self-assemble into highly uniform nanofibers in water with diameters of 11-13(±1) nm and micron-size lengths. Spectroscopic characterization studies demonstrate the J-type π-π interactions among the BTBT molecules within the hydrophobic core of the self-assembled nanofibers yielding an electrical conductivity as high as 6.0 × 10-6 S cm-1. The BTBT π-core is demonstrated, for the first time, in the formation of self-assembled peptide 1D nanostructures in aqueous media for potential use in tissue engineering, bioelectronics and (opto)electronics. The conductivity achieved here is one of the highest reported to date in a non-doped state.

  8. THE ORION FINGERS: NEAR-IR SPECTRAL IMAGING OF AN EXPLOSIVE OUTFLOW

    Energy Technology Data Exchange (ETDEWEB)

    Youngblood, Allison; Bally, John [Department of Astrophysical and Planetary Sciences, University of Colorado, UCB 389, Boulder, CO 80309 (United States); Ginsburg, Adam, E-mail: allison.youngblood@colorado.edu [ESO Headquarters, Karl-Schwarzschild-Strasse 2, D-85748 Garching bei München (Germany)

    2016-06-01

    We present near-IR (1.1–2.4 μ m) position–position–velocity cubes of the 500 year old Orion BN/KL explosive outflow with spatial resolution 1″ and spectral resolution 86 km s{sup −1}. We construct integrated intensity maps free of continuum sources of 15 H{sub 2} and [Fe ii] lines while preserving kinematic information of individual outflow features. Included in the detected H{sub 2} lines are the 1-0 S(1) and 1-0 Q(3) transitions, allowing extinction measurements across the outflow. Additionally, we present dereddened flux ratios for over two dozen outflow features to allow for the characterization of the true excitation conditions of the BN/KL outflow. All of the ratios show the dominance of the shock excitation of the H{sub 2} emission, although some features exhibit signs of fluorescent excitation from stellar radiation or J-type shocks. We also detect tracers of the PDR/ionization front north of the Trapezium stars in [O i] and [Fe ii] and analyze other observed outflows not associated with the BN/KL outflow.

  9. Communication: The ground electronic state of Si2C: Rovibrational level structure, quantum monodromy, and astrophysical implications

    International Nuclear Information System (INIS)

    Reilly, Neil J.; Kokkin, Damian L.; McCarthy, Michael C.; Changala, P. Bryan; Baraban, Joshua H.; Stanton, John F.

    2015-01-01

    We report the gas-phase optical detection of Si 2 C near 390 nm and the first experimental investigation of the rovibrational structure of its 1 A 1 ground electronic state using mass-resolved and fluorescence spectroscopy and variational calculations performed on a high-level ab initio potential. From this joint study, it is possible to assign all observed K a = 1 vibrational levels up to 3800 cm −1 with confidence, as well as a number of levels in the K a = 0, 2,  and 3 manifolds. Dixon-dip plots for the bending coordinate (ν 2 ) allow an experimental determination of a barrier to linearity of 783(48) cm −1 (2σ), in good agreement with theory (802(9) cm −1 ). The calculated (K a , ν 2 ) eigenvalue lattice shows an archetypal example of quantum monodromy (absence of a globally valid set of quantum numbers) that is reflected by the experimentally observed rovibrational levels. The present study provides a solid foundation for infrared and optical surveys of Si 2 C in astronomical objects, particularly in the photosphere of N- and J-type carbon stars where the isovalent SiC 2 molecule is known to be abundant

  10. Wide-range light-harvesting donor-acceptor assemblies through specific intergelator interactions via self-assembly.

    Science.gov (United States)

    Samanta, Suman K; Bhattacharya, Santanu

    2012-12-03

    We have synthesized two new low-molecular-mass organogelators based on tri-p-phenylene vinylene derivatives, one of which could be designated as the donor whereas the other one is an acceptor. These were prepared specifically to show the intergelator interactions at the molecular level by using donor-acceptor self-assembly to achieve appropriate control over their macroscopic properties. Intermolecular hydrogen-bonding, π-stacking, and van der Waals interactions operate for both the individual components and the mixtures, leading to the formation of gels in the chosen organic solvents. Evidence for intergelator interactions was acquired from various spectroscopic, microscopic, thermal, and mechanical investigations. Due to the photochromic nature of these molecules, interesting photophysical properties, such as solvatochromism and J-type aggregation, were clearly observed. An efficient energy transfer was exhibited by the mixture of donor-acceptor assemblies. An array of four chromophores was built up by inclusion of two known dyes (anthracene and rhodamine 6G) for the energy-transfer studies. Interestingly, an energy-transfer cascade was observed in the assembly of four chromophores in a particular order (anthracene-donor-acceptor-rhodamine 6G), and if one of the components was removed from the assembly the energy transfer process was discontinued. This allowed the build up of a light-harvesting process with a wide range. Excitation at one end produces an emission at the other end of the assembly. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Interaction of sodium dodecyl sulfate with watermelon chromoplasts and examination of the organization of lycopene within the chromoplasts.

    Science.gov (United States)

    Fish, Wayne W

    2006-10-18

    The properties of plant-derived precipitates of watermelon lycopene were examined in aqueous sodium dodecyl sulfate (SDS) as part of an ongoing effort to develop simpler, more economical ways to quantify carotenoids in melon fruit. Levels of SDS >0.2% were found to increase the water solubility of lycopene in the state in which it was isolated from watermelon. Electron microscopy and chemical analyses suggested that the watermelon lycopene as isolated is packaged inside a membrane to form a chromoplast. Spectral peaks in the visible region of the watermelon chromoplasts in SDS exhibited a bathochromic shift from those in organic solvent. Watermelon chromoplasts in SDS exhibited pronounced circular dichroic activity in the visible region. Binding measurements indicated that about 120 molecules of SDS were bound per molecule of lycopene inside the chromoplast; likely, the detergent molecules are bound to the chromoplast membrane. Around 80% of the chromoplast-SDS complexes were retained on a 0.45 mum membrane filter. Together, these observations are consistent with lycopene in a J-type chiral arrangement inside a membrane to form a chromoplast. The binding of SDS molecules to the chromoplast membrane form a complex that is extensively more water-soluble than the chromoplast alone.

  12. Retinal neurodegeneration may precede microvascular changes characteristic of diabetic retinopathy in diabetes mellitus.

    Science.gov (United States)

    Sohn, Elliott H; van Dijk, Hille W; Jiao, Chunhua; Kok, Pauline H B; Jeong, Woojin; Demirkaya, Nazli; Garmager, Allison; Wit, Ferdinand; Kucukevcilioglu, Murat; van Velthoven, Mirjam E J; DeVries, J Hans; Mullins, Robert F; Kuehn, Markus H; Schlingemann, Reinier Otto; Sonka, Milan; Verbraak, Frank D; Abràmoff, Michael David

    2016-05-10

    Diabetic retinopathy (DR) has long been recognized as a microvasculopathy, but retinal diabetic neuropathy (RDN), characterized by inner retinal neurodegeneration, also occurs in people with diabetes mellitus (DM). We report that in 45 people with DM and no to minimal DR there was significant, progressive loss of the nerve fiber layer (NFL) (0.25 μm/y) and the ganglion cell (GC)/inner plexiform layer (0.29 μm/y) on optical coherence tomography analysis (OCT) over a 4-y period, independent of glycated hemoglobin, age, and sex. The NFL was significantly thinner (17.3 μm) in the eyes of six donors with DM than in the eyes of six similarly aged control donors (30.4 μm), although retinal capillary density did not differ in the two groups. We confirmed significant, progressive inner retinal thinning in streptozotocin-induced "type 1" and B6.BKS(D)-Lepr(db)/J "type 2" diabetic mouse models on OCT; immunohistochemistry in type 1 mice showed GC loss but no difference in pericyte density or acellular capillaries. The results suggest that RDN may precede the established clinical and morphometric vascular changes caused by DM and represent a paradigm shift in our understanding of ocular diabetic complications.

  13. Synthesis, structure, and electrical conductivity of A'[A(sub 2)B(sub 3)O(sub 10)] (A'=Rb, Cs A=Sr, Ba; B=Nb, Ta)

    International Nuclear Information System (INIS)

    Thangadurai, Venkataraman; Schmid-Beurmann, Peter; Weppner, Werner

    2001-01-01

    New members of Dion-Jacobson (D-J)-type layered perovskites of the general formula A'[A(sub 2)B(sub 3)O(sub 10)] (A'=Rb, Cs; A=Sr, Ba; B=Nb, Ta) were prepared by solid-state reactions between the metal oxides, nitrates, and carbonates under specific conditions. The crystal structures were determined using powder X-ray diffraction data in combination with Rietveld analysis. The c-axis lattice parameter increases as the size of the alkaline earth ion increases. Both Sr and Ba compounds crystallize in a tetragonal cell, while the corresponding Ca compounds crystallize in an orthorhombic cell. The electrical conductivity of the layered perovskites was determined using AC impedance analysis and DC methods. Among the compounds with the same alkali ion and different alkaline earth ions, the Ca compound exhibits a higher electrical conductivity than the corresponding Sr and Ba compounds. DC measurements reveal that the layered perovskites exhibit both ionic and electronic conduction

  14. System Design of a Cheetah Robot Toward Ultra-high Speed

    Directory of Open Access Journals (Sweden)

    Mantian Li

    2014-05-01

    Full Text Available High-speed legged locomotion pushes the limits of the most challenging problems of design and development of the mechanism, also the control and the perception method. The cheetah is an existence proof of concept of what we imitate for high-speed running, and provides us lots of inspiration on design. In this paper, a new model of a cheetah-like robot is developed using anatomical analysis and design. Inspired by a biological neural mechanism, we propose a novel control method for controlling the muscles' flexion and extension, and simulations demonstrate good biological properties and leg's trajectory. Next, a cheetah robot prototype is designed and assembled with pneumatic muscles, a musculoskeletal structure, an antagonistic muscle arrangement and a J-type cushioning foot. Finally, experiments of the robot legs swing and kick ground tests demonstrate its natural manner and validate the design of the robot. In the future, we will test the bounding behaviour of a real legged system.

  15. Zinc chlorophyll aggregates as hole transporters for biocompatible, natural-photosynthesis-inspired solar cells

    Science.gov (United States)

    Li, Yue; Sasaki, Shin-ichi; Tamiaki, Hitoshi; Liu, Cheng-Liang; Song, Jiaxing; Tian, Wenjing; Zheng, Enqiang; Wei, Yingjin; Chen, Gang; Fu, Xueqi; Wang, Xiao-Feng

    2015-11-01

    The intriguing properties of extremely efficient delocalization and migration of excitons in chlorophyll (Chl) J-type aggregates have inspired intense research activities toward their structural understanding, functional interpretation and mimicry synthesis. Herein, we demonstrated the J-aggregates of zinc methyl 3-devinyl-3-hydroxymethyl-pyropheophorbide a (ZnChl-1) generated by spin-coating method for the application as a hole transporter in titania-based solar cells using methyl trans-32-carboxypyropheophorbide a (H2Chl-2) or its zinc complex (ZnChl-2) as the sensitizer. The effective carrier mobility of the J-aggregates films was determined by the organic field-effect transistor to be 6.2 × 10-4 cm2 V-1 s-1. Solar cells sharing the architecture of FTO/H2Chl-2 or ZnChl-2 on TiO2/(ZnChl-1)n/Ag were fabricated and the factors that presumably determine their photovoltaic performances were discussed. The photovoltaic devices studied herein employing inexpensive and pollution-free biomaterials provide a unique solution of utilizing solar energy with a care of the important environmental issue.

  16. Two-fluid dusty shocks: simple benchmarking problems and applications to protoplanetary discs

    Science.gov (United States)

    Lehmann, Andrew; Wardle, Mark

    2018-05-01

    The key role that dust plays in the interstellar medium has motivated the development of numerical codes designed to study the coupled evolution of dust and gas in systems such as turbulent molecular clouds and protoplanetary discs. Drift between dust and gas has proven to be important as well as numerically challenging. We provide simple benchmarking problems for dusty gas codes by numerically solving the two-fluid dust-gas equations for steady, plane-parallel shock waves. The two distinct shock solutions to these equations allow a numerical code to test different forms of drag between the two fluids, the strength of that drag and the dust to gas ratio. We also provide an astrophysical application of J-type dust-gas shocks to studying the structure of accretion shocks on to protoplanetary discs. We find that two-fluid effects are most important for grains larger than 1 μm, and that the peak dust temperature within an accretion shock provides a signature of the dust-to-gas ratio of the infalling material.

  17. Chlorophyll J-aggregates: from bioinspired dye stacks to nanotubes, liquid crystals, and biosupramolecular electronics.

    Science.gov (United States)

    Sengupta, Sanchita; Würthner, Frank

    2013-11-19

    Among the natural light-harvesting (LH) systems, those of green sulfur and nonsulfur photosynthetic bacteria are exceptional because they lack the support of a protein matrix. Instead, these so-called chlorosomes are based solely on "pigments". These are self-assembled bacteriochlorophyll c, d, and e derivatives, which consist of a chlorophyll skeleton bearing a 3(1)-hydroxy functional group. Chemists consider the latter as an essential structural unit to direct the formation of light-harvesting self-assembled dye aggregates with J-type excitonic coupling. The intriguing properties of chlorosomal J-type aggregates, particularly narrow red-shifted absorption bands, compared with monomers and their ability to delocalize and migrate excitons, have inspired intense research activities toward synthetic analogues in this field. The ultimate goal of this research field is the development of (opto-)electronic devices based on the architectural principle of chlorosomal LH systems. In this regard, the challenge is to develop small, functional building blocks with appropriate substituents that are preprogrammed to self-assemble across different length scales and to emulate functions of natural LH systems or to realize entirely new functions beyond those found in nature. In this Account, we highlight our achievements in the past decade with semisynthetic zinc chlorins (ZnChls) as model compounds of bacteriochlorophylls obtained from the naturally most abundant chlorin precursor: chlorophyll a. To begin, we explore how supramolecular strategies involving π-stacking, hydrogen bonding, and metal-oxygen coordination can be used to design ZnChl-based molecular stack, tube, and liquid crystalline assemblies conducive to charge and energy transport. Our design principle is based on the bioinspired functionalization of the 3(1)-position of ZnChl with a hydroxy or methoxy group; the former gives rise to tubular assemblies, whereas the latter induces stack assemblies. Functionalization

  18. Layer-by-layer films and colloidal dispersions of graphene oxide nanosheets for efficient control of the fluorescence and aggregation properties of the cationic dye acridine orange.

    Science.gov (United States)

    Hansda, Chaitali; Chakraborty, Utsav; Hussain, Syed Arshad; Bhattacharjee, Debajyoti; Paul, Pabitra Kumar

    2016-03-15

    Chemically derived graphene oxide (GO) nanosheets have received great deal of interest for technological application such as optoelectronic and biosensors. Aqueous dispersions of GO become an efficient template to induce the association of cationic dye namely Acridine Orange (AO). Interactions of AO with colloidal GO was governed by both electrostatic and π-π stacking cooperative interactions. The type of dye aggregations was found to depend on the concentration of GO in the mixed ensemble. Spectroscopic calculations revealed the formation of both H and J-type dimers, but H-type aggregations were predominant. Preparation of layer-by-layer (LbL) electrostatic self-assembled films of AO and GO onto poly (allylamine hydrochloride) (PAH) coated quartz substrate is also reported in this article. UV-Vis absorption, steady state and time resolve fluorescence and Raman spectroscopic techniques have been employed to explore the detail photophysical properties of pure AO, AO/GO mixed solution and AO/GO LbL films. Scanning electron microscopy was also used for visual evidence of the synthesized nanodimensional GO sheets. The fluorescence quenching of AO in the presence of GO in aqueous solution was due to the interfacial photoinduced electron transfer (PET) from photoexcited AO to GO i.e. GO acts as an efficient quenching agent for the fluorescence emission of AO. The quenching is found to be static in nature. Raman spectroscopic results also confirmed the interaction of AO with GO and the electron transfer. The formation of AO/GO complex via very fast excited state electron transfer mechanism may be proposed as to prepare GO-based fluorescence sensor for biomolecular detection without direct labeling the biomolecules by fluorescent probe. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Regulation of human Nfu activity in Fe-S cluster delivery-characterization of the interaction between Nfu and the HSPA9/Hsc20 chaperone complex.

    Science.gov (United States)

    Wachnowsky, Christine; Liu, Yushi; Yoon, Taejin; Cowan, J A

    2018-01-01

    Iron-sulfur cluster biogenesis is a complex, but highly regulated process that involves de novo cluster formation from iron and sulfide ions on a scaffold protein, and subsequent delivery to final targets via a series of Fe-S cluster-binding carrier proteins. The process of cluster release from the scaffold/carrier for transfer to the target proteins may be mediated by a dedicated Fe-S cluster chaperone system. In human cells, the chaperones include heat shock protein HSPA9 and the J-type chaperone Hsc20. While the role of chaperones has been somewhat clarified in yeast and bacterial systems, many questions remain over their functional roles in cluster delivery and interactions with a variety of human Fe-S cluster proteins. One such protein, Nfu, has recently been recognized as a potential interaction partner of the chaperone complex. Herein, we examined the ability of human Nfu to function as a carrier by interacting with the human chaperone complex. Human Nfu is shown to bind to both chaperone proteins with binding affinities similar to those observed for IscU binding to the homologous HSPA9 and Hsc20, while Nfu can also stimulate the ATPase activity of HSPA9. Additionally, the chaperone complex was able to promote Nfu function by enhancing the second-order rate constants for Fe-S cluster transfer to target proteins and providing directionality in cluster transfer from Nfu by eliminating promiscuous transfer reactions. Together, these data support a hypothesis in which Nfu can serve as an alternative carrier protein for chaperone-mediated cluster release and delivery in Fe-S cluster biogenesis and trafficking. © 2017 Federation of European Biochemical Societies.

  20. ANTS-anchored Zn-Al-CO3-LDH particles as fluorescent probe for sensing of folic acid

    International Nuclear Information System (INIS)

    Liu, Pengfei; Liu, Dan; Liu, Yanhuan; Li, Lei

    2016-01-01

    A novel fluorescent nanosensor for detecting folic acid (FA) in aqueous media has been developed based on 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) anchored to the surface of Zn-Al-CO 3 -layered double hydroxides (LDH) particles. The nanosensor showed high fluorescence intensity and good photostability due to a strong coordination interaction between surface Zn 2+ ions of Zn-Al-CO 3 -LDH and N atoms of ANTS, which were verified by result of X-ray photoelectron spectroscopy (XPS). ANTS-anchored on the surface of Zn-Al-CO 3 -LDH restricted the intra-molecular rotation leading to ANTS-anchored J-type aggregation emission enhancement. ANTS-anchored Zn-Al-CO 3 -LDH particles exhibited highly sensitive and selective response to FA over other common metal ions and saccharides present in biological fluids. The proposed mechanism was that oxygen atoms of -SO 3 groups in ANTS-anchored on the surface of Zn-Al-CO 3 -LDH were easily collided by FA molecules to form potential hydrogen bonds between ANTS-anchored and FA molecules, which could effectively quench the ANTS-anchored fluorescence. Under the simulated physiological conditions (pH of 7.4), the fluorescence quenching was fitted to Stern-Volmer equation with a linear response in the concentration range of 1 μM to 200 μM with a limit of detection of 0.1 μM. The results indicate that ANTS-anchored Zn-Al-CO 3 -LDH particles can afford a very sensitive system for the sensing FA in aqueous solution. - Highlights: • A novel fluorescent nanosensor has been developed. • The sensor exhibited highly sensitive and selective response to FA. • The fluorescence quenching was fitted to Stern–Volmer equation. • The linear response range was 1–200 μM with a limit of detection of 0.1 μM.

  1. MHOs toward HMOs: A Search for Molecular Hydrogen Emission-Line Objects toward High-mass Outflows

    Energy Technology Data Exchange (ETDEWEB)

    Wolf-Chase, Grace [Astronomy Department Adler Planetarium 1300 S. Lake Shore Drive Chicago, IL 60605 (United States); Arvidsson, Kim [Trull School of Sciences and Mathematics Schreiner University 2100 Memorial Blvd. Kerrville, TX 78028 (United States); Smutko, Michael, E-mail: gwolfchase@adlerplanetarium.org [Center for Interdisciplinary Exploration and Research in Astrophysics (CIERA), and Dept. of Physics and Astronomy, Northwestern University, 2131 Tech Drive, Evanston, IL 60208 (United States)

    2017-07-20

    We present the results of a narrow-band near-infrared imaging survey for Molecular Hydrogen emission-line Objects (MHOs) toward 26 regions containing high-mass protostellar candidates and massive molecular outflows. We have detected a total of 236 MHOs, 156 of which are new detections, in 22 out of the 26 regions. We use H{sub 2} 2.12 μ m/H{sub 2} 2.25 μ m flux ratios, together with morphology, to separate the signatures of fluorescence associated with photo-dissociation regions (PDRs) from shocks associated with outflows in order to identify the MHOs. PDRs have typical low flux ratios of ∼1.5–3, while the vast majority of MHOs display flux ratios typical of C-type shocks (∼6–20). A few MHOs exhibit flux ratios consistent with expected values for J-type shocks (∼3–4), but these are located in regions that may be contaminated with fluorescent emission. Some previously reported MHOs have low flux ratios, and are likely parts of PDRs rather than shocks indicative of outflows. We identify a total of 36 outflows across the 22 target regions where MHOs were detected. In over half these regions, MHO arrangements and fluorescent structures trace features present in CO outflow maps, suggesting that the CO emission traces a combination of dynamical effects, which may include gas entrained in expanding PDRs as well as bipolar outflows. Where possible, we link MHO complexes to distinct outflows and identify candidate driving sources.

  2. Assessment of bone biopsy needles for sample size, specimen quality and ease of use

    International Nuclear Information System (INIS)

    Roberts, C.C.; Liu, P.T.; Morrison, W.B.; Leslie, K.O.; Carrino, J.A.; Lozevski, J.L.

    2005-01-01

    To assess whether there are significant differences in ease of use and quality of samples among several bone biopsy needles currently available. Eight commonly used, commercially available bone biopsy needles of different gauges were evaluated. Each needle was used to obtain five consecutive samples from a lamb lumbar pedicle. Subjective assessment of ease of needle use, ease of sample removal from the needle and sample quality, before and after fixation, was graded on a 5-point scale. The number of attempts necessary to reach a 1 cm depth was recorded. Each biopsy specimen was measured in the gross state and after fixation. The RADI Bonopty 15 g and Kendall Monoject J-type 11 g needles were rated the easiest to use, while the Parallax Core-Assure 11 g and the Bard Ostycut 16 g were rated the most difficult. Parallax Core-Assure and Kendall Monoject needles had the highest quality specimen in the gross state; Cook Elson/Ackerman 14 g and Bard Ostycut 16 g needles yielded the lowest. The MD Tech without Trap-Lok 11 g needle had the highest quality core after fixation, while the Bard Ostycut 16 g had the lowest. There was a significant difference in pre-fixation sample length between needles (P<0.0001), despite acquiring all cores to a standard 1 cm depth. Core length and width decrease in size by an average of 28% and 42% after fixation. Bone biopsy needles vary significantly in performance. Detailed knowledge of the strengths and weaknesses of different needles is important to make an appropriate selection for each individual's practice. (orig.)

  3. Microwave and millimeter wave astrochemistry: Laboratory studies of transition metal-containing free radicals and spectroscopic observations of molecular interstellar environments

    Science.gov (United States)

    Adande, Gilles Rapotchombo

    Progress in our understanding of the chemical composition of the interstellar medium leans both on laboratory analyses of high resolution rotational spectra from molecules that may be present in these regions, and on radio astronomical observations of molecular tracers to constrain astrochemical models. Due to the thermodynamic conditions in outer space, some molecules likely to be found in interstellar regions in relevant abundances are open shell radicals. In a series of laboratory studies, the pure rotational spectra of the transition metal containing radicals sulfur species ScS, YS, VS and ZnSH were obtained for the first time. In addition to accurate and precise rest frequencies for these species, bonding characteristics were determined from fine and hyperfine molecular parameters. It was found that these sulfides have a higher degree of covalent bonding than their mostly ionic oxide counterparts. Isomers and isotope ratios are excellent diagnostic tools for a variety of astrochemical models. From radio observations of isotopes of nitrile species, the galactic gradient of 14N/15N was accurately established. A further study of this ratio in carbon rich asymptotic giant branch stars provided observational evidence for an unknown process in J type carbon stars, and highlighted the need to update stellar nucleosynthesis models. Proper radiative transfer modeling of the emission spectra of interstellar molecules can yield a wealth of information about the abundance and distribution of these species within the observed sources. To model the asymmetric emission of SO and SO2 in oxygen-rich supergiants, an in-house code was developed, and successfully applied to gain insight into circumstellar sulfur chemistry of VY Canis Majoris. It was concluded that current astrochemistry kinetic models, based on spherical symmetry assumptions, need to be revisited.

  4. Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor.

    Science.gov (United States)

    Knight, Martha; Finn, Thomas M; Zehmer, John; Clayton, Adam; Pilon, Aprile

    2011-09-09

    An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid-liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane-ethyl acetate-methanol-water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol-potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. π–π-Induced aggregation and single-crystal fluorescence anisotropy of 5,6,10b-triazaacephenanthrylene

    Directory of Open Access Journals (Sweden)

    Katarzyna Ostrowska

    2018-05-01

    Full Text Available The structural origin of absorption and fluorescence anisotropy of the single crystal of the π-conjugated heterocyclic system 5,6,10b-triazaacephenanthrylene, TAAP, is presented in this study. X-ray analysis shows that the crystal framework in the space group P\\overline{1} is formed by centrosymmetric dimers of face-to-face mutually oriented TAAP molecules joined by π–π non-covalent interactions. The conformation of the TAAP molecule is stabilized by intramolecular C—H...N(sp2, N(sp2H...π(CN, and C—H...O(sp2 hydrogen bonds. The presence of weak π–π interactions is confirmed by quantum theory of atoms in molecules (QTAIM and non-covalent interaction (NCI analysis. The analysis of the optical spectra of TAAP in solution and in the solid state does not allow the specification of the aggregation type. DFT calculations for the dimer in the gas phase indicate that the lowest singlet excitation is forbidden by symmetry, suggesting H-type aggregation, even though the overall absorption spectrum is bathochromically shifted as for the J-type. The experimental determination of the permanent dipole moment of a TAAP molecule in 1,4-dioxane solution indicates the presence of the monomer form. The calculated absorption and emission spectra of the crystal in a simple approximation are consistent with the experimentally determined orientation of the absorption and emission transition dipole moments in TAAP single crystals. The electrostatic interaction between monomers with a permanent dipole moment (ca 4 D each could result in the unusual spectroscopic JH-aggregate behaviour of the TAAP dimer.

  6. Dual localized AtHscB involved in iron sulfur protein biogenesis in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Xiang Ming Xu

    2009-10-01

    Full Text Available Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.In this study we demonstrate that AtHscB from Arabidopsis can rescue the Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol.

  7. The Orion Fingers: H2 Temperatures and Excitation in an Explosive Outflow

    Science.gov (United States)

    Youngblood, Allison; France, Kevin; Ginsburg, Adam; Hoadley, Keri; Bally, John

    2018-04-01

    We measure H2 temperatures and column densities across the Orion Becklin-Neugebauer/Kleinmann-Low (BN/KL) explosive outflow from a set of 13 near-infrared (IR) H2 rovibrational emission lines observed with the TripleSpec spectrograph on Apache Point Observatory’s 3.5 m telescope. We find that most of the region is well characterized by a single temperature (∼2000–2500 K), which may be influenced by the limited range of upper-energy levels (6000–20,000 K) probed by our data set. The H2 column density maps indicate that warm H2 comprises 10‑5–10‑3 of the total H2 column density near the center of the outflow. Combining column density measurements for co-spatial H2 and CO at T = 2500 K, we measure a CO/H2 fractional abundance of 2 × 10‑3 and discuss possible reasons why this value is in excess of the canonical 10‑4 value, including dust attenuation, incorrect assumptions on co-spatiality of the H2 and CO emission, and chemical processing in an extreme environment. We model the radiative transfer of H2 in this region with ultraviolet (UV) pumping models to look for signatures of H2 fluorescence from H I Lyα pumping. Dissociative (J-type) shocks and nebular emission from the foreground Orion H II region are considered as possible Lyα sources. From our radiative transfer models, we predict that signatures of Lyα pumping should be detectable in near-IR line ratios given a sufficiently strong source, but such a source is not present in the BN/KL outflow. The data are consistent with shocks as the H2 heating source.

  8. Racial Disparities in the Pathogenesis of Type 2 Diabetes and its Subtypes in the African Diaspora: A New Paradigm.

    Science.gov (United States)

    Gaillard, Trudy R; Osei, Kwame

    2016-03-01

    The global epidemic of diabetes has extended to the developing countries including Sub-Sahara Africa. In this context, blacks with type 2 diabetes in the African Diaspora continue to manifest 1.5-2 times higher prevalent rates than in their white counterparts. Previous studies have demonstrated that blacks with and without type 2 diabetes have alterations in hepatic and peripheral insulin sensitivity, beta-cell function, and hepatic insulin clearance as well as hepatic glucose dysregulation when compared to whites. In addition, non-diabetic blacks in the African Diaspora manifest multiple metabolic mediators that predict type 2 diabetes and its subtypes. These pathogenic modifiers include differences in subclinical inflammation, oxidative stress burden, and adipocytokines in blacks in the African Diaspora prior to clinical diagnosis. Consequently, blacks in the African Diaspora manifest subtypes of type 2 diabetes, including ketosis-prone diabetes and J type diabetes. Given the diversity of type 2 diabetes in blacks in the African Diaspora, we hypothesize that blacks manifest multiple early pathogenic defects prior to the diagnosis of type 2 diabetes and its subtypes. These metabolic alterations have strong genetic component, which appears to play pivotal and primary role in the pathogenesis of type 2 diabetes and its subtypes in blacks in the African Diaspora. However, environmental factors must also be considered as major contributors to the higher prevalence of type 2 diabetes and its subtypes in blacks in the African Diaspora. These multiple alterations should be targets for early prevention of type 2 diabetes in blacks in the African Diaspora.

  9. [Posttraumatic stress disorder (PTSD) as a consequence of the interaction between an individual genetic susceptibility, a traumatogenic event and a social context].

    Science.gov (United States)

    Auxéméry, Y

    2012-10-01

    the type 2 dopaminergic receptor is associated with a severe comorbidity of PTSD with the presence of somatic disorders, anxiety, social change and depression. For noradrenergic neuromodulation, an interaction between the polymorphism of gene GABRA2 and the occurrence of PTSD is described whereas an interaction between the number of traumatic events and Val(158)Met polymorphism of the gene coding for catecholamine-o-methyltransferase has also been found. The role of polymorphisms in FKBP5 (a co-chaperone of hsp 90 which binds to the glucocorticoid receptor) in predicting PTSD too, with a gene-by-environment point of view. These gene-by-environment studies are needed to focus more on distinct endophenotypes and influences from environmental factors. If several candidate genes are involved, a weighting of susceptibility to such and such a neurological regulation system will imply various endophenotypes. According to the monoamine predominantly incriminated, PTSD can take on a more hyper-vegetative clinical expression linked with noradrenergic overuse. Differently, avoidance behaviour and the depressive aspect invoke more a modification of the serotoninergic modulation whilst posttraumatic psychotic reactions question the role of dopaminergic pathways. Neuroscientific discoveries interesting the biological support of PTSD can thus modify our view of the conception of the disorder in relation to different therapeutic prospects. Chronic PTSD can manifest itself in different clinical forms. The repetition syndrome can appear a long time after the traumatic event, following a paucisymptomatic latency period, which can last several years or even decades. The absence of complaints from the patient is common, the latter suffering in silence. Often other comorbid disorders and other complaints arise sooner than the clinical picture. Thus a depressive episode characterised as drug-seeking behaviour is frequently encountered. The therapeutic accompaniment traditionally combines a

  10. Evaluation of Flat Surface Temperature Probes

    Science.gov (United States)

    Beges, G.; Rudman, M.; Drnovsek, J.

    2011-01-01

    The objective of this paper is elaboration of elements related to metrological analysis in the field of surface temperature measurement. Surface temperature measurements are applicable in many fields. As examples, safety testing of electrical appliances and a pharmaceutical production line represent case studies for surface temperature measurements. In both cases correctness of the result of the surface temperature has an influence on final product safety and quality and thus conformity with specifications. This paper deals with the differences of flat surface temperature probes in measuring the surface temperature. For the purpose of safety testing of electrical appliances, surface temperature measurements are very important for safety of the user. General requirements are presented in European standards, which support requirements in European directives, e.g., European Low Voltage Directive 2006/95/EC and pharmaceutical requirements, which are introduced in official state legislation. This paper introduces a comparison of temperature measurements of an attached thermocouple on the measured surface and measurement with flat surface temperature probes. As a heat generator, a so called temperature artifact is used. It consists of an aluminum plate with an incorporated electrical heating element with very good temperature stability in the central part. The probes and thermocouple were applied with different forces to the surface in horizontal and vertical positions. The reference temperature was measured by a J-type fine-wire (0.2 mm) thermocouple. Two probes were homemade according to requirements in the European standard EN 60335-2-9/A12, one with a fine-wire (0.2 mm) thermocouple and one with 0.5mm of thermocouple wire diameter. Additional commercially available probes were compared. Differences between probes due to thermal conditions caused by application of the probe were found. Therefore, it can happen that measurements are performed with improper equipment or

  11. Temperature increase beneath etched dentin discs during composite polymerization.

    Science.gov (United States)

    Karaarslan, Emine Sirin; Secilmis, Asli; Bulbul, Mehmet; Yildirim, Cihan; Usumez, Aslihan

    2011-01-01

    The purpose of this in vitro study was to measure the temperature increase during the polymerization of a composite resin beneath acid-etched or laser-etched dentin discs. The irradiation of dentin with an Er:YAG laser may have a positive effect on the thermal conductivity of dentin. This technique has not been studied extensively. Forty dentin discs (5 mm in diameter and 0.5 or 1 mm in height) were prepared from extracted permanent third molars. These dentin discs were etched with 20% orthophosphoric acid or an Er:YAG laser, and were then placed on an apparatus developed to measure temperature increases. The composite resin was polymerized with a high-intensity quartz tungsten halogen (HQTH) or light-emitting diode unit (LED). The temperature increase was measured under the dentin disc with a J-type thermocouple wire that was connected to a data logger. Five measurements were made for each dentin disc, curing unit, and etching system combination. Differences between the initial and the highest temperature readings were taken, and the five calculated temperature changes were averaged to determine the value of the temperature increase. Statistical analysis was performed with a three-way ANOVA and Tukey HSD tests at a 0.05 level of significance. Further SEM examinations were performed. The temperature increase values varied significantly, depending on etching systems (p < 0.05), dentin thicknesses (p < 0.05), and curing units (p < 0.05). Temperature increases measured beneath laser-etched discs were significantly higher than those for acid-etched dentin discs (p < 0.05). The HQTH unit induced significantly higher temperature increases than the LED unit (p < 0.05). The LED unit induced the lowest temperature change (5.2°C) in the 1-mm, acid-etched dentin group. The HQTH unit induced the highest temperature change (10.4°C) for the 0.5-mm, laser-etched dentin group. The risk of heat-induced pulpal damage should be taken into consideration

  12. ANTS-anchored Zn-Al-CO{sub 3}-LDH particles as fluorescent probe for sensing of folic acid

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Pengfei; Liu, Dan; Liu, Yanhuan [State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029 (China); Li, Lei, E-mail: lilei@mail.buct.edu.cn [State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029 (China)

    2016-09-15

    A novel fluorescent nanosensor for detecting folic acid (FA) in aqueous media has been developed based on 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) anchored to the surface of Zn-Al-CO{sub 3}-layered double hydroxides (LDH) particles. The nanosensor showed high fluorescence intensity and good photostability due to a strong coordination interaction between surface Zn{sup 2+} ions of Zn-Al-CO{sub 3}-LDH and N atoms of ANTS, which were verified by result of X-ray photoelectron spectroscopy (XPS). ANTS-anchored on the surface of Zn-Al-CO{sub 3}-LDH restricted the intra-molecular rotation leading to ANTS-anchored J-type aggregation emission enhancement. ANTS-anchored Zn-Al-CO{sub 3}-LDH particles exhibited highly sensitive and selective response to FA over other common metal ions and saccharides present in biological fluids. The proposed mechanism was that oxygen atoms of -SO{sub 3} groups in ANTS-anchored on the surface of Zn-Al-CO{sub 3}-LDH were easily collided by FA molecules to form potential hydrogen bonds between ANTS-anchored and FA molecules, which could effectively quench the ANTS-anchored fluorescence. Under the simulated physiological conditions (pH of 7.4), the fluorescence quenching was fitted to Stern-Volmer equation with a linear response in the concentration range of 1 μM to 200 μM with a limit of detection of 0.1 μM. The results indicate that ANTS-anchored Zn-Al-CO{sub 3}-LDH particles can afford a very sensitive system for the sensing FA in aqueous solution. - Highlights: • A novel fluorescent nanosensor has been developed. • The sensor exhibited highly sensitive and selective response to FA. • The fluorescence quenching was fitted to Stern–Volmer equation. • The linear response range was 1–200 μM with a limit of detection of 0.1 μM.

  13. Pollution and energy management in tanning industry

    International Nuclear Information System (INIS)

    Zaman, N.U.

    2005-01-01

    Tanning industry uses a number of chemicals such as Common Salt, Lime (Calcium Hydroxide), Sodium Sulfide and Basic Chromium Sulfate etc. During process, only a part of the chemical is consumed and the rest ends up in the effluent as pollutant. This paper deals with the techniques, locally developed or published in literature to recycle these chemicals and also discusses some energy saving techniques which can be used in tanning industry. Basic Chromium Sulfate (BCS) is one of the expensive chemicals used in 'Chrome Tanning'. By precipitating d filtering basic chromium sulfate, the recovery is nearly complete and the effluent obtained contains less than 1ppm Chromium. Dried raw hides contain up to 15% sodium chloride (w/w) and this can be removed in solid form by using mechanical brushes and can be re-used. The recovered salt contains foreign matter as impurities. After dissolution in water, the salt solution is filtered through cartridge filters and can be used in pickle bath. Liming slurry containing sodium sulfide is wasted as it contains fleshing and hair etc. A self cleaning 'J' type screen has no moving parts and removes fleshing and hair from the lime suspension. 'Counter Current Washing Technique,' reduces the wash water quantity by a factor of five to six. Air born pollution generated during buffing and dyeing can be captured by properly designed air filters. The solvents released in atmosphere during dyeing and finishing can be recovered by absorption. Fat, gelatin and protein can be recovered from waste fleshing. In tanning industry, drying of hides is the major consumer of thermal energy. Hot air can be produced by steam, hot water or solar energy. Advantages and disadvantages of these options are discussed. Wastage of thermal energy in dryers can be reduced by improving the existing designs. Hot water for tanning purposes can be generated by recovering waste heat present in the boiler flue gases. Boiler efficiency can also be improved by cycling heat

  14. Hepatoprotective Activity of Easter Lily (Lilium longiflorum Thunb.) Bulb Extracts.

    Science.gov (United States)

    Tang, Wenping; Munafo, John P; Palatini, Kimberly; Esposito, Debora; Huang, Mou-Tuan; Komarnytsky, Slavko; Ho, Chi-Tang; Gianfagna, Thomas J

    2015-11-11

    The hepatoprotective activities of two different extracts, a hydroethanolic crude bulb extract (CB) and a steroidal glycoside-rich 1-butanol extract (BuOH), prepared from the bulbs of Easter lily (Lilium longiflorum Thunb.), were evaluated in a 24 week study in the female KK.Cg-A(y)/J Type 2 diabetic mouse model. Animals were divided into six groups (n = 16): control mice received Easter lily bulb extract-free drinking water together with a low- or high-fat diet (diabetic control); drinking water for the remaining groups was supplemented with CB extract (1%), BuOH extract (0.1 or 0.2%), and reference drug Metformin (0.001%), together with a high-fat diet. Both CB and BuOH extract treatment groups exhibited significantly improved liver function based on comparisons of triglycerides [diabetic 219 ± 34 mg/dL, CB 131 ± 27 mg/dL, BuOH(0.2%) 114 ± 35 mg/dL], CB total cholesterol (TC) (diabetic 196 ± 12 mg/dL, CB 159 ± 5 mg/dL), average liver mass [diabetic 2.96 ± 0.13 g, CB 2.58 ± 0.08 g, BuOH(0.1%) 2.48 ± 0.13 g], alanine transferase [diabetic 74 ± 5 units/L, CB 25 ± 1 units/L, BuOH(0.1%) 45 ± 1 units/L], and histological examinations. Glucose metabolism was improved only in CB, which was confirmed by oral glucose tolerance tests (OGTT) in diet-induced obese C57BL/6J mice exposed to CB extract. These data suggest that steroidal glycosides 1-5 might play a role in the hepatoprotective activity of the BuOH extracts, while the results of the TC measurements and OGTT study indicate that other constituents present in the CB extract are responsible for its hypocholesterolemic and hypoglycemic activity.

  15. The puzzle of the CNO isotope ratios in asymptotic giant branch carbon stars

    Science.gov (United States)

    Abia, C.; Hedrosa, R. P.; Domínguez, I.; Straniero, O.

    2017-03-01

    Context. The abundance ratios of the main isotopes of carbon, nitrogen and oxygen are modified by the CNO-cycle in the stellar interiors. When the different dredge-up events mix the burning material with the envelope, valuable information on the nucleosynthesis and mixing processes can be extracted by measuring these isotope ratios. Aims: Previous determinations of the oxygen isotopic ratios in asymptotic giant branch (AGB) carbon stars were at odds with the existing theoretical predictions. We aim to redetermine the oxygen ratios in these stars using new spectral analysis tools and further develop discussions on the carbon and nitrogen isotopic ratios in order to elucidate this problem. Methods: Oxygen isotopic ratios were derived from spectra in the K-band in a sample of galactic AGB carbon stars of different spectral types and near solar metallicity. Synthetic spectra calculated in local thermodynamic equillibrium (LTE) with spherical carbon-rich atmosphere models and updated molecular line lists were used. The CNO isotope ratios derived in a homogeneous way, were compared with theoretical predictions for low-mass (1.5-3 M⊙) AGB stars computed with the FUNS code assuming extra mixing both during the RGB and AGB phases. Results: For most of the stars the 16O/17O/18O ratios derived are in good agreement with theoretical predictions confirming that, for AGB stars, are established using the values reached after the first dredge-up (FDU) according to the initial stellar mass. This fact, as far as the oxygen isotopic ratios are concerned, leaves little space for the operation of any extra mixing mechanism during the AGB phase. Nevertheless, for a few stars with large 16O/17O/18O, the operation of such a mechanism might be required, although their observed 12C/13C and 14N/15N ratios would be difficult to reconcile within this scenario. Furthermore, J-type stars tend to have lower 16O/17O ratios than the normal carbon stars, as already indicated in previous studies

  16. Development of zwitterionic chromophores for electro-optic applications

    Science.gov (United States)

    Xiong, Ying

    -dendrons anchored at both the donor and acceptor parts can be poled (r33 = 63 pm/V). (4) Crosslinkable NLO polymers can be prepared by grafting PeQDM-C3OH and 5-aminobenzocyclobutenone as a thermal crosslinker onto acid-containing polyethersulfone. The EO coefficient of a crosslinkable NLO polyethersulfone with 4.8 wt% chromophore core content is 37 pm/V. (5) The use of a polymer with a high dielectric constant to host PeQDM gives rise to the largest EO coefficient (r33 = 110 pm/V), due to the well dispersed monomeric chromophores. The J-type chromophore aggregates formed in a less polar polymer host could still contribute to EO activity, if the dissociated monomer intermediate during the J-H aggregate transformation could be oriented under the poling conditions.

  17. Developments in gamma-ray spectrometry: systems, software, and methods-II. 3. Low-Energy Gamma-Ray Spectrometry Using a Compton-Suppressed Telescope Detector

    International Nuclear Information System (INIS)

    Sigg, R.A.; DiPrete, D.P.

    2001-01-01

    The Savannah River Technology Center (SRTC) utilizes gamma-ray spectrometry in studying numerous areas of applied interest to the Savannah River Site (SRS). For example, analyses of long-lived gamma-ray-emitting fission products and actinides are required to meet waste characterization, process holdup, environmental restoration, and decontamination and decommissioning efforts. A significant portion of the overall effort centers on measurements of gamma rays having energies below several hundred kilo-electron-volts. To assist these efforts, the SRTC recently acquired a spectrometer system that provides lower natural and Compton scattered background levels while achieving relatively high counting efficiencies for low-energy gamma rays. The combination of high efficiency and low background provides factor-of- 2-to-4 improvements in minimum detectable activities and allows meeting programmatic objectives with shorter measurement times. Numerous Compton-suppression spectrometers have been reported since the concept was first advanced. The spectrometer consists of two high-purity germanium detectors in a telescope configuration surrounded by a background /Compton-suppression sodium iodide detector. The front germanium detector is a 20-mm-thick x 60-mm-diam broad energy spectrometer, and the rear detector is a 40% efficient 61- mm-diam x 60-cm-thick closed-end coaxial spectrometer. The cryostat housing the germanium detectors (a) includes a carbon composite window for transmitting low-energy gamma rays, (b) is in a J-type configuration to mask the germanium detectors from natural activities in the cryo-pumping media, and (c) is fabricated from materials selected for low background. The telescope detector is in the 8.6-cm-inside-diameter annulus of a 22.9- x 22.9-cm sodium iodide detector encased in a 10-cm-thick lead shield. The counting system is located in a basement counting room having ∼60-cm-thick concrete walls. Initial tests show that the low-energy segment of

  18. IRS SCAN-MAPPING OF THE WASP-WAIST NEBULA (IRAS 16253-2429). I. DERIVATION OF SHOCK CONDITIONS FROM H2 EMISSION AND DISCOVERY OF 11.3 μm PAH ABSORPTION

    International Nuclear Information System (INIS)

    Barsony, Mary; Wolf-Chase, Grace A.; Ciardi, David R.; O'Linger, JoAnn

    2010-01-01

    The outflow driven by the Class 0 protostar, IRAS 16253-2429, is associated with bipolar cavities visible in scattered mid-infrared light, which we refer to as the Wasp-Waist Nebula. InfraRed Spectometer (IRS) scan mapping with the Spitzer Space Telescope of a ∼1' x 2' area centered on the protostar was carried out. The outflow is imaged in six pure rotational (0-0 S(2) through 0-0 S(7)) H 2 lines, revealing a distinct, S-shaped morphology in all maps. A source map in the 11.3 μm polycyclic aromatic hydrocarbon (PAH) feature is presented in which the protostellar envelope appears in absorption. This is the first detection of absorption in the 11.3 μm PAH feature. Spatially resolved excitation analysis of positions in the blue- and redshifted outflow lobes, with extinction-corrections determined from archival Spitzer 8 μm imaging, shows remarkably constant temperatures of ∼1000 K in the shocked gas. The radiated luminosity in the observed H 2 transitions is found to be 1.94 ± 0.05 x 10 -5 L sun in the redshifted lobe and 1.86 ± 0.04 x 10 -5 L sun in the blueshifted lobe. These values are comparable to the mechanical luminosity of the flow. By contrast, the mass of hot (T ∼ 1000 K) H 2 gas is 7.95 ± 0.19 x 10 -7 M sun in the redshifted lobe and 5.78 ± 0.17 x 10 -7 M sun in the blueshifted lobe. This is just a tiny fraction, of order 10 -3 , of the gas in the cold (30 K), swept-up gas mass derived from millimeter CO observations. The H 2 ortho/para ratio of 3:1 found at all mapped points in this flow suggests previous passages of shocks through the gas. Comparison of the H 2 data with detailed shock models of Wilgenbus et al. shows the emitting gas is passing through Jump (J-type) shocks. Pre-shock densities of 10 4 cm -3 ≤ n H ≤ 10 5 cm -3 are inferred for the redshifted lobe and n H ≤ 10 3 cm -3 for the blueshifted lobe. Shock velocities are 5 km s -1 ≤ v s ≤ 10 km s -1 for the redshifted gas and v s = 10 km s -1 for the blueshifted gas

  19. Radiation Tolerance of Controlled Fusion Welds in High Temperature Oxidation Resistant FeCrAl Alloys

    Energy Technology Data Exchange (ETDEWEB)

    Gussev, Maxim N. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Field, Kevin G. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2017-08-01

    High temperature oxidation resistant iron-chromium-aluminum (FeCrAl) alloys are candidate alloys for nuclear applications due to their exceptional performance during off-normal conditions such as a loss-of-coolant accident (LOCA) compared to currently deployed zirconium-based claddings [1]. A series of studies have been completed to determine the weldability of the FeCrAl alloy class and investigate the weldment performance in the as-received (non-irradiated) state [2,3]. These initial studies have shown the general effects of composition and microstructure on the weldability of FeCrAl alloys. Given this, limited details on the radiation tolerance of FeCrAl alloys and their weldments exist. Here, the highest priority candidate FeCrAl alloys and their weldments have been investigated after irradiation to enable a better understanding of FeCrAl alloy weldment performance within a high-intensity neutron field. The alloys examined include C35M (Fe-13%Cr-5% Al) and variants with aluminum (+2%) or titanium carbide (+1%) additions. Two different sub-sized tensile geometries, SS-J type and SS-2E (or SS-mini), were neutron irradiated in the High Flux Isotope Reactor to 1.8-1.9 displacements per atom (dpa) in the temperature range of 195°C to 559°C. Post irradiation examination of the candidate alloys was completed and included uniaxial tensile tests coupled with digital image correlation (DIC), scanning electron microscopy-electron back scattered diffraction analysis (SEM-EBSD), and SEM-based fractography. In addition to weldment testing, non-welded parent material was examined as a direct comparison between welded and non-welded specimen performance. Both welded and non-welded specimens showed a high degree of radiation-induced hardening near irradiation temperatures of 200°C, moderate radiation-induced hardening near temperatures of 360°C, and almost no radiation-induced hardening at elevated temperatures near 550°C. Additionally, low-temperature irradiations showed