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Sample records for adaptor protein appl1

  1. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa [Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Yang, Jun-Mo [Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul 135-710 (Korea, Republic of); Kang, Jong-Sun, E-mail: kangj01@skku.edu [Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Hahn, Myong-Joon, E-mail: hahnmj@skku.edu [Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  2. The fifth adaptor protein complex.

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    Jennifer Hirst

    2011-10-01

    Full Text Available Adaptor protein (AP complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.

  3. Role of adaptor proteins in secretory granule biogenesis and maturation

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    Mathilde L Bonnemaison

    2013-08-01

    Full Text Available In the regulated secretory pathway, secretory granules (SGs store peptide hormones that are released on demand. SGs are formed at the trans-Golgi network (TGN and must undergo a maturation process to become responsive to secretagogues. The production of mature SGs requires concentrating newly synthesized soluble content proteins in granules whose membranes contain the appropriate integral membrane proteins. The mechanisms underlying the sorting of soluble and integral membrane proteins destined for SGs from other proteins are not yet well understood. For soluble proteins, luminal pH and divalent metals can affect aggregation and interaction with surrounding membranes. The trafficking of granule membrane proteins can be controlled by both luminal and cytosolic factors. Cytosolic adaptor proteins, which recognize the cytosolic domains of proteins that span the SG membrane, have been shown to play essential roles in the assembly of functional SGs. Adaptor protein 1A (AP-1A is known to interact with specific motifs in its cargo proteins and with the clathrin heavy chain, contributing to the formation of a clathrin coat. AP-1A is present in patches on immature SG membranes, where it removes cargo and facilitates SG maturation. AP-1A recruitment to membranes can be modulated by PACS-1 (Phosphofurin Acidic Cluster Sorting protein 1, a cytosolic protein which interacts with both AP-1A and cargo that has been phosphorylated by casein kinase II. A cargo/PACS-1/AP-1A complex is necessary to drive the appropriate transport of several cargo proteins within the regulated secretory pathway. The GGA (Golgi-localized, -ear containing, ADP-ribosylation factor binding family of adaptor proteins serve a similar role. We review the functions of AP-1A, PACS-1 and GGAs in facilitating the retrieval of proteins from immature SGs and review examples of cargo proteins whose trafficking within the regulated secretory pathway is governed by adaptor proteins.

  4. XB130: A novel adaptor protein in cancer signal transduction

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    ZHANG, RUIYAO; ZHANG, JINGYAO; WU, QIFEI; MENG, FANDI; LIU, CHANG

    2016-01-01

    Adaptor proteins are functional proteins that contain two or more protein-binding modules to link signaling proteins together, which affect cell growth and shape and have no enzymatic activity. The actin filament-associated protein (AFAP) family is an important member of the adaptor proteins, including AFAP1, AFAP1L1 and AFAP1L2/XB130. AFAP1 and AFAP1L1 share certain common characteristics and function as an actin-binding protein and a cSrc-activating protein. XB130 exhibits certain unique features in structure and function. The mRNA of XB130 is expressed in human spleen, thyroid, kidney, brain, lung, pancreas, liver, colon and stomach, and the most prominent disease associated with XB130 is cancer. XB130 has a controversial effect on cancer. Studies have shown that XB130 can promote cancer progression and downregulation of XB130-reduced growth of tumors derived from certain cell lines. A higher mRNA level of XB130 was shown to be associated with a better survival in non-small cell lung cancer. Previous studies have shown that XB130 can regulate cell growth, migration and invasion and possibly has the effect through the cAMP-cSrc-phosphoinositide 3-kinase/Akt pathway. Except for cancer, XB130 is also associated with other pathological or physiological procedures, such as airway repair and regeneration. PMID:26998266

  5. Modulation of lipoprotein receptor functions by intracellular adaptor proteins.

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    Stolt, Peggy C; Bock, Hans H

    2006-10-01

    Members of the low density lipoprotein (LDL) receptor gene family are critically involved in a wide range of physiological processes including lipid and vitamin homeostasis, cellular migration, neurodevelopment, and synaptic plasticity, to name a few. Lipoprotein receptors exert these diverse biological functions by acting as cellular uptake receptors or by inducing intracellular signaling cascades. It was discovered that a short sequence in the intracellular region of all lipoprotein receptors, Asn-Pro-X-Tyr (NPXY) is important for mediating either endocytosis or signal transduction events, and that this motif serves as a binding site for phosphotyrosine-binding (PTB) domain containing scaffold proteins. These molecular adaptors connect the transmembrane receptors with the endocytosis machinery and regulate cellular trafficking, or function as assembly sites for dynamic multi-protein signaling complexes. Whereas the LDL receptor represents the archetype of an endocytic lipoprotein receptor, the structurally closely related apolipoprotein E receptor 2 (apoER2) and very low density lipoprotein (VLDL) receptor activate a kinase-dependent intracellular signaling cascade after binding to the neuronal signaling molecule Reelin. This review focuses on two related PTB domain containing adaptor proteins that mediate these divergent lipoprotein receptor responses, ARH (autosomal recessive hypercholesterolemia protein) and Dab1 (disabled-1), and discusses the structural and molecular basis of this different behaviour.

  6. Negative regulation of lymphocyte activation by the adaptor protein LAX.

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    Zhu, Minghua; Granillo, Olivia; Wen, Renren; Yang, Kaiyong; Dai, Xuezhi; Wang, Demin; Zhang, Weiguo

    2005-05-01

    The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.

  7. Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry

    DEFF Research Database (Denmark)

    Al-Eryani, Yusra; Ib Rasmussen, Morten; Kjellström, Sven;

    2016-01-01

    Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxida......Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol......-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress...... and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby...

  8. APPL1 potentiates insulin sensitivity by facilitating the binding of IRS1/2 to the insulin receptor.

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    Ryu, Jiyoon; Galan, Amanda K; Xin, Xiaoban; Dong, Feng; Abdul-Ghani, Muhammad A; Zhou, Lijun; Wang, Changhua; Li, Cuiling; Holmes, Bekke M; Sloane, Lauren B; Austad, Steven N; Guo, Shaodong; Musi, Nicolas; DeFronzo, Ralph A; Deng, Chuxia; White, Morris F; Liu, Feng; Dong, Lily Q

    2014-05-22

    Binding of insulin receptor substrate proteins 1 and 2 (IRS1/2) to the insulin receptor (IR) is essential for the regulation of insulin sensitivity and energy homeostasis. However, the mechanism of IRS1/2 recruitment to the IR remains elusive. Here, we identify adaptor protein APPL1 as a critical molecule that promotes IRS1/2-IR interaction. APPL1 forms a complex with IRS1/2 under basal conditions, and this complex is then recruited to the IR in response to insulin or adiponectin stimulation. The interaction between APPL1 and IR depends on insulin- or adiponectin-stimulated APPL1 phosphorylation, which is greatly reduced in insulin target tissues in obese mice. appl1 deletion in mice consistently leads to systemic insulin resistance and a significant reduction in insulin-stimulated IRS1/2, but not IR, tyrosine phosphorylation, indicating that APPL1 sensitizes insulin signaling by acting at a site downstream of the IR. Our study uncovers a mechanism regulating insulin signaling and crosstalk between the insulin and adiponectin pathways.

  9. APPL1 Potentiates Insulin Sensitivity by Facilitating the Binding of IRS1/2 to the Insulin Receptor

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    Jiyoon Ryu

    2014-05-01

    Full Text Available Binding of insulin receptor substrate proteins 1 and 2 (IRS1/2 to the insulin receptor (IR is essential for the regulation of insulin sensitivity and energy homeostasis. However, the mechanism of IRS1/2 recruitment to the IR remains elusive. Here, we identify adaptor protein APPL1 as a critical molecule that promotes IRS1/2-IR interaction. APPL1 forms a complex with IRS1/2 under basal conditions, and this complex is then recruited to the IR in response to insulin or adiponectin stimulation. The interaction between APPL1 and IR depends on insulin- or adiponectin-stimulated APPL1 phosphorylation, which is greatly reduced in insulin target tissues in obese mice. appl1 deletion in mice consistently leads to systemic insulin resistance and a significant reduction in insulin-stimulated IRS1/2, but not IR, tyrosine phosphorylation, indicating that APPL1 sensitizes insulin signaling by acting at a site downstream of the IR. Our study uncovers a mechanism regulating insulin signaling and crosstalk between the insulin and adiponectin pathways.

  10. The Emerging and Diverse Roles of Src-Like Adaptor Proteins in Health and Disease

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    Nikolett Marton

    2015-01-01

    Full Text Available Although Src-like adaptor proteins (SLAP-1 and SLAP-2 were mainly studied in lymphocytes, where they act as negative regulators and provide fine control of receptor signaling, recently, several other functions of these proteins were discovered. In addition to the well-characterized immunoregulatory functions, SLAP proteins appear to have an essential role in the pathogenesis of type I hypersensitivity, osteoporosis, and numerous malignant diseases. Both adaptor proteins are expressed in a wide variety of tissues, where they have mostly inhibitory effects on multiple intracellular signaling pathways. In this review, we summarize the diverse effects of SLAP proteins.

  11. Distinct adaptor proteins assist exit of Kre2-family proteins from the yeast ER

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    Yoichi Noda

    2014-07-01

    Full Text Available The Svp26 protein of S. cerevisiae is an ER- and Golgi-localized integral membrane protein with 4 potential membrane-spanning domains. It functions as an adaptor protein that facilitates the ER exit of Ktr3, a mannosyltransferase required for biosynthesis of O-linked oligosaccharides, and the ER exit of Mnn2 and Mnn5, mannosyltransferases, which participate in the biosynthesis of N-linked oligosaccharides. Ktr3 belongs to the Kre2 family, which consists of 9 members of type-II membrane proteins sharing sequence similarities. In this report, we examined all Kre2 family members and found that the Golgi localizations of two others, Kre2 and Ktr1, were dependent on Svp26 by immunofluorescence microscopy and cell fractionations in sucrose density gradients. We show that Svp26 functions in facilitating the ER exit of Kre2 and Ktr1 by an in vitro COPII budding assay. Golgi localization of Ktr4 was not dependent on Svp26. Screening null mutants of the genes encoding abundant COPII membrane proteins for those showing mislocalization of Ktr4 in the ER revealed that Erv41 and Erv46 are required for the correct Golgi localization of Ktr4. We provide biochemical evidence that the Erv41-Erv46 complex functions as an adaptor protein for ER exit of Ktr4. This is the first demonstration of the molecular function of this evolutionally conserved protein complex. The domain switching experiments show that the lumenal domain of Ktr4 is responsible for recognition by the Erv41-Erv46 complex. Thus, ER exit of Kre2-family proteins is dependent on distinct adaptor proteins and our results provide new insights into the traffic of Kre2-family mannosyltransferases.

  12. APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.

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    Heidi J Chial

    Full Text Available BACKGROUND: Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR domain, a central pleckstrin homology (PH domain, and a C-terminal phosphotyrosine binding (PTB domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported. METHODOLOGY: Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species. CONCLUSIONS: All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2 and heterotypic (i.e., APPL1-APPL2 manner on curved cell membranes

  13. The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin β1 endocytosis.

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    Teckchandani, Anjali; Mulkearns, Erin E; Randolph, Timothy W; Toida, Natalie; Cooper, Jonathan A

    2012-08-01

    Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin β1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain-binding sites. This mutant localized to clathrin structures with integrin β1, AP2, and reduced amounts of Eps15. Of interest, although integrin β1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin β1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin β1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.

  14. Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling.

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    Pertl-Obermeyer, Heidi; Wu, Xu Na; Schrodt, Jens; Müdsam, Christina; Obermeyer, Gerhard; Schulze, Waltraud X

    2016-09-01

    Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3β and ap-4β knock-out mutants. In ap-3β mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking.

  15. The adapter protein APPL1 links FSH receptor to inositol 1,4,5-trisphosphate production and is implicated in intracellular Ca(2+) mobilization.

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    Thomas, Richard M; Nechamen, Cheryl A; Mazurkiewicz, Joseph E; Ulloa-Aguirre, Alfredo; Dias, James A

    2011-04-01

    FSH binds to its receptor (FSHR) on target cells in the ovary and testis, to regulate oogenesis and spermatogenesis, respectively. The signaling cascades activated after ligand binding are extremely complex and have been shown to include protein kinase A, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/protein kinase B, and inositol 1,4,5-trisphosphate-mediated calcium signaling pathways. The adapter protein APPL1 (Adapter protein containing Pleckstrin homology domain, Phosphotyrosine binding domain and Leucine zipper motif), which has been linked to an assortment of other signaling proteins, was previously identified as an interacting protein with FSHR. Thus, alanine substitution mutations in the first intracellular loop of FSHR were generated to determine which residues are essential for FSHR-APPL1 interaction. Three amino acids were essential; when any one of them was altered, APPL1 association with FSHR mutants was abrogated. Two of the mutants (L377A and F382A) that displayed poor cell-surface expression were not studied further. Substitution of FSHR-K376A did not affect FSH binding or agonist-stimulated cAMP production in either transiently transfected human embryonic kidney cells or virally transduced human granulosa cells (KGN). In the KGN line, as well as primary cultures of rat granulosa cells transduced with wild type or mutant receptor, FSH-mediated progesterone or estradiol production was not affected by the mutation. However, in human embryonic kidney cells inositol 1,4,5-trisphosphate production was curtailed and KGN cells transduced with FSHR-K376A evidenced reduced Ca(2+) mobilization from intracellular stores after FSH treatment.

  16. DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor prote... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 recep

  17. Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry.

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    Al-Eryani, Yusra; Ib Rasmussen, Morten; Kjellström, Sven; Højrup, Peter; Emanuelsson, Cecilia; von Wachenfeldt, Claes

    2016-09-01

    Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234-1245. © 2016 Wiley Periodicals, Inc.

  18. Role of SRC-like adaptor protein (SLAP) in immune and malignant cell signaling.

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    Kazi, Julhash U; Kabir, Nuzhat N; Rönnstrand, Lars

    2015-07-01

    SRC-like adaptor protein (SLAP) is an adaptor protein structurally similar to the SRC family protein kinases. Like SRC, SLAP contains an SH3 domain followed by an SH2 domain but the kinase domain has been replaced by a unique C-terminal region. SLAP is expressed in a variety of cell types. Current studies suggest that it regulates signaling of various cell surface receptors including the B cell receptor, the T cell receptor, cytokine receptors and receptor tyrosine kinases which are important regulator of immune and cancer cell signaling. SLAP targets receptors, or its associated components, by recruiting the ubiquitin machinery and thereby destabilizing signaling. SLAP directs receptors to ubiquitination-mediated degradation and controls receptors turnover as well as signaling. Thus, SLAP appears to be an important component in regulating signal transduction required for immune and malignant cells.

  19. The Lnk adaptor protein: a key regulator of normal and pathological hematopoiesis.

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    Velazquez, Laura

    2012-12-01

    The development and function of blood cells are regulated by specific growth factors/cytokines and their receptors' signaling pathways. In this way, these factors influence cell survival, proliferation and differentiation of hematopoietic cells. Central to this positive and/or negative control are the adaptor proteins. Since their identification 10 years ago, members of the Lnk adaptor protein family have proved to be important activators and/or inhibitors in the hematopoietic, immune and vascular system. In particular, the generation of animal and cellular models for the Lnk and APS proteins has helped establish the physiological role of these molecules through the identification of their specific signaling pathways and the characterization of their binding partners. Moreover, the recent identification of mutations in the LNK gene in myeloproliferative disorders, as well as the correlation of a single nucleotide polymorphism on LNK with hematological, immune and vascular diseases have suggested its involvement in the pathophysiology of these malignancies. The latter findings have thus raised the possibility of addressing Lnk signaling for the treatment of certain human diseases. This review therefore describes the pathophysiological role of this adaptor protein in hematological malignancies and the potential benefits of Lnk therapeutic targeting.

  20. Science Signaling Podcast for 12 July 2016: Adaptor proteins limit signaling.

    Science.gov (United States)

    Wiley, H Steven; VanHook, Annalisa M

    2016-07-12

    This Podcast features an interview with Steven Wiley, senior author of a Research Article that appears in the 12 July 2016 issue of Science Signaling, about how the abundance of adaptor proteins and feedback regulators affect the flow of information downstream of the epidermal growth factor receptor (EGFR). Information flows through a signaling pathway by sequential interactions between core components of the pathway, many of which have enzymatic activity. Adaptor proteins do not directly participate in relaying the signal and do not have enzymatic activity, but are important for signaling because they facilitate interactions between the core components. Using quantitative methods, Shi et al demonstrated that core components of the EGFR pathway were highly abundant in both normal cells and cancer cells. However, adaptor proteins were present in much lower abundance in both cell types, indicating that it is the abundance of these proteins that limit signaling downstream of EGFR. The authors also found that differences in EGFR signaling between different cell types likely resulted from the variable abundance of feedback regulators.Listen to Podcast.

  1. Selective autophagy of non-ubiquitylated targets in plants: looking for cognate receptor/adaptor proteins

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    Vasko eVeljanovski

    2014-06-01

    Full Text Available Cellular homeostasis is essential for the physiology of eukaryotic cells. Eukaryotic cells, including plant cells, utilize two main pathways to adjust the level of cytoplasmic components, namely the proteasomal and the lysosomal/vacuolar pathways. Macroautophagy is a lysosomal/vacuolar pathway which, until recently, was thought to be non-specific and a bulk degradation process. However, selective autophagy which can be activated in the cell under various physiological conditions, involves the specific degradation of defined macromolecules or organelles by a conserved molecular mechanism. For this process to be efficient, the mechanisms underlying the recognition and selection of the cargo to be engulfed by the double-membrane autophagosome are critical, and not yet well understood. Ubiquitin (poly-ubiquitin conjugation to the target appears to be a conserved ligand mechanism in many types of selective autophagy, and defined receptors/adaptors recognizing and regulating the autophagosomal capture of the ubiquitylated target have been characterized. However, non-proteinaceous and non-ubiquitylated cargoes are also selectively degraded by this pathway. This ubiquitin-independent selective autophagic pathway also involves receptor and/or adaptor proteins linking the cargo to the autophagic machinery. Some of these receptor/adaptor proteins including accessory autophagy-related (Atg and non-Atg proteins have been described in yeast and animal cells but not yet in plants. In this review we discuss the ubiquitin-independent cargo selection mechanisms in selective autophagy degradation of organelles and macromolecules and speculate on potential plant receptor/adaptor proteins.

  2. Versatile modes of peptide recognition by the AAA+ adaptor protein SspB

    Energy Technology Data Exchange (ETDEWEB)

    Levchenko, Igor; Grant, Robert A.; Flynn, Julia M.; Sauer, Robert T.; Baker, Tania A. (MIT)

    2010-07-19

    Energy-dependent proteases often rely on adaptor proteins to modulate substrate recognition. The SspB adaptor binds peptide sequences in the stress-response regulator RseA and in ssrA-tagged proteins and delivers these molecules to the AAA+ ClpXP protease for degradation. The structure of SspB bound to an ssrA peptide is known. Here, we report the crystal structure of a complex between SspB and its recognition peptide in RseA. Notably, the RseA sequence is positioned in the peptide-binding groove of SspB in a direction opposite to the ssrA peptide, the two peptides share only one common interaction with the adaptor, and the RseA interaction site is substantially larger than the overlapping ssrA site. This marked diversity in SspB recognition of different target proteins indicates that it is capable of highly flexible and dynamic substrate delivery.

  3. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  4. Role of adaptor proteins in motor regulation and membrane transport

    NARCIS (Netherlands)

    M.A. Schlager (Max)

    2010-01-01

    markdownabstract__Abstract__ Active transport along the cytoskeleton is a process essential for proper cellular function. Although much is known about the motor proteins that generate the necessary force and the cytoskeleton that provides the cellular infrastructure, many questions still remain. Fo

  5. The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb.

    Science.gov (United States)

    Sun, Miao; Asghar, Suwaiba Z; Zhang, Huaye

    2016-09-01

    The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking.

  6. The interaction between the adaptor protein APS and Enigma is involved in actin organisation

    DEFF Research Database (Denmark)

    Barres, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick

    2005-01-01

    APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein...... that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma...... and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin...

  7. Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

    Science.gov (United States)

    Unterweger, Daniel; Kostiuk, Benjamin; Ötjengerdes, Rina; Wilton, Ashley; Diaz-Satizabal, Laura; Pukatzki, Stefan

    2015-08-13

    Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.

  8. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    Science.gov (United States)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  9. Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes.

    Science.gov (United States)

    Cheung, Giselle; Cousin, Michael A

    2012-04-25

    Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.

  10. Structural basis for the interaction of the adaptor protein grb14 with activated ras.

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    Rohini Qamra

    Full Text Available Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA domain, a pleckstrin-homology (PH domain, a family-specific BPS (between PH and SH2 region, and a C-terminal Src-homology-2 (SH2 domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V. The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.

  11. A highly versatile adaptor protein for the tethering of growth factors to gelatin-based biomaterials.

    Science.gov (United States)

    Addi, Cyril; Murschel, Frédéric; Liberelle, Benoît; Riahi, Nesrine; De Crescenzo, Gregory

    2017-03-01

    In the field of tissue engineering, the tethering of growth factors to tissue scaffolds in an oriented manner can enhance their activity and increase their half-life. We chose to investigate the capture of the basic Fibroblast Growth Factor (bFGF) and the Epidermal Growth Factor (EGF) on a gelatin layer, as a model for the functionalization of collagen-based biomaterials. Our strategy relies on the use of two high affinity interactions, that is, the one between two distinct coil peptides as well as the one occurring between a collagen-binding domain (CBD) and gelatin. We expressed a chimeric protein to be used as an adaptor that comprises one of the coil peptides and a CBD derived from the human fibronectin. We proved that it has the ability to bind simultaneously to a gelatin substrate and to form a heterodimeric coiled-coil domain with recombinant growth factors being tagged with the complementary coil peptide. The tethering of the growth factors was characterized by ELISA and surface plasmon resonance-based biosensing. The bioactivity of the immobilized bFGF and EGF was evaluated by a human umbilical vein endothelial cell proliferation assay and a vascular smooth muscle cell survival assay. We found that the tethering of EGF preserved its mitogenic and anti-apoptotic activity. In the case of bFGF, when captured via our adaptor protein, changes in its natural mode of interaction with gelatin were observed.

  12. Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity

    Directory of Open Access Journals (Sweden)

    Anselm H.C. Horn

    2015-11-01

    Full Text Available The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

  13. Src-like-adaptor protein (SLAP) differentially regulates normal and oncogenic c-Kit signaling.

    Science.gov (United States)

    Kazi, Julhash U; Agarwal, Shruti; Sun, Jianmin; Bracco, Enrico; Rönnstrand, Lars

    2014-02-01

    The Src-like-adaptor protein (SLAP) is an adaptor protein sharing considerable structural homology with Src. SLAP is expressed in a variety of cells and regulates receptor tyrosine kinase signaling by direct association. In this report, we show that SLAP associates with both wild-type and oncogenic c-Kit (c-Kit-D816V). The association involves the SLAP SH2 domain and receptor phosphotyrosine residues different from those mediating Src interaction. Association of SLAP triggers c-Kit ubiquitylation which, in turn, is followed by receptor degradation. Although SLAP depletion potentiates c-Kit downstream signaling by stabilizing the receptor, it remains non-functional in c-Kit-D816V signaling. Ligand-stimulated c-Kit or c-Kit-D816V did not alter membrane localization of SLAP. Interestingly oncogenic c-Kit-D816V, but not wild-type c-Kit, phosphorylates SLAP on residues Y120, Y258 and Y273. Physical interaction between c-Kit-D816V and SLAP is mandatory for the phosphorylation to take place. Although tyrosine-phosphorylated SLAP does not affect c-Kit-D816V signaling, mutation of these tyrosine sites to phenylalanine can restore SLAP activity. Taken together the data demonstrate that SLAP negatively regulates wild-type c-Kit signaling, but not its oncogenic counterpart, indicating a possible mechanism by which the oncogenic c-Kit bypasses the normal cellular negative feedback control.

  14. Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

    Energy Technology Data Exchange (ETDEWEB)

    Román-Hernández, Giselle; Grant, Robert A.; Sauer, Robert T.; Baker, Tania A.; (HHMI)

    2009-06-19

    The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids. In each case, the alpha-amino group and side chain of the N-terminal residue are the major determinants of recognition. The binding pocket for the N-end residue is preformed in the free adaptor, and only small adjustments are needed to accommodate N-end rule residues having substantially different sizes and shapes. M53A ClpS is known to mediate degradation of an expanded repertoire of substrates, including those with N-terminal valine or isoleucine. A structure of Met53A ClpS engaged with an N-end rule tryptophan reveals an essentially wild-type mechanism of recognition, indicating that the Met(53) side chain directly enforces specificity by clashing with and excluding beta-branched side chains. Finally, experimental and structural data suggest mechanisms that make proteins with N-terminal methionine bind very poorly to ClpS, explaining why these high-abundance proteins are not degraded via the N-end rule pathway in the cell.

  15. Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rynditch A. V.

    2012-12-01

    Full Text Available Intersectin 1 (ITSN1 is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S co-precipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also co-localize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells.

  16. Unexpected diversity in Shisa-like proteins suggests the importance of their roles as transmembrane adaptors.

    Science.gov (United States)

    Pei, Jimin; Grishin, Nick V

    2012-03-01

    The Shisa family of single-transmembrane proteins is characterized by an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Its founding member, Xenopus Shisa, promotes head development by antagonizing Wnt and FGF signaling. Recently, a mouse brain-specific Shisa protein CKAMP44 (Shisa9) was shown to play an important role in AMPA receptor desensitization. We used sequence similarity searches against protein, genome and EST databases to study the evolutionary origin and phylogenetic distribution of Shisa homologs. In addition to nine Shisa subfamilies in vertebrates, we detected distantly related Shisa homologs that possess an N-terminal domain with six conserved cysteines. These Shisa-like proteins include FAM159 and KIAA1644 mainly from vertebrates, and members from various bilaterian invertebrates and Porifera, suggesting their presence in the last common ancestor of Metazoa. Shisa-like genes have undergone large expansions in Branchiostoma floridae and Saccoglossus kowalevskii, and appear to have been lost in certain insects. Pattern-based searches against eukaryotic proteomes also uncovered several other families of predicted single-transmembrane proteins with a similar cysteine-rich domain. We refer to these proteins (Shisa/Shisa-like, WBP1/VOPP1, CX, DUF2650, TMEM92, and CYYR1) as STMC6 proteins (single-transmembrane proteins with conserved 6 cysteines). STMC6 genes are widespread in Metazoa, with the human genome containing 17 members. Frequent occurrences of PY motifs in STMC6 proteins suggest that most of them could interact with WW-domain-containing proteins, such as the NEDD4 family E3 ubiquitin ligases, and could play critical roles in protein degradation and sorting. STMC6 proteins are likely transmembrane adaptors that regulate membrane proteins such as cell surface receptors.

  17. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine-phos...

  18. PHF6 Degrees of Separation: The Multifaceted Roles of a Chromatin Adaptor Protein

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    Matthew A.M. Todd

    2015-06-01

    Full Text Available The importance of chromatin regulation to human disease is highlighted by the growing number of mutations identified in genes encoding chromatin remodeling proteins. While such mutations were first identified in severe developmental disorders, or in specific cancers, several genes have been implicated in both, including the plant homeodomain finger protein 6 (PHF6 gene. Indeed, germline mutations in PHF6 are the cause of the Börjeson–Forssman–Lehmann X-linked intellectual disability syndrome (BFLS, while somatic PHF6 mutations have been identified in T-cell acute lymphoblastic leukemia (T-ALL and acute myeloid leukemia (AML. Studies from different groups over the last few years have made a significant impact towards a functional understanding of PHF6 protein function. In this review, we summarize the current knowledge of PHF6 with particular emphasis on how it interfaces with a distinct set of interacting partners and its functional roles in the nucleoplasm and nucleolus. Overall, PHF6 is emerging as a key chromatin adaptor protein critical to the regulation of neurogenesis and hematopoiesis.

  19. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    Science.gov (United States)

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion.

  20. Highly pathogenic avian influenza virus nucleoprotein interacts with TREX complex adaptor protein Aly/REF.

    Science.gov (United States)

    Balasubramaniam, Vinod R M T; Hong Wai, Tham; Ario Tejo, Bimo; Omar, Abdul Rahman; Syed Hassan, Sharifah

    2013-01-01

    We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.

  1. Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus

    Directory of Open Access Journals (Sweden)

    Skrypkina I. Ya.

    2011-06-01

    Full Text Available Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN122a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the novel isoform. Methods. Dimerization ability of ITSN1-22a was tested by immunoprecipitation and subsequent Western blot analysis. To specify the region responsible for dimerization, site-directed mutagenesis and truncation analysis were carried out. Inhibition of endocytosis by potassium depletion and EGF stimulation of HEK293 were performed. Results. We have found that ITSN1-22a forms dimers in HEK293 cells. The dimerization of ITSN1-22a was mediated by C-terminal domain. We showed that cysteines C1016 and C1019 were involved in homodimerization. Inhibition of clathrin-mediated endocytosis and mitogen stimulation did not affect ITSN1-22a dimer formation. Conclusions. ITSN1-22a is the only one known ITSN1 isoform, which is capable to form homodimers via disulphide bonds. This could be important for the formation of protein complexes containing ITSN1 molecules.

  2. The adaptor protein FHL2 enhances the cellular innate immune response to influenza A virus infection.

    Science.gov (United States)

    Nordhoff, Carolin; Hillesheim, Andrea; Walter, Beate M; Haasbach, Emanuel; Planz, Oliver; Ehrhardt, Christina; Ludwig, Stephan; Wixler, Viktor

    2012-07-01

    The innate immune response of influenza A virus-infected cells is predominantly mediated by type I interferon-induced proteins. Expression of the interferon β (IFNβ) itself is initiated by accumulating viral RNA and is transmitted by different signalling cascades that feed into activation of the three transcriptional elements located in the IFNβ promoter, AP-1, IRF-3 and NF-κB. FHL2 (four-and-a-half LIM domain protein 2) is an adaptor molecule that shuttles between membrane and nucleus regulating signalling cascades and gene transcription. Here we describe FHL2 as a novel regulator of influenza A virus propagation. Using mouse FHL2 wild-type, knockout and rescued cells and human epithelial cells with different expression levels of FHL2 we showed that FHL2 decreases influenza A virus propagation by regulating the intrinsic cellular antiviral immune response. On virus infection FHL2 translocates into the nucleus, potentiating the IRF-3-dependent transcription of the IFNβ gene.

  3. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.

    Science.gov (United States)

    Zhong, Bo; Yang, Yan; Li, Shu; Wang, Yan-Yi; Li, Ying; Diao, Feici; Lei, Caoqi; He, Xiao; Zhang, Lu; Tien, Po; Shu, Hong-Bing

    2008-10-17

    Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.

  4. Dengue virus targets the adaptor protein MITA to subvert host innate immunity.

    Directory of Open Access Journals (Sweden)

    Chia-Yi Yu

    Full Text Available Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN. We found that activation of interferon regulatory factor 3 (IRF3 triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓(96G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.

  5. Dengue virus targets the adaptor protein MITA to subvert host innate immunity.

    Science.gov (United States)

    Yu, Chia-Yi; Chang, Tsung-Hsien; Liang, Jian-Jong; Chiang, Ruei-Lin; Lee, Yi-Ling; Liao, Ching-Len; Lin, Yi-Ling

    2012-01-01

    Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN). We found that activation of interferon regulatory factor 3 (IRF3) triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA) but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓(96)G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT) stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.

  6. The motogenic and mitogenic responses to HGF are amplified by the Shc adaptor protein

    DEFF Research Database (Denmark)

    Pelicci, G; Giordano, S; Zhen, Z

    1995-01-01

    The receptor of Hepatocyte Growth Factor-Scatter Factor (HGF) is a tyrosine kinase which regulates cell motility and growth. After ligand-induced tyrosine phosphorylation, the HGF receptor associates with the Shc adaptor, via the SH2 domain. Site-directed mutagenesis of the HGF receptor indicates...

  7. Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING.

    Science.gov (United States)

    Shi, Heping; Wu, Jiaxi; Chen, Zhijian J; Chen, Chuo

    2015-07-21

    Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2'3'-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2'3'-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2'3'-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein-ligand interactions.

  8. Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor. Dual Src homology (SH) 2/SH3 domain requirement and functional consequences

    DEFF Research Database (Denmark)

    Su, J; Yang, L T; Sap, J

    1996-01-01

    Receptor protein-tyrosine phosphatase RPTPalpha is found associated in vivo with the adaptor protein Grb2. Formation of this complex, which contains no detectable levels of Sos, is known to depend on a C-terminal phosphorylated tyrosine residue (Tyr798) in RPTPalpha and on the Src homology (SH) 2...

  9. Src-like adaptor protein (SLAP) is upregulated in antigen-stimulated mast cells and acts as a negative regulator.

    Science.gov (United States)

    Park, Seung-Kiel; Qiao, Huihong; Beaven, Michael A

    2009-06-01

    Our studies in the RBL-2H3 mast cell line suggest that responses to antigen (Ag) are negatively modulated through upregulation of Src-like adaptor protein (SLAP). Ag stimulation of RBL-2H3 cells leads to increased levels of SLAP (but not SLAP2) transcripts and protein over a period of several hours. The effects of pharmacologic inhibitors indicate that the upregulation of SLAP is dependent on multiple signaling pathways. Knockdown of SLAP with anti-SLAP siRNA is associated with enhanced phosphorylation of Syk, the linker for activation of T cells (LAT), phospholipase C gamma, MAP kinases, and various transcription factors. Production of IL-3 and MCP-1, but not degranulation, is also enhanced. The upregulation of SLAP may thus serve to limit the duration of cytokine production in Ag-stimulated cells.

  10. Brain-derived neurotrophic factor modulation of Kv1.3 channel is disregulated by adaptor proteins Grb10 and nShc

    Directory of Open Access Journals (Sweden)

    Marks David R

    2009-01-01

    Full Text Available Abstract Background Neurotrophins are important regulators of growth and regeneration, and acutely, they can modulate the activity of voltage-gated ion channels. Previously we have shown that acute brain-derived neurotrophic factor (BDNF activation of neurotrophin receptor tyrosine kinase B (TrkB suppresses the Shaker voltage-gated potassium channel (Kv1.3 via phosphorylation of multiple tyrosine residues in the N and C terminal aspects of the channel protein. It is not known how adaptor proteins, which lack catalytic activity, but interact with members of the neurotrophic signaling pathway, might scaffold with ion channels or modulate channel activity. Results We report the co-localization of two adaptor proteins, neuronal Src homology and collagen (nShc and growth factor receptor-binding protein 10 (Grb10, with Kv1.3 channel as demonstrated through immunocytochemical approaches in the olfactory bulb (OB neural lamina. To further explore the specificity and functional ramification of adaptor/channel co-localization, we performed immunoprecipitation and Western analysis of channel, kinase, and adaptor transfected human embryonic kidney 293 cells (HEK 293. nShc formed a direct protein-protein interaction with Kv1.3 that was independent of BDNF-induced phosphorylation of Kv1.3, whereas Grb10 did not complex with Kv1.3 in HEK 293 cells. Both adaptors, however, co-immunoprecipitated with Kv1.3 in native OB. Grb10 was interestingly able to decrease the total expression of Kv1.3, particularly at the membrane surface, and subsequently eliminated the BDNF-induced phosphorylation of Kv1.3. To examine the possibility that the Src homology 2 (SH2 domains of Grb10 were directly binding to basally phosphorylated tyrosines in Kv1.3, we utilized point mutations to substitute multiple tyrosine residues with phenylalanine. Removal of the tyrosines 111–113 and 449 prevented Grb10 from decreasing Kv1.3 expression. In the absence of either adaptor protein

  11. Regulation of in vitro and in vivo immune functions by the cytosolic adaptor protein SKAP-HOM.

    Science.gov (United States)

    Togni, M; Swanson, K D; Reimann, S; Kliche, S; Pearce, A C; Simeoni, L; Reinhold, D; Wienands, J; Neel, B G; Schraven, B; Gerber, A

    2005-09-01

    SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca(2+) responses, are normal in SKAP-HOM(-/-) animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM(-/-). In addition, adhesion of activated B cells to fibronectin (a ligand for beta1 integrins) as well as to ICAM-1 (a ligand for beta2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

  12. A novel method for evaluating microglial activation using ionized calcium-binding adaptor protein-1 staining: cell body to cell size ratio

    NARCIS (Netherlands)

    Hovens, Iris; Nyakas, Csaba; Schoemaker, Regina

    2014-01-01

    Aim: The aim was to validate a newly developed methodology of semi-automatic image analysis to analyze microglial morphology as marker for microglial activation in ionized calcium-binding adaptor protein-1 (IBA-1) stained brain sections. Methods: The novel method was compared to currently used analy

  13. A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins.

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    Ricardo Núñez Miguel

    Full Text Available The Toll-like receptor 4 (TLR4 is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3 and nuclear factor kappaB (NFkappaB respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.

  14. The ubiquitin ligase RNF5 regulates antiviral responses by mediating degradation of the adaptor protein MITA.

    Science.gov (United States)

    Zhong, Bo; Zhang, Lu; Lei, Caoqi; Li, Ying; Mao, Ai-Ping; Yang, Yan; Wang, Yan-Yi; Zhang, Xiao-Lian; Shu, Hong-Bing

    2009-03-20

    Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

  15. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    Science.gov (United States)

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein.

  16. Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues

    Institute of Scientific and Technical Information of China (English)

    va Korpos; Ferenc Dek; Ibolya Kiss

    2015-01-01

    The extracellular matrix (ECM) performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic chang-es during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2) in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junc-tion formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth.

  17. Rap1-GTP-interacting Adaptor Molecule (RIAM) Protein Controls Invasion and Growth of Melanoma Cells*

    Science.gov (United States)

    Hernández-Varas, Pablo; Coló, Georgina P.; Bartolomé, Ruben A.; Paterson, Andrew; Medraño-Fernández, Iria; Arellano-Sánchez, Nohemí; Cabañas, Carlos; Sánchez-Mateos, Paloma; Lafuente, Esther M.; Boussiotis, Vassiliki A.; Strömblad, Staffan; Teixidó, Joaquin

    2011-01-01

    The Mig-10/RIAM/lamellipodin (MRL) family member Rap1-GTP-interacting adaptor molecule (RIAM) interacts with active Rap1, a small GTPase that is frequently activated in tumors such as melanoma and prostate cancer. We show here that RIAM is expressed in metastatic human melanoma cells and that both RIAM and Rap1 are required for BLM melanoma cell invasion. RIAM silencing in melanoma cells led to inhibition of tumor growth and to delayed metastasis in a severe combined immunodeficiency xenograft model. Defective invasion of RIAM-silenced melanoma cells arose from impairment in persistent cell migration directionality, which was associated with deficient activation of a Vav2-RhoA-ROCK-myosin light chain pathway. Expression of constitutively active Vav2 and RhoA in cells depleted for RIAM partially rescued their invasion, indicating that Vav2 and RhoA mediate RIAM function. These results suggest that inhibition of cell invasion in RIAM-silenced melanoma cells is likely based on altered cell contractility and cell polarization. Furthermore, we show that RIAM depletion reduces β1 integrin-dependent melanoma cell adhesion, which correlates with decreased activation of both Erk1/2 MAPK and phosphatidylinositol 3-kinase, two central molecules controlling cell growth and cell survival. In addition to causing inhibition of cell proliferation, RIAM silencing led to higher susceptibility to cell apoptosis. Together, these data suggest that defective activation of these kinases in RIAM-silenced cells could account for inhibition of melanoma cell growth and that RIAM might contribute to the dissemination of melanoma cells. PMID:21454517

  18. Positive and negative regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL.

    Science.gov (United States)

    Zhu, Minghua; Liu, Yan; Koonpaew, Surapong; Granillo, Olivia; Zhang, Weiguo

    2004-10-18

    Linker for activation of B cells (LAB, also called NTAL; a product of wbscr5 gene) is a newly identified transmembrane adaptor protein that is expressed in B cells, NK cells, and mast cells. Upon BCR activation, LAB is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT-deficient mice. To study the in vivo function of LAB, LAB-deficient mice were generated. Although disruption of the Lab gene did not affect lymphocyte development, it caused mast cells to be hyperresponsive to stimulation via the FcepsilonRI, evidenced by enhanced Erk activation, calcium mobilization, degranulation, and cytokine production. These data suggested that LAB negatively regulates mast cell function. However, mast cells that lacked both linker for activation of T cells (LAT) and LAB proteins had a more severe block in FcepsilonRI-mediated signaling than LAT(-/-) mast cells, demonstrating that LAB also shares a redundant function with LAT to play a positive role in FcepsilonRI-mediated signaling.

  19. The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development.

    Science.gov (United States)

    Lin, Wai W; Yi, Zuoan; Stunz, Laura L; Maine, Christian J; Sherman, Linda A; Bishop, Gail A

    2015-09-01

    Tumor necrosis factor receptor-associated factor 3 (TRAF3) is an adaptor protein that inhibits signaling by CD40 and by the receptor for B cell-activating factor (BAFF) and negatively regulates homeostatic B cell survival. Loss-of-function mutations in TRAF3 are associated with human B cell malignancies, in particular multiple myeloma. The cytokine interleukin-6 (IL-6) supports the differentiation and survival of normal and neoplastic plasma cells. We found that mice with a deficiency in TRAF3 specifically in B cells (B-Traf3(-/-) mice) had about twice as many plasma cells as did their littermate controls. TRAF3-deficient B cells had enhanced responsiveness to IL-6, and genetic loss of IL-6 in B-Traf3(-/-) mice restored their plasma cell numbers to normal. TRAF3 inhibited IL-6 receptor (IL-6R)-mediated signaling by facilitating the association of PTPN22 (a nonreceptor protein tyrosine phosphatase) with the kinase Janus-activated kinase 1 (Jak1), which in turn blocked phosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Consistent with these results, the number of plasma cells in the PTPN22-deficient mice was increased compared to that in the wild-type mice. Our findings identify TRAF3 and PTPN22 as inhibitors of IL-6R signaling in B cells and reveal a previously uncharacterized role for TRAF3 in the regulation of plasma cell differentiation.

  20. A transgenic Drosophila model demonstrates that the Helicobacter pylori CagA protein functions as a eukaryotic Gab adaptor.

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    Crystal M Botham

    2008-05-01

    Full Text Available Infection with the human gastric pathogen Helicobacter pylori is associated with a spectrum of diseases including gastritis, peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. The cytotoxin-associated gene A (CagA protein of H. pylori, which is translocated into host cells via a type IV secretion system, is a major risk factor for disease development. Experiments in gastric tissue culture cells have shown that once translocated, CagA activates the phosphatase SHP-2, which is a component of receptor tyrosine kinase (RTK pathways whose over-activation is associated with cancer formation. Based on CagA's ability to activate SHP-2, it has been proposed that CagA functions as a prokaryotic mimic of the eukaryotic Grb2-associated binder (Gab adaptor protein, which normally activates SHP-2. We have developed a transgenic Drosophila model to test this hypothesis by investigating whether CagA can function in a well-characterized Gab-dependent process: the specification of photoreceptors cells in the Drosophila eye. We demonstrate that CagA expression is sufficient to rescue photoreceptor development in the absence of the Drosophila Gab homologue, Daughter of Sevenless (DOS. Furthermore, CagA's ability to promote photoreceptor development requires the SHP-2 phosphatase Corkscrew (CSW. These results provide the first demonstration that CagA functions as a Gab protein within the tissue of an organism and provide insight into CagA's oncogenic potential. Since many translocated bacterial proteins target highly conserved eukaryotic cellular processes, such as the RTK signaling pathway, the transgenic Drosophila model should be of general use for testing the in vivo function of bacterial effector proteins and for identifying the host genes through which they function.

  1. Downregulation of the NHE3-binding PDZ-adaptor protein PDZK1 expression during cytokine-induced inflammation in interleukin-10-deficient mice.

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    Henrike Lenzen

    Full Text Available BACKGROUND: Impaired salt and water absorption is an important feature in the pathogenesis of diarrhea in inflammatory bowel disease (IBD. We analyzed the expression of proinflammatory cytokines in the infiltrating immune cells and the function and expression of the Na(+/H(+ exchanger isoform 3 (NHE3 and its regulatory PDZ-adaptor proteins NHERF1, NHERF2, and PDZK1 in the colon of interleukin-10-deficient (IL-10(-/- mice. METHODOLOGY/PRINCIPAL FINDINGS: Gene and protein expression were analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR, in situ RT-PCR, and immunohistochemistry. NHE3 activity was measured fluorometrically in apical enterocytes within isolated colonic crypts. Mice developed chronic colitis characterized by a typical immune cell infiltration composed of T-lymphocytes and macrophages, with high levels of gene and protein expression of the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α. In parallel, inducible nitric oxide synthase expression was increased while procaspase 3 expression was unaffected. Interferon-γ expression remained low. Although acid-activated NHE3 activity was significantly decreased, the inflammatory process did not affect its gene and protein expression or its abundance and localization in the apical membrane. However, expression of the PDZ-adaptor proteins NHERF2 and PDZK1 was downregulated. NHERF1 expression was unchanged. In a comparative analysis we observed the PDZK1 downregulation also in the DSS (dextran sulphate sodium model of colitis. CONCLUSIONS/SIGNIFICANCE: The impairment of the absorptive function of the inflamed colon in the IL-10(-/- mouse, in spite of unaltered NHE3 expression and localization, is accompanied by the downregulation of the NHE3-regulatory PDZ adaptors NHERF2 and PDZK1. We propose that the downregulation of PDZ-adaptor proteins may be an important factor leading to NHE3 dysfunction and diarrhea in the course of the cytokine

  2. The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer

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    Natsume Hiroko

    2012-07-01

    Full Text Available Abstract Background Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer. Methods and Results A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2 in five highly amplified chromosome regions (with a copy number of more than 6 were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3 domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH, and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360 of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. Conclusion These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

  3. RECOMBINANT LENTIVIRUS-MEDIATED SILENCING OF ADAPTOR PROTEIN RUK/CIN85 EXPRESSION INFLUENCES BIOLOGICAL RESPONSES OF TUMOR CELLS

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    A. A. Samoylenko

    2013-08-01

    Full Text Available Ruk/CIN85 is an adaptor protein that plays important roles in the regulation of cellular processes such as cell death, proliferation and motility. It was recently shown that overexpression of Ruk/CIN85 increases the oncogenic potential of human breast adenocarcinoma MCF-7 cells. It was the aim of the present study to investigate whether inhibition of Ruk/CIN85 expression has an effect on the biological properties of the cells. In order to down-regulate Ruk/CIN85 expression of small interfering RNA-based approach was used. For down-regulation of Ruk/CIN85 lentiviral constructs encoding Ruk/CIN85-specific small hairpin RNA sequences were generated. By using the obtained recombinant lentiviruses it was shown that inhibition of Ruk/CIN85 expression influences biological properties (motility, proliferation, ABCG2 expression, and ROS generation of various tumour cell types such as human breast adenocarcinoma MCF-7, human colorectal adenocarcinoma HT-29, and Lewis mouse lung carcinoma cells.

  4. Nuclear IKKbeta is an adaptor protein for IkappaBalpha ubiquitination and degradation in UV-induced NF-kappaB activation.

    Science.gov (United States)

    Tsuchiya, Yoshihiro; Asano, Tomoichiro; Nakayama, Keiko; Kato, Tomohisa; Karin, Michael; Kamata, Hideaki

    2010-08-27

    Proinflammatory cytokines activate NF-kappaB using the IkappaB kinase (IKK) complex that phosphorylates inhibitory proteins (IkappaBs) at N-terminal sites resulting in their ubiquitination and degradation in the cytoplasm. Although ultraviolet (UV) irradiation does not lead to IKK activity, it activates NF-kappaB by an unknown mechanism through IkappaBalpha degradation without N-terminal phosphorylation. Here, we describe an adaptor function of nuclear IKKbeta in UV-induced IkappaBalpha degradation. UV irradiation induces the nuclear translocation of IkappaBalpha and association with IKKbeta, which constitutively interacts with beta-TrCP through heterogeneous ribonucleoprotein-U (hnRNP-U) leading to IkappaBalpha ubiquitination and degradation. Furthermore, casein kinase 2 (CK2) and p38 associate with IKKbeta and promote IkappaBalpha degradation by phosphorylation at C-terminal sites. Thus, nuclear IKKbeta acts as an adaptor protein for IkappaBalpha degradation in UV-induced NF-kappaB activation. NF-kappaB activated by the nuclear IKKbeta adaptor protein suppresses anti-apoptotic gene expression and promotes UV-induced cell death.

  5. Molecular analysis of the prostacyclin receptor's interaction with the PDZ1 domain of its adaptor protein PDZK1.

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    Gabriel Birrane

    Full Text Available The prostanoid prostacyclin, or prostaglandin I2, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1 of PDZK1, we generated a nine residue peptide (KK(411IAACSLC(417 containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP ((411IAACSLC(417. Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.

  6. Role of Crk Adaptor Proteins in Cellular Migration and Invasion in Human Breast Cancer

    Science.gov (United States)

    2007-03-01

    adapter protein. Mol Cell Biol. 19(12):8169-79. 22. Cabodi S, Tinnirello A, Di Stefano P, Bisaro B, Ambrosino E, Castellano I, Sapino A, Arisio R...Vande Woude GF. Met, metastasis, motility and more. Nat Rev Mol Cell Biol 2003;4:915 –25. 3. Rosario M, Birchmeier W. How to make tubes: signaling by

  7. CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection.

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    Daniel Pérez-Núñez

    Full Text Available African swine fever virus (ASFV CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity.

  8. Invertebrate and vertebrate class III myosins interact with MORN repeat-containing adaptor proteins.

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    Kirk L Mecklenburg

    Full Text Available In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP. Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A. In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior.

  9. The Shc family protein adaptor, Rai, negatively regulates T cell antigen receptor signaling by inhibiting ZAP-70 recruitment and activation.

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    Micol Ferro

    Full Text Available Rai/ShcC is a member of the Shc family of protein adaptors expressed with the highest abundance in the central nervous system, where it exerts a protective function by coupling neurotrophic receptors to the PI3K/Akt survival pathway. Rai is also expressed, albeit at lower levels, in other cell types, including T and B lymphocytes. We have previously reported that in these cells Rai attenuates antigen receptor signaling, thereby impairing not only cell proliferation but also, opposite to neurons, cell survival. Here we have addressed the mechanism underlying the inhibitory activity of Rai on TCR signaling. We show that Rai interferes with the TCR signaling cascade one of the earliest steps--recruitment of the initiating kinase ZAP-70 to the phosphorylated subunit of the TCR/CD3 complex, which results in a generalized dampening of the downstream signaling events. The inhibitory activity of Rai is associated to its inducible recruitment to phosphorylated CD3, which occurs in the physiological signaling context of the immune synapse. Rai is moreover found as a pre-assembled complex with ZAP-70 and also constitutively interacts with the regulatory p85 subunit of PI3K, similar to neuronal cells, notwithstanding the opposite biological outcome, i.e. impairment of PI-3K/Akt activation. The data highlight the ability of Rai to establish interactions with the TCR and key signaling mediators which, either directly (e.g. by inhibiting ZAP-70 recruitment to the TCR or sequestering ZAP-70/PI3K in the cytosol or indirectly (e.g. by promoting the recruitment of effectors responsible for signal extinction prevent full triggering of the TCR signaling cascade.

  10. New function of the adaptor protein SH2B1 in brain-derived neurotrophic factor-induced neurite outgrowth.

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    Chien-Hung Shih

    Full Text Available Neurite outgrowth is an essential process for the establishment of the nervous system. Brain-derived neurotrophic factor (BDNF binds to its receptor TrkB and regulates axonal and dendritic morphology of neurons through signal transduction and gene expression. SH2B1 is a signaling adaptor protein that regulates cellular signaling in various physiological processes. The purpose of this study is to investigate the role of SH2B1 in the development of the central nervous system. In this study, we show that knocking down SH2B1 reduces neurite formation of cortical neurons whereas overexpression of SH2B1β promotes the development of hippocampal neurons. We further demonstrate that SH2B1β promotes BDNF-induced neurite outgrowth and signaling using the established PC12 cells stably expressing TrkB, SH2B1β or SH2B1β mutants. Our data indicate that overexpressing SH2B1β enhances BDNF-induced MEK-ERK1/2, and PI3K-AKT signaling pathways. Inhibition of MEK-ERK1/2 and PI3K-AKT pathways by specific inhibitors suggest that these two pathways are required for SH2B1β-promoted BDNF-induced neurite outgrowth. Moreover, SH2B1β enhances BDNF-stimulated phosphorylation of signal transducer and activator of transcription 3 at serine 727. Finally, our data indicate that the SH2 domain and tyrosine phosphorylation of SH2B1β contribute to BDNF-induced signaling pathways and neurite outgrowth. Taken together, these findings demonstrate that SH2B1β promotes BDNF-induced neurite outgrowth through enhancing pathways involved MEK-ERK1/2 and PI3K-AKT.

  11. Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

    Science.gov (United States)

    Ross, Breyan H.; Lin, Yimo; Corales, Esteban A.; Burgos, Patricia V.; Mardones, Gonzalo A.

    2014-01-01

    Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non

  12. Role of Crk Adaptor Proteins in Cellular Migration and Invasion in Human Breast Cancer

    Science.gov (United States)

    2009-03-01

    0.0001349 1.280209 0.00657 cholesterol 25- hydroxylase ZNF539 2.225650467 0.0042638 1.295457 0.03133 zinc finger protein 254 PRSS7 2.233104326 0.0239284...4.98281  6.27E‐07  Toll‐like receptor signaling pathway  ‐3.51463  0.00044  Caprolactam degradation  ‐0.99768  0.318433  Phenylalanine , tyrosine and...Escherichia coli infection – EHEC  ‐1.38214  0.166928  Phenylalanine  metabolism  ‐2.08135  0.037401  Pathogenic Escherichia coli infection – EPEC  ‐0.48134

  13. Src-like adaptor protein 2 (SLAP2) binds to and inhibits FLT3 signaling

    Science.gov (United States)

    Moharram, Sausan A.; Chougule, Rohit A.; Su, Xianwei; Li, Tianfeng; Sun, Jianmin; Zhao, Hui; Rönnstrand, Lars; Kazi, Julhash U.

    2016-01-01

    Fms-like tyrosine kinase (FLT3) is a frequently mutated oncogene in acute myeloid leukemia (AML). FLT3 inhibitors display promising results in a clinical setting, but patients relapse after short-term treatment due to the development of resistant disease. Therefore, a better understanding of FLT3 downstream signal transduction pathways will help to identify an alternative target for the treatment of AML patients carrying oncogenic FLT3. Activation of FLT3 results in phosphorylation of FLT3 on several tyrosine residues that recruit SH2 domain-containing signaling proteins. We screened a panel of SH2 domain-containing proteins and identified SLAP2 as a potent interacting partner of FLT3. We demonstrated that interaction occurs when FLT3 is activated, and also, an intact SH2 domain of SLAP2 is required for binding. SLAP2 binding sites in FLT3 mainly overlap with those of SRC. SLAP2 over expression in murine proB cells or myeloid cells inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation in vitro, and tumor formation in vivo. Microarray analysis suggests that higher SLAP2 expression correlates with a gene signature similar to that of loss of oncogene function. Furthermore, FLT3-ITD positive AML patients with higher SLAP2 expression displayed better prognosis compared to those with lower expression of SLAP2. Expression of SLAP2 blocked FLT3 downstream signaling cascades including AKT, ERK, p38 and STAT5. Finally, SLAP2 accelerated FLT3 degradation through enhanced ubiquitination. Collectively, our data suggest that SLAP2 acts as a negative regulator of FLT3 signaling and therefore, modulation of SLAP2 expression levels may provide an alternative therapeutic approach for FLT3-ITD positive AML. PMID:27458164

  14. Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling.

    Science.gov (United States)

    Houtman, Jon C D; Brown, Patrick H; Bowden, Brent; Yamaguchi, Hiroshi; Appella, Ettore; Samelson, Lawrence E; Schuck, Peter

    2007-01-01

    Multisite interactions and the formation of ternary or higher-order protein complexes are ubiquitous features of protein interactions. Cooperativity between different ligands is a hallmark for information transfer, and is frequently critical for the biological function. We describe a new computational platform for the global analysis of isothermal titration calorimetry (ITC) data for the study of binary and ternary multisite interactions, implemented as part of the public domain multimethod analysis software SEDPHAT. The global analysis of titrations performed in different orientations was explored, and the potential for unraveling cooperativity parameters in multisite interactions was assessed in theory and experiment. To demonstrate the practical potential and limitations of global analyses of ITC titrations for the study of cooperative multiprotein interactions, we have examined the interactions of three proteins that are critical for signal transduction after T-cell activation, LAT, Grb2, and Sos1. We have shown previously that multivalent interactions between these three molecules promote the assembly of large multiprotein complexes important for T-cell receptor activation. By global analysis of the heats of binding observed in sets of ITC injections in different orientations, which allowed us to follow the formation of binary and ternary complexes, we observed negative and positive cooperativity that may be important to control the pathway of assembly and disassembly of adaptor protein particles.

  15. Cargo adaptors: structures illuminate mechanisms regulating vesicle biogenesis.

    Science.gov (United States)

    Paczkowski, Jon E; Richardson, Brian C; Fromme, J Christopher

    2015-07-01

    Cargo adaptors sort transmembrane protein cargos into nascent vesicles by binding directly to their cytosolic domains. Recent studies have revealed previously unappreciated roles for cargo adaptors and regulatory mechanisms governing their function. The adaptor protein (AP)-1 and AP-2 clathrin adaptors switch between open and closed conformations that ensure they function at the right place at the right time. The exomer cargo adaptor has a direct role in remodeling the membrane for vesicle fission. Several different cargo adaptors functioning in distinct trafficking pathways at the Golgi are similarly regulated through bivalent binding to the ADP-ribosylation factor 1 (Arf1) GTPase, potentially enabling regulation by a threshold concentration of Arf1. Taken together, these studies highlight that cargo adaptors do more than just adapt cargos.

  16. The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis.

    Directory of Open Access Journals (Sweden)

    Deysi V T Wong

    Full Text Available Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL-1 and IL-18. These molecules and toll-like receptors (TLRs activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days. On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50% and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%, TLR9 (400%, TRAF6 (236%, IL-1β (405%, IL-18 (365%, COX-2 (2,777% and NF-κB (245% in the WT animals when compared with saline-injected group (P<0.05. Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05. In contrast to the MyD88-/- and TLR2-/- mice, the irinotecan-injected TLR9-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL-18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2-/- or TLR9-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis.

  17. Involvement of β3A Subunit of Adaptor Protein-3 in Intracellular Trafficking of Receptor-like Protein Tyrosine Phosphatase PCP-2

    Institute of Scientific and Technical Information of China (English)

    Hui DONG; Hong YUAN; Weirong JIN; Yan SHEN; Xiaojing XU; Hongyang WANG

    2007-01-01

    PCP-2 is a human receptor-like protein tyrosine phosphatase and a member of the MAM domain family cloned in human pancreatic adenocarcinoma cells. Previous studies showed that PCP-2 directly interacted with β-catenin through the juxtamembrane domain, dephosphorylated β-catenin and played an important role in the regulation of cell adhesion. Recent study showed that PCP-2 was also involved in the repression of β-catenin-induced transcriptional activity. Here we describe the interactions of PCP-2 with the β3A subunit of adaptor protein (AP)-3 and sorting nexin (SNX) 3. These protein complexes were detected using the yeast two-hybrid assay with the juxtamembrane and membrane-proximal catalytic domain of PCP-2 as "bait". Both AP-3 and SNX3 are molecules involved in intracellular trafficking of membrane receptors. The association between the β3A subunit of AP-3 and PCP-2 was further confirmed in mammalian cells. Our results suggested a possible mechanism of intracellular trafficking of PCP-2 mediated by AP-3 and SNX3 which might participate in the regulation of PCP-2 functions.

  18. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    Science.gov (United States)

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

  19. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor

    DEFF Research Database (Denmark)

    Chen, Y; Grall, D; Salcini, A E

    1996-01-01

    The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have...... kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors....

  20. Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis.

    Science.gov (United States)

    Lacruz, Rodrigo S; Brookes, Steven J; Wen, Xin; Jimenez, Jaime M; Vikman, Susanna; Hu, Ping; White, Shane N; Lyngstadaas, S Petter; Okamoto, Curtis T; Smith, Charles E; Paine, Michael L

    2013-03-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data

  1. Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration.

    Science.gov (United States)

    Kuropka, Benno; Witte, Amelie; Sticht, Jana; Waldt, Natalie; Majkut, Paul; Hackenberger, Christian P R; Schraven, Burkhart; Krause, Eberhard; Kliche, Stefanie; Freund, Christian

    2015-11-01

    Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.

  2. The cell signaling adaptor protein EPS-8 is essential for C. elegans epidermal elongation and interacts with the ankyrin repeat protein VAB-19.

    Directory of Open Access Journals (Sweden)

    Mei Ding

    Full Text Available The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation. The cytoskeletal ankyrin repeat protein VAB-19 is required for cell shape changes and localizes to cell-matrix attachment structures. The molecular functions of VAB-19 in this process are obscure, as no previous interactors for VAB-19 have been described.In screens for VAB-19 binding proteins we identified the signaling adaptor EPS-8. Within C. elegans epidermal cells, EPS-8 and VAB-19 colocalize at cell-matrix attachment structures. The central domain of EPS-8 is necessary and sufficient for its interaction with VAB-19. eps-8 null mutants, like vab-19 mutants, are defective in epidermal elongation and in epidermal-muscle attachment. The eps-8 locus encodes two isoforms, EPS-8A and EPS-8B, that appear to act redundantly in epidermal elongation. The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains. VAB-19 appears to act earlier in the biogenesis of attachment structures and may recruit EPS-8 to these structures.EPS-8 and VAB-19 define a novel pathway acting at cell-matrix attachments to regulate epithelial cell shape. This is the first report of a role for EPS-8 proteins in cell-matrix attachments. The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms.

  3. Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN.

    Science.gov (United States)

    Takayanagi, Hiroki; Yuzawa, Satoru; Sumimoto, Hideki

    2015-02-01

    The adaptor protein LGN interacts via the N-terminal domain comprising eight tetratricopeptide-repeat (TPR) motifs with its partner proteins mInsc, NuMA, Frmpd1 and Frmpd4 in a mutually exclusive manner. Here, the crystal structure of the LGN TPR domain in complex with human Frmpd4 is described at 1.5 Å resolution. In the complex, the LGN-binding region of Frmpd4 (amino-acid residues 990-1011) adopts an extended structure that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPR motifs. Comparison with the previously determined structures of the LGN-Frmpd1, LGN-mInsc and LGN-NuMA complexes reveals that these partner proteins interact with LGN TPR1-6 via a common core binding region with consensus sequence (E/Q)XEX4-5(E/D/Q)X1-2(K/R)X0-1(V/I). In contrast to Frmpd1, Frmpd4 makes additional contacts with LGN via regions N- and C-terminal to the core sequence. The N-terminal extension is replaced by a specific α-helix in mInsc, which drastically increases the direct contacts with LGN TPR7/8, consistent with the higher affinity of mInsc for LGN. A crystal structure of Frmpd4-bound LGN in an oxidized form is also reported, although oxidation does not appear to strongly affect the interaction with Frmpd4.

  4. The 3A Protein from Multiple Picornaviruses Utilizes the Golgi Adaptor Protein ACBD3 To Recruit PI4KIIIβ

    OpenAIRE

    Greninger, Alexander L.; Giselle M Knudsen; Betegon, Miguel; Burlingame, Alma L.; Joseph L Derisi

    2012-01-01

    The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIβ) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIβ and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIβ by affinity purification of Strep-Tagged transiently transfected constructs...

  5. Expression of the neuronal adaptor protein X11alpha protects against memory dysfunction in a transgenic mouse model of Alzheimer's disease.

    LENUS (Irish Health Repository)

    Mitchell, Jacqueline C

    2010-01-01

    X11alpha is a neuronal-specific adaptor protein that binds to the amyloid-beta protein precursor (AbetaPP). Overexpression of X11alpha reduces Abeta production but whether X11alpha also protects against Abeta-related memory dysfunction is not known. To test this possibility, we crossed X11alpha transgenic mice with AbetaPP-Tg2576 mice. AbetaPP-Tg2576 mice produce high levels of brain Abeta and develop age-related defects in memory function that correlate with increasing Abeta load. Overexpression of X11alpha alone had no detectable adverse effect upon behavior. However, X11alpha reduced brain Abeta levels and corrected spatial reference memory defects in aged X11alpha\\/AbetaPP double transgenics. Thus, X11alpha may be a therapeutic target for Alzheimer\\'s disease.

  6. RECOMBINANT FLUORESCENT SENSOR OF HYDROGEN PEROXIDE HyPer FUSED WITH ADAPTOR PROTEIN Ruk/CIN85: DESIGNING OF EXPRESSION VECTOR AND ITS FUNCTIONAL CHARACTERIZATION

    Directory of Open Access Journals (Sweden)

    А. V. Bazalii

    2015-10-01

    Full Text Available The aim of this study was to design the expression vector encoding fluorescent sensor of hydrogen peroxide HyPer fused with adaptor protein Ruk/CIN85 as well as to check its subcellular distribution and ability to sense hydrogen peroxide. It was demonstrated that in transiently transfected HEK293 and MCF-7 cells Ruk/CIN85-HyPer is concentrated in dot-like vesicular structures of different size while HyPer is diffusely distributed throughout the cell. Using live cell fluorescence microscopy we observed gradual increase in hydrogen peroxide concentration in representative vesicular structures during the time of experiment. Thus, the developed genetic construction encoding the chimeric Ruk/CIN85-HyPer fluorescent protein represents a new tool to study localized H2O2 production in living cells.

  7. Protein modifications regulate the role of 14-3-3γ adaptor protein in cAMP-induced steroidogenesis in MA-10 Leydig cells.

    Science.gov (United States)

    Aghazadeh, Yasaman; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-09-19

    The 14-3-3 protein family comprises adaptors and scaffolds that regulate intracellular signaling pathways. The 14-3-3γ isoform is a negative regulator of steroidogenesis that is hormonally induced and transiently functions at the initiation of steroidogenesis by delaying maximal steroidogenesis in MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3γ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels. Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. The 14-3-3γ/STAR pattern of interaction was found to be opposite that of the 14-3-3γ homodimerization pattern. Phosphorylation and acetylation of 14-3-3γ showed similar patterns to homodimerization and STAR binding, respectively. 14-3-3γ Ser(58) phosphorylation and 14-3-3γ Lys(49) acetylation were blocked using trans-activator of HIV transcription factor 1 peptides coupled to 14-3-3γ sequences containing Ser(58) or Lys(49). Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation. Taken together, these results indicate that Ser(58) phosphorylation and Lys(49) acetylation of 14-3-3γ occur in a coordinated time-dependent manner to regulate 14-3-3γ homodimerization. 14-3-3γ Ser(58) phosphorylation is required for STAR interactions under control conditions, and 14-3-3γ Lys(49) acetylation is important for the cAMP-dependent induction of these interactions.

  8. Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

    Institute of Scientific and Technical Information of China (English)

    Takuya Watanabe; Masumi Tsuda; Yoshinori Makino; Tassos Konstantinou; Hiroshi Nishihara; Tokifumi Majima; Akio Minami; Stephan M Feller; Shinya Tanaka

    2009-01-01

    Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extraceilular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of Crkll demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of Crkll, is critical for the induction of Gabl-Y307 phosphorylation. SH2 mutation of Crkll also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gabl, whereas Crk-SH3(N) interacted with the Gabl mutant, which lacks the clus-tered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The GabI-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxiilin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory-lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

  9. Inflammasome adaptor protein Apoptosis-associated speck-like protein containing CARD (ASC) is critical for the immune response and survival in west Nile virus encephalitis.

    Science.gov (United States)

    Kumar, Mukesh; Roe, Kelsey; Orillo, Beverly; Muruve, Daniel A; Nerurkar, Vivek R; Gale, Michael; Verma, Saguna

    2013-04-01

    West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis in humans. The WNV-induced innate immune response, including production of antiviral cytokines, is critical for controlling virus infection. The adaptor protein ASC mediates a critical step in innate immune signaling by bridging the interaction between the pathogen recognition receptors and caspase 1 in inflammasome complexes, but its role in WNV immunopathogenesis is not defined. Here, we demonstrate that ASC is essential for interleukin-1β (IL-1β) production and development of effective host immunity against WNV. ASC-deficient mice exhibited increased susceptibility to WNV infection, and reduced survival was associated with enhanced virus replication in the peripheral tissues and central nervous system (CNS). Infection of cultured bone marrow-derived dendritic cells showed that ASC was essential for the activation of caspase 1, a key component of inflammasome assembly. ASC(-/-) mice exhibited attenuated levels of proinflammatory cytokines in the serum. Intriguingly, infected ASC(-/-) mice also displayed reduced levels of alpha interferon (IFN-α) and IgM in the serum, indicating the overall protective role of ASC in restricting WNV infection. However, brains from ASC(-/-) mice displayed unrestrained inflammation, including elevated levels of proinflammatory cytokines and chemokines, such as IFN-γ, CCL2, and CCL5, which correlated with more pronounced activation of the astrocytes, enhanced infiltration of peripheral immune cells in the CNS, and increased neuronal cell death. Collectively, our data provide new insights into the role of ASC as an essential modulator of inflammasome-dependent and -independent immune responses to effectively control WNV infection.

  10. RTK SLAP down: the emerging role of Src-like adaptor protein as a key player in receptor tyrosine kinase signaling.

    Science.gov (United States)

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2015-02-01

    SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.

  11. Differential association of the Na+/H+ exchanger regulatory factor (NHERF) family of adaptor proteins with the raft- and the non-raft brush border membrane fractions of NHE3

    NARCIS (Netherlands)

    A. Sultan (Ayesha); M. Luo (Ma); Q. Yu (Qingbao); B. Riederer (Beat Michel); W. Xia (Weiliang); M. Chen (Mingmin); S. Lissner (Simone); J.E. Gessner (Johannes); M. Donowitz (Mark); C. Chris Yun (C.); H. deJonge (Hugo); G. Lamprecht (Georg); U. Seidler (Ursula)

    2013-01-01

    textabstractBackground/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is uncl

  12. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

    Energy Technology Data Exchange (ETDEWEB)

    Sawasdee, Nunghathai; Junking, Mutita [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Ngaojanlar, Piengpaga [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Immunology and Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Sukomon, Nattakan; Ungsupravate, Duangporn [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Limjindaporn, Thawornchai [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Akkarapatumwong, Varaporn [Institute of Molecular Biosciences, Mahidol University at Salaya Campus, Nakorn Pathom 73170 (Thailand); Noisakran, Sansanee [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)

    2010-10-08

    Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

  13. Induction of Androgen Formation in the Male by a TAT-VDAC1 Fusion Peptide Blocking 14-3-3ɛ Protein Adaptor and Mitochondrial VDAC1 Interactions

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    Aghazadeh, Yasaman; Martinez-Arguelles, Daniel B; Fan, Jinjiang; Culty, Martine; Papadopoulos, Vassilios

    2014-01-01

    Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production. PMID:24947306

  14. Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

    Science.gov (United States)

    Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2014-07-01

    Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells.

  15. Involvement of Grb2 adaptor protein in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated signaling and anaplastic large cell lymphoma growth.

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    Riera, Ludovica; Lasorsa, Elena; Ambrogio, Chiara; Surrenti, Nadia; Voena, Claudia; Chiarle, Roberto

    2010-08-20

    Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.

  16. Interaction of ubiquitin ligase CBL with LMP2A protein of Epstein-Barr virus occurs via PTB domain of CBL and does not depend on adaptor ITSN1

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    Dergai O. V.

    2013-03-01

    Full Text Available Aim. Previously Latent membrane protein 2A (LMP2A of Epstein-Barr virus was found to be ubiquitylated by CBL ubiquitin ligase but no direct interaction of LMP2A with CBL was reported. We aimed to explore this interaction and study a possibility of adaptor protein involvement. Taking into consideration that both LMP2A and CBL were shown to interact with endocytic adaptor protein intersectin 1 (ITSN1, we assumed that the latter could serve as a scaffold for LMP2A/CBL complex. Methods. We used an immunofluorescence and coimmuno- precipitation approaches to test a mutual complex formation of ITSN1, CBL and LMP2A proteins. Results. LMP2A coimmunoprecipitated with CBL while LMP2A did not interact with CBL G306E mutant harboring inactive phosphotyrosine-binding domain. We observed a triple colocalization of ITSN1, CBL and LMP2A signals in MCF-7 cells as well as coprecipitation of all mentioned proteins. Overexpression of ITSN1 did not affect the efficiency of complex formation of LMP2A with CBL. Moreover, LMP2A mutant unable to interact with ITSN1 was readily precipitated with CBL. Conclusions. LMP2A can be engaged in the complex together with endocytic adaptor ITSN1 and ubiquitin ligase CBL. We show that PTB domain of CBL is responsible for interaction with LMP2A. ITSN1 is not required for LMP2A recruiting to CBL.

  17. The adaptor protein SAP directly associates with PECAM-1 and regulates PECAM-1-mediated-cell adhesion in T-like cell lines.

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    Proust, Richard; Crouin, Catherine; Gandji, Leslie Yewakon; Bertoglio, Jacques; Gesbert, Franck

    2014-04-01

    SAP is a small cytosolic adaptor protein expressed in hematopoietic lineages whose main function is to regulate intracellular signaling pathways induced by the triggering of members of the SLAM receptor family. In this paper, we have identified the adhesion molecule PECAM-1 as a new partner for SAP in a conditional yeast two-hybrid screen. PECAM-1 is an immunoglobulin-like molecule expressed by endothelial cells and leukocytes, which possesses both pro- and anti-inflammatory properties. However, little is known about PECAM-1 functions in T cells. We show that SAP directly and specifically interacts with the cytosolic tyrosine 686 of PECAM-1. We generated different T-like cell lines in which SAP or PECAM-1 are expressed or down modulated and we demonstrate that a diminished SAP expression correlates with a diminished PECAM-1-mediated adhesion. Although SAP has mainly been shown to associate with SLAM receptors, we evidence here that SAP is a new actor downstream of PECAM-1.

  18. Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

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    Kazi, Julhash U; Rönnstrand, Lars

    2012-01-01

    Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  19. Src-Like adaptor protein (SLAP binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    Directory of Open Access Journals (Sweden)

    Julhash U Kazi

    Full Text Available Fms-like tyrosine kinase 3 (Flt3 is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML. Src-like adaptor protein (SLAP is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  20. Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors.

    Science.gov (United States)

    Semerdjieva, Sophia; Abdul-Razak, Hayder H; Salim, Sharifah S; Yáñez-Muñoz, Rafael J; Chen, Philip E; Tarabykin, Victor; Alifragis, Pavlos

    2013-04-01

    Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

  1. The interaction of the cellular export adaptor protein Aly/REF with ICP27 contributes to the efficiency of herpes simplex virus 1 mRNA export.

    Science.gov (United States)

    Tian, Xiaochen; Devi-Rao, Gayathri; Golovanov, Alexander P; Sandri-Goldin, Rozanne M

    2013-07-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.

  2. SH2 domain–containing adaptor protein B expressed in dendritic cells is involved in T-cell homeostasis by regulating dendritic cell–mediated Th2 immunity

    Science.gov (United States)

    2017-01-01

    Purpose The Src homology 2 domain–containing adaptor protein B (SHB) is widely expressed in immune cells and acts as an important regulator for hematopoietic cell function. SHB silencing induces Th2 immunity in mice. SHB is also involved in T-cell homeostasis in vivo. However, SHB has not yet been studied and addressed in association with dendritic cells (DCs). Materials and Methods The effects of SHB expression on the immunogenicity of DCs were assessed by Shb gene silencing in mouse bone marrow–derived DCs (BMDCs). After silencing, surface phenotype, cytokine expression profile, and T-cell stimulation capacity of BMDCs were examined. We investigated the signaling pathways involved in SHB expression during BMDC development. We also examined the immunogenicity of SHB-knockdown (SHBKD) BMDCs in a mouse atopic dermatitis model. Results SHB was steadily expressed in mouse splenic DCs and in in vitro–generated BMDCs in both immature and mature stages. SHB expression was contingent on activation of the mitogen- activated protein kinase/Foxa2 signaling pathway during DC development. SHBKD increased the expression of MHC class II and costimulatory molecules without affecting the cytokine expression of BMDCs. When co-cultured with T cells, SHBKD in BMDCs significantly induced CD4+ T-cell proliferation and the expression of Th2 cytokines, while the regulatory T cell (Treg) population was downregulated. In mouse atopic dermatitis model, mice inoculated with SHBKD DCs developed more severe symptoms of atopic dermatitis compared with mice injected with control DCs. Conclusion SHB expression in DCs plays an important role in T-cell homeostasis in vivo by regulating DC-mediated Th2 polarization. PMID:28168174

  3. Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin.

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    Wood, Stephen; Goldufsky, Josef; Shafikhani, Sasha H

    2015-05-01

    Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38β and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell.

  4. SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways

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    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G.; Dissous, Colette

    2016-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis. PMID:27636711

  5. SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways.

    Science.gov (United States)

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G; Dissous, Colette

    2016-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis.

  6. Role of the Caenorhabditis elegans Shc adaptor protein in the c-Jun N-terminal kinase signaling pathway.

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    Mizuno, Tomoaki; Fujiki, Kota; Sasakawa, Aya; Hisamoto, Naoki; Matsumoto, Kunihiro

    2008-12-01

    Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like mitogen-activated protein kinase (MAPK) signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPK kinase (MAPKK), and KGB-1 JNK-like MAPK. In this study, we identify the shc-1 gene, which encodes a C. elegans homolog of Shc, as a factor that specifically interacts with MEK-1. The shc-1 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. A specific tyrosine residue in the NPXY motif of MLK-1 creates a docking site for SHC-1 with the phosphotyrosine binding (PTB) domain. Introduction of a mutation that perturbs binding to the PTB domain or the NPXY motif abolishes the function of SHC-1 or MLK-1, respectively, thereby abolishing the resistance to heavy metal stress. These results suggest that SHC-1 acts as a scaffold to link MAPKKK to MAPKK activation in the KGB-1 MAPK signal transduction pathway.

  7. The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

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    Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L; Stein, Paul L; Wang, Chyung-Ru

    2014-12-01

    CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.

  8. The Mu subunit of Plasmodium falciparum clathrin-associated adaptor protein 2 modulates in vitro parasite response to artemisinin and quinine.

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    Henriques, Gisela; van Schalkwyk, Donelly A; Burrow, Rebekah; Warhurst, David C; Thompson, Eloise; Baker, David A; Fidock, David A; Hallett, Rachel; Flueck, Christian; Sutherland, Colin J

    2015-05-01

    The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance.

  9. Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO.

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    Marcos Rodrigo Alborghetti

    Full Text Available Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans, SCOCO (short coiled-coil protein / UNC-69 and kinesins (e.g. kinesin heavy chain / UNC116 are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth, we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance, cross-linking coupled with mass spectrometry (MS, SAXS (Small Angle X-ray Scattering and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance studies of the region involved in this process, corresponding to FEZ1 (92-194. Through studies involving the protein in its monomeric configuration (reduced and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

  10. Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.

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    Leonie Unterholzner

    2011-09-01

    Full Text Available Recognition of viruses by pattern recognition receptors (PRRs causes interferon-β (IFN-β induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1 and IκB kinase-ε (IKKε, which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

  11. The influenza virus protein PB1-F2 inhibits the induction of type I interferon at the level of the MAVS adaptor protein.

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    Zsuzsanna T Varga

    2011-06-01

    Full Text Available PB1-F2 is a 90 amino acid protein that is expressed from the +1 open reading frame in the PB1 gene of some influenza A viruses and has been shown to contribute to viral pathogenicity. Notably, a serine at position 66 (66S in PB1-F2 is known to increase virulence compared to an isogenic virus with an asparagine (66N at this position. Recently, we found that an influenza virus expressing PB1-F2 N66S suppresses interferon (IFN-stimulated genes in mice. To characterize this phenomenon, we employed several in vitro assays. Overexpression of the A/Puerto Rico/8/1934 (PR8 PB1-F2 protein in 293T cells decreased RIG-I mediated activation of an IFN-β reporter and secretion of IFN as determined by bioassay. Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype. Similar observations were found in the context of viral infection with a PR8 PB1-F2 N66S virus. To understand the relationship between NS1, a previously described influenza virus protein involved in suppression of IFN synthesis, and PB1-F2, we investigated the induction of IFN when NS1 and PB1-F2 were co-expressed in an in vitro transfection system. In this assay we found that PB1-F2 N66S further reduced IFN induction in the presence of NS1. By inducing the IFN-β reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS. Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS. In summary, we have characterized the anti-interferon function of PB1-F2 and we suggest that this activity contributes to the enhanced pathogenicity seen with PB1-F2 N66S- expressing influenza viruses.

  12. Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells by activating the APPL1-AMPK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Tong; Wu, Yu-wei; Lu, Hui; Guo, Yuan [Second Dental Center, Peking University School and Hospital of Stomatology, Beijing (China); National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing (China); Tang, Zhi-hui, E-mail: tang_zhihui@live.cn [Second Dental Center, Peking University School and Hospital of Stomatology, Beijing (China); National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing (China)

    2015-05-29

    Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with multi-lineage differentiation potential including osteogenesis and adipogenesis. While significant progress has been made in understanding the transcriptional control of hASC fate, little is known about how hASC differentiation is regulated by the autocrine loop. The most abundant adipocytokine secreted by adipocytes, adiponectin (APN) plays a pivotal role in glucose metabolism and energy homeostasis. Growing evidence suggests a positive association between APN and bone formation yet little is known regarding the direct effects of APN on hASC osteogenesis. Therefore, this study was designed to investigate the varied osteogenic effects and regulatory mechanisms of APN in the osteogenic commitment of hASCs. We found that APN enhanced the expression of osteoblast-related genes in hASCs, such as osteocalcin, alkaline phosphatase, and runt-related transcription factor-2 (Runx2, also known as CBFa1), in a dose- and time-dependent manner. This was further confirmed by the higher expression levels of alkaline phosphatase and increased formation of mineralization nodules, along with the absence of inhibition of cell proliferation. Importantly, APN at 1 μg/ml was the optimal concentration, resulting in maximum deposition of calcium nodules, and was significant superior to bone morphogenetic protein 2. Mechanistically, we found for the first time that APN increased nuclear translocation of the leucine zipper motif (APPL)-1 as well as AMP-activated protein kinase (AMPK) phosphorylation, which were reversed by pretreatment with APPL1 siRNA. Our results indicate that APN promotes the osteogenic differentiation of hASCs by activating APPL1-AMPK signaling, suggesting that manipulation of APN is a novel therapeutic target for controlling hASC fate. - Highlights: • Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells. • The knock-down of APPL1 block the enhancement of

  13. Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C on the regulation of Toll-like receptors, adaptor proteins and inflammatory response

    Directory of Open Access Journals (Sweden)

    Weith Andreas

    2005-11-01

    Full Text Available Abstract Background Bacterial and viral exacerbations play a crucial role in a variety of lung diseases including COPD or asthma. Since the lung epithelium is a major source of various inflammatory mediators that affect the immune response, we analyzed the inflammatory reaction of primary lung epithelial cells to different microbial molecules that are recognized by Toll-like receptors (TLR. Methods The effects of TLR ligands on primary small airway epithelial cells were analyzed in detail with respect to cytokine, chemokine and matrix metalloproteinase secretion. In addition, the regulation of the expression of TLRs and their adaptor proteins in small airway epithelial cells was investigated. Results Our data demonstrate that poly(I:C, a synthetic analog of viral dsRNA, mediated the strongest proinflammatory effects among the tested ligands, including an increased secretion of IL-6, IL-8, TNF-α, GM-CSF, GRO-α, TARC, MCP-1, MIP-3α, RANTES, IFN-β, IP-10 and ITAC as well as an increased release of MMP-1, MMP-8, MMP-9, MMP-10 and MMP-13. Furthermore, our data show that poly(I:C as well as type-1 and type-2 cytokines have a pronounced effect on the expression of TLRs and molecules involved in TLR signaling in small airway epithelial cells. Poly(I:C induced an elevated expression of TLR1, TLR2 and TLR3 and increased the gene expression of the general TLR adaptor MyD88 and IRAK-2. Simultaneously, poly(I:C decreased the expression of TLR5, TLR6 and TOLLIP. Conclusion Poly(I:C, an analog of viral dsRNA and a TLR3 ligand, triggers a strong inflammatory response in small airway epithelial cells that is likely to contribute to viral exacerbations of pulmonary diseases like asthma or COPD. The pronounced effects of poly(I:C on the expression of Toll-like receptors and molecules involved in TLR signaling is assumed to influence the immune response of the lung epithelium to viral and bacterial infections. Likewise, the regulation of TLR expression by type

  14. The cellular RNA export receptor TAP/NXF1 is required for ICP27-mediated export of herpes simplex virus 1 RNA, but the TREX complex adaptor protein Aly/REF appears to be dispensable.

    Science.gov (United States)

    Johnson, Lisa A; Li, Ling; Sandri-Goldin, Rozanne M

    2009-07-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 has been shown to shuttle between the nucleus and cytoplasm and to bind viral RNA during infection. ICP27 was found to interact with the cellular RNA export adaptor protein Aly/REF, which is part of the TREX complex, and to relocalize Aly/REF to viral replication sites. ICP27 is exported to the cytoplasm through the export receptor TAP/NXF1, and ICP27 must be able to interact with TAP/NXF1 for efficient export of HSV-1 early and late transcripts. We examined the dynamics of ICP27 movement and its localization with respect to Aly/REF and TAP/NXF1 in living cells during viral infection. Recombinant viruses with a yellow fluorescent protein (YFP) tag on the N or C terminus of ICP27 were constructed. While the N-terminally tagged ICP27 virus behaved like wild-type HSV-1, the C-terminally tagged virus was defective in viral replication and gene expression, and ICP27 was confined to the nucleus, suggesting that the C-terminal YFP tag interfered with ICP27's C-terminal interactions, including the interaction with TAP/NXF1. To assess the role of Aly/REF and TAP/NXF1 in viral RNA export, these factors were knocked down using small interfering RNA. Knockdown of Aly/REF had little effect on the export of ICP27 or poly(A)(+) RNA during infection. In contrast, a decrease in TAP/NXF1 levels severely impaired export of ICP27 and poly(A)(+) RNA. We conclude that TAP/NXF1 is essential for ICP27-mediated export of RNA during HSV-1 infection, whereas Aly/REF may be dispensable.

  15. The microRNA mir-71 inhibits calcium signaling by targeting the TIR-1/Sarm1 adaptor protein to control stochastic L/R neuronal asymmetry in C. elegans.

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    Yi-Wen Hsieh

    Full Text Available The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWC(ON and AWC(OFF, by inhibiting a calcium-mediated signaling pathway in the future AWC(ON cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWC(ON fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWC(ON cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWC(ON identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWC(ON neurons. tir-1 expression is downregulated through its 3' UTR in AWC(ON, in which mir-71 is expressed at a higher level than in AWC(OFF. In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3' UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWC(ON identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.

  16. The microRNA mir-71 inhibits calcium signaling by targeting the TIR-1/Sarm1 adaptor protein to control stochastic L/R neuronal asymmetry in C. elegans.

    Science.gov (United States)

    Hsieh, Yi-Wen; Chang, Chieh; Chuang, Chiou-Fen

    2012-01-01

    The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWC(ON) and AWC(OFF), by inhibiting a calcium-mediated signaling pathway in the future AWC(ON) cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWC(ON) fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWC(ON) cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWC(ON) identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWC(ON) neurons. tir-1 expression is downregulated through its 3' UTR in AWC(ON), in which mir-71 is expressed at a higher level than in AWC(OFF). In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3' UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWC(ON) identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.

  17. The Fe65 adaptor protein interacts through its PID1 domain with the transcription factor CP2/LSF/LBP1.

    Science.gov (United States)

    Zambrano, N; Minopoli, G; de Candia, P; Russo, T

    1998-08-07

    The neural protein Fe65 possesses three putative protein-protein interaction domains: one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1 and PID2); the most C-terminal of these domains (PID2) interacts in vivo with the Alzheimer's beta-amyloid precursor protein, whereas the WW domain binds to Mena, the mammalian homolog of Drosophila-enabled protein. By the interaction trap procedure, we isolated a cDNA clone encoding a possible ligand of the N-terminal PID/PTB domain of Fe65 (PID1). Sequence analysis of this clone revealed that this ligand corresponded to the previously identified transcription factor CP2/LSF/LBP1. Co-immunoprecipitation experiments demonstrated that the interaction between Fe65 and CP2/LSF/LBP1 also takes place in vivo between the native molecules. The localization of both proteins was studied using fractionated cellular extracts. These experiments demonstrated that the various isoforms of CP2/LSF/LBP1 are differently distributed among subcellular fractions. At least one isoform, derived from alternative splicing (LSF-ID), is present outside the nucleus; Fe65 was found in both fractions. Furthermore, transfection experiments with an HA-tagged CP2/LSF/LBP1 cDNA demonstrated that Fe65 interacts also with the nuclear form of CP2/LSF/LBP1. Considering that the analysis of Fe65 distribution in fractionated cell extracts demonstrated that this protein is present both in nuclear and non-nuclear fractions, we examined the expression of Fe65 deletion mutants in the two fractions. This analysis allowed us to observe that a small region N-terminal to the WW domain is phosphorylated and is necessary for the presence of Fe65 in the nuclear fraction.

  18. The adaptor molecule signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is essential in mechanisms involving the Fyn tyrosine kinase for induction and progression of collagen-induced arthritis.

    Science.gov (United States)

    Zhong, Ming-Chao; Veillette, André

    2013-11-01

    Signaling lymphocytic activation molecule-associated protein (SAP) is an Src homology 2 domain-only adaptor involved in multiple immune cell functions. It has also been linked to immunodeficiencies and autoimmune diseases, such as systemic lupus erythematosus. Here, we examined the role and mechanism of action of SAP in autoimmunity using a mouse model of autoimmune arthritis, collagen-induced arthritis (CIA). We found that SAP was essential for development of CIA in response to collagen immunization. It was also required for production of collagen-specific antibodies, which play a key role in disease pathogenesis. These effects required SAP expression in T cells, not in B cells. In mice immunized with a high dose of collagen, the activity of SAP was nearly independent of its ability to bind the protein tyrosine kinase Fyn and correlated with the capacity of SAP to promote full differentiation of follicular T helper (TFH) cells. However, with a lower dose of collagen, the role of SAP was more dependent on Fyn binding, suggesting that additional mechanisms other than TFH cell differentiation were involved. Further studies suggested that this might be due to a role of the SAP-Fyn interaction in natural killer T cell development through the ability of SAP-Fyn to promote Vav-1 activation. We also found that removal of SAP expression during progression of CIA attenuated disease severity. However, it had no effect on disease when CIA was clinically established. Together, these results indicate that SAP plays an essential role in CIA because of Fyn-independent and Fyn-dependent effects on TFH cells and, possibly, other T cell types.

  19. Defective jejunal and colonic salt absorption and alteredNa +/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice

    NARCIS (Netherlands)

    N. Broere (Nellie); M. Chen (Min); A. Cinar (Ayhan); A.K. Singh (Arbind); J. Hillesheim (Jutta); B. Riederer (Beat Michel); M. Lunnemann; I. Rottinghaus (Ingrid); A. Krabbenhöft (Anja); R. Engelhardt (Regina); B. Rausch; E.J. Weinman (Edward); M. Donowitz (Mark); A. Hubbard; O. Kocher (Olivier); H.R. de Jonge (Hugo); B.M. Hogema (Boris); U. Seidler (Ursula)

    2009-01-01

    textabstractWe investigated the role of the Na+/H+ exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice display

  20. SLAM-family receptors: immune regulators with or without SAP-family adaptors.

    Science.gov (United States)

    Veillette, André

    2010-03-01

    The signaling lymphocytic activation molecule (SLAM) family of receptors and the SLAM-associated protein (SAP) family of intracellular adaptors are expressed in immune cells. By way of their cytoplasmic domain, SLAM-related receptors physically associate with SAP-related adaptors. Evidence is accumulating that the SLAM and SAP families play crucial roles in multiple immune cell types. Moreover, the prototype of the SAP family, that is SAP, is mutated in a human immunodeficiency, X-linked lymphoproliferative (XLP) disease. In the presence of SAP-family adaptors, the SLAM family usually mediates stimulatory signals that promote immune cell activation or differentiation. In the absence of SAP-family adaptors, though, the SLAM family undergoes a "switch-of-function," thereby mediating inhibitory signals that suppress immune cell functions. The molecular basis and significance of this mechanism are discussed herein.

  1. The Neuron-Specific Rai (ShcC) Adaptor Protein Inhibits Apoptosis by Coupling Ret to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

    Science.gov (United States)

    Pelicci, Giuliana; Troglio, Flavia; Bodini, Alessandra; Melillo, Rosa Marina; Pettirossi, Valentina; Coda, Laura; De Giuseppe, Antonio; Santoro, Massimo; Pelicci, Pier Giuseppe

    2002-01-01

    Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals. PMID:12242309

  2. Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF Family of Adaptor Proteins with the Raft- and the Non-Raft Brush Border Membrane Fractions of NHE3

    Directory of Open Access Journals (Sweden)

    Ayesha Sultan

    2013-11-01

    Full Text Available Background/Aims: Trafficking, brush border membrane (BBM retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50, NHERF2 (E3KARP, and NHERF3 (PDZK1 with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes (“lipid rafts” were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3- mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

  3. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation

    DEFF Research Database (Denmark)

    Barres, Romain; Grémeaux, Thierry; Gual, Philippe

    2006-01-01

    a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma m......RNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport...... and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin...

  4. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.

    Science.gov (United States)

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J; West, Christopher M

    2016-02-26

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.

  5. Role of polymorphisms of toll-like receptor (TLR 4, TLR9, toll-interleukin 1 receptor domain containing adaptor protein (TIRAP and FCGR2A genes in malaria susceptibility and severity in Burundian children

    Directory of Open Access Journals (Sweden)

    Esposito Susanna

    2012-06-01

    Full Text Available Abstract Background Malaria caused by Plasmodium falciparum is one of the leading causes of human morbidity and mortality from infectious diseases, predominantly in tropical and sub-tropical countries. As genetic variations in the toll-like receptors (TLRs-signalling pathway have been associated with either susceptibility or resistance to several infectious and inflammatory diseases, the supposition is that single nucleotide polymorphisms (SNPs of TLR2, TLR4, TLR9, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP and FCGR2A could modulate malaria susceptibility and severity. Methods This study was planned to make a further contribution to solving the problem of the real role of the most common polymorphisms of TLR4, TLR9, TIRAP and FCGR2A genes in modulating the risk of malaria and disease severity in children from Burundi, Central Africa. All the paediatric patients aged six months to 10 years admitted to the hospital of Kiremba, Burundi, between February 2011 and September 2011, for fever and suspicion of acute malaria were screened for malaria parasitaemia by light microscopy of thick and thin blood smears. In children with malaria and in uninfected controls enrolled during the study period in the same hospital, blood samples were obtained on filter paper and TLR4 Asp299Gly rs4986790, TLR9 G1174A rs352139, T-1486 C rs187084 TLR9 T-1237 C rs5743836, TIRAP Ser180Leu rs8177374 and the FCGR2A His131Arg rs1801274 polymorphisms were studied using an ABI PRISM 7900 HT Fast Real-time instrument. Results A total of 602 patients and 337 controls were enrolled. Among the malaria cases, 553 (91.9 % were considered as suffering from uncomplicated and 49 (8.1 % from severe malaria. TLR9 T1237C rs5743836CC was associated with an increased risk of developing malaria (p = 0.03, although it was found with the same frequency in uncomplicated and severe malaria cases. No other differences were found in all alleles studied and in

  6. Adaptor protein Crk Ⅰ mediates malignant potential of human ovarian cancer%接合物蛋白CrkⅠ在卵巢癌恶性潜能中的作用

    Institute of Scientific and Technical Information of China (English)

    车亚玲; 王瑾; 令狐华

    2011-01-01

    Objective To explore the role of adaptor protein Crk Ⅰ in malignant potential of human ovarian cancer. Methods Crk and Dock180 expression were detected by Western blotting in ovarian cancer tissues (EOC, n =28), benign ovarian tumors (BOT, n =13) and normal ovary tissues (Normal, n =10). Co-precipitation was performed to evaluate the in vivo protein-protein interaction of Dock180 and Crk Ⅰn 3 different ovarian cancer cell lines ( SKOV3, MCAS and RMUG-L cells). The expression of Crk Ⅰn SKOV3 cells were silenced by using siRNA interference, and then Rac1 activity and cell invasion in the transfected cells were observed. Results The intensity of Crk Ⅰ and Dock180 expression was consistent with each other in EOC tissues. Both were observed to be significantly higher in EOC than those in the BOT and normal ovary tissues(P < 0. 05 ). No significant difference was found between BOT and Normal group either for Crk Ⅰ or Dock180 expression (P > 0. 05 ). In consistent with this result, Dock180 preferred to combine with Crk Ⅰ rather than with Crk Ⅱ in all 3 ovarian cancer cell lines. Furthermore, Crk knockdown celIs presented with sustainable Crk Ⅰ expression depletion, significantly decreased Rac1 activity and cell invasion. Conclusion Crk might be involved in malignant potential of human EOC mainly through Crk Ⅰ/Dock180/Rac1 pathway.%目的 证实接合物蛋白CrkⅠ在卵巢癌恶性潜能中的作用.方法 采用Western blot法检测卵巢癌组织、卵巢良性肿瘤组织、正常卵巢组织中Crk和Dock180蛋白的表达;用免疫沉淀法检测3种卵巢癌细胞株中Crk与Dock180蛋白的内源性结合;用小干扰RNA敲低SKOV3细胞中内源性的Crk,检测Crk表达缺失性细胞Crk蛋白表达水平、Rac1酶活性和侵袭力的变化.结果 Dock180与CrkⅠ的表达强度呈现明显的一致性,卵巢癌组织中二者的表达均显著高于卵巢良性肿瘤组织和正常卵巢组织(P0.05).3个卵巢癌细胞株中Dock180主要

  7. Styles of Creativity: Adaptors and Innovators in a Singapore Context

    Science.gov (United States)

    Ee, Jessie; Seng, Tan Oon; Kwang, Ng Aik

    2007-01-01

    Kirton (1976) described two creative styles, namely adaptors and innovators. Adaptors prefer to "do things better" whilst, innovators prefer to "do things differently". This study explored the relationship between two creative styles (adaptor and innovator) and the Big Five personality traits (extraversion, agreeableness, conscientiousness,…

  8. SLAM family receptors and SAP adaptors in immunity.

    Science.gov (United States)

    Cannons, Jennifer L; Tangye, Stuart G; Schwartzberg, Pamela L

    2011-01-01

    The signaling lymphocyte activation molecule (SLAM)-associated protein, SAP, was first identified as the protein affected in most cases of X-linked lymphoproliferative (XLP) syndrome, a rare genetic disorder characterized by abnormal responses to Epstein-Barr virus infection, lymphoproliferative syndromes, and dysgammaglobulinemia. SAP consists almost entirely of a single SH2 protein domain that interacts with the cytoplasmic tail of SLAM and related receptors, including 2B4, Ly108, CD84, Ly9, and potentially CRACC. SLAM family members are now recognized as important immunomodulatory receptors with roles in cytotoxicity, humoral immunity, autoimmunity, cell survival, lymphocyte development, and cell adhesion. In this review, we cover recent findings on the roles of SLAM family receptors and the SAP family of adaptors, with a focus on their regulation of the pathways involved in the pathogenesis of XLP and other immune disorders.

  9. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    Science.gov (United States)

    Lam, Tonika; Thomas, Lisa M; White, Clayton A; Li, Guideng; Pone, Egest J; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  10. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    Directory of Open Access Journals (Sweden)

    Tonika Lam

    Full Text Available Class switch DNA recombination (CSR of the immunoglobulin heavy chain (IgH locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID expression and AID targeting to switch (S regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit and PKA-RIα (regulatory inhibitory subunit and uracil DNA glycosylase (Ung. 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198 or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR. 14-3-3 adaptors colocalized with AID and replication protein A (RPA in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr, an accessory protein of human immunodeficiency virus type-1 (HIV-1, which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  11. An organized co-assembly of clathrin adaptors is essential for endocytosis.

    Science.gov (United States)

    Skruzny, Michal; Desfosses, Ambroise; Prinz, Simone; Dodonova, Svetlana O; Gieras, Anna; Uetrecht, Charlotte; Jakobi, Arjen J; Abella, Marc; Hagen, Wim J H; Schulz, Joachim; Meijers, Rob; Rybin, Vladimir; Briggs, John A G; Sachse, Carsten; Kaksonen, Marko

    2015-04-20

    Clathrin-mediated endocytosis, the main trafficking route from the plasma membrane to the cytoplasm, is critical to many fundamental cellular processes. Clathrin, coupled to the membrane by adaptor proteins, is thought to play a major structural role in endocytosis by self-assembling into a cage-like lattice around the forming vesicle. Although clathrin adaptors are essential for endocytosis, little is known about their structural role in this process. Here we show that the membrane-binding domains of two conserved clathrin adaptors, Sla2 and Ent1, co-assemble in a PI(4,5)P2-dependent manner to form organized lattices on membranes. We determined the structure of the co-assembled lattice by electron cryo-microscopy and designed mutations that specifically impair the lattice formation in vitro. We show that these mutations block endocytosis in vivo. We suggest that clathrin adaptors not only link the polymerized clathrin to the membrane but also form an oligomeric structure, which is essential for membrane remodeling during endocytosis.

  12. TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88.

    Directory of Open Access Journals (Sweden)

    Hidenori Ohnishi

    Full Text Available MyD88, a Toll/interleukin-1 receptor homology (TIR domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

  13. Optimization of multiplexed RADseq libraries using low-cost adaptors.

    Science.gov (United States)

    Henri, Hélène; Cariou, Marie; Terraz, Gabriel; Martinez, Sonia; El Filali, Adil; Veyssiere, Marine; Duret, Laurent; Charlat, Sylvain

    2015-04-01

    Reduced representation genomics approaches, of which RADseq is currently the most popular form, offer the possibility to produce genome wide data from potentially any species, without previous genomic information. The application of RADseq to highly multiplexed libraries (including numerous specimens, and potentially numerous different species) is however limited by technical constraints. First, the cost of synthesis of Illumina adaptors including molecular identifiers (MIDs) becomes excessive when numerous specimens are to be multiplexed. Second, the necessity to empirically adjust the ratio of adaptors to genomic DNA concentration impedes the high throughput application of RADseq to heterogeneous samples, of variable DNA concentration and quality. In an attempt to solve these problems, we propose here some adjustments regarding the adaptor synthesis. First, we show that the common and unique (MID) parts of adaptors can be synthesized separately and subsequently ligated, which drastically reduces the synthesis cost, and thus allows multiplexing hundreds of specimens. Second, we show that self-ligation of adaptors, which makes the adaptor concentration so critical, can be simply prevented by using unphosphorylated adaptors, which significantly improves the ligation and sequencing yield.

  14. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

    Science.gov (United States)

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-08-31

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability.

  15. The myxoma virus m-t5 ankyrin repeat host range protein is a novel adaptor that coordinately links the cellular signaling pathways mediated by Akt and Skp1 in virus-infected cells.

    Science.gov (United States)

    Werden, Steven J; Lanchbury, Jerry; Shattuck, Donna; Neff, Chris; Dufford, Max; McFadden, Grant

    2009-12-01

    Most poxviruses express multiple proteins containing ankyrin (ANK) repeats accounting for a large superfamily of related but unique determinants of poxviral tropism. Recently, select members of this novel family of poxvirus proteins have drawn considerable attention for their potential roles in modulating intracellular signaling networks during viral infection. The rabbit-specific poxvirus, myxoma virus (MYXV), encodes four unique ANK repeat proteins, termed M-T5, M148, M149, and M150, all of which include a carboxy-terminal PRANC domain which closely resembles a cellular protein motif called the F-box domain. Here, we show that each MYXV-encoded ANK repeat protein, including M-T5, interacts directly with the Skp1 component of the host SCF ubiquitin ligase complex, and that the binding of M-T5 to cullin 1 is indirect via binding to Skp1 in the host SCF complex. To understand the significance of these virus-host protein interactions, the various binding domains of M-T5 were mapped. The N-terminal ANK repeats I and II were identified as being important for interaction with Akt, whereas the C-terminal PRANC/F-box-like domain was essential for binding to Skp1. We also report that M-T5 can bind Akt and the host SCF complex (via Skp1) simultaneously in MYXV-infected cells. Finally, we report that M-T5 specifically mediates the relocalization of Akt from the nucleus to the cytoplasm during infection with the wild-type MYXV, but not the M-T5 knockout version of the virus. These results indicate that ANK/PRANC proteins play a critical role in reprogramming disparate cellular signaling cascades to establish a new cellular environment more favorable for virus replication.

  16. 生活方式对高脂肥胖大鼠APPL1和GLUT4的影响%The effects of life style on APPL1 and glucose transporter 4 in high fat induced obese rats

    Institute of Scientific and Technical Information of China (English)

    童俊露; 王佑民

    2012-01-01

    目的 探讨饮食、运动对高脂饲料喂养的肥胖大鼠肌肉组织中细胞内转接蛋白(APPL1)、葡萄糖转运蛋白4(GLUT4)的影响,以及APPL1与GLUT4的关系.方法 70只4周龄SD大鼠随机分为5组:正常对照组(NC组)、高脂肥胖组(HF组)、饮食控制组(DC组)、运动锻炼组(EC组)、饮食运动结合组(DE组).NC组给予普通饮食,其余各组给予高脂饮食.12周后,DC组饮食限制,EC组进行游泳运动,DE组两者结合,共 6周,空腹取血检测相关代谢指标,取骨骼肌组织检测甘油三脂(TG)的含量和APPL1、GLUT4的表达.结果 HF组大鼠体重、TG、总胆固醇(TCH)、空腹血糖(FBG)、空腹胰岛素(FINS)明显高于NC组(P<0.05),APPL1、GLUT4的水平明显低于NC组(P<0.05),骨骼肌组织中的TG水平明显增加(P<0.05).饮食、运动干预后能减轻体重(P<0.05),降低TG、TCH水平(P<0.05),增加胰岛素敏感指数(P<0.05),降低骨骼肌组织中的TG水平(P<0.05),上调APPL1、GLUT4的表达(P<0.05).结论 高脂饮食可诱导大鼠的胰岛素抵抗模型.通过饮食控制和(或)运动锻炼均可降低高脂喂养的脂质水平,改善胰岛素抵抗,上调骨骼肌组织中APPL1、GLUT4水平.%Objective To investigate the effects of diet control and exercise on APPL1, glucose transporter 4 ( GLUT4 ) and their correlation in high fat diet induced obese rats. Methods 70 SD rats of 4 weeks, age were randomly divided into five groups: normal control group ( NC group ), high-fat diet group( HF group ), diet control group ( DC group ),exercise control group( EC group )and diet and exercise control group( DE group ). The animals of NC group were fed with normal chow and the others with high-fat diet. After 12 weeks, the rats of DC group were given diet control for 6 weeks, at the same time, EC group swam everyday, DE group had to do both two interventions. Then the blood of rats in fasting was obtained to measure metabolic indexs, triglyceride lipid and expression of AP-PL1

  17. Ubc2, an Ortholog of the Yeast Ste50p Adaptor, Possesses a Basidiomycete-Specific Carboxy terminal Extension Essential for Pathogenicity Independent of Pheromone Response.

    Science.gov (United States)

    Proteins involved in the MAP kinase pathway controlling mating, morphogenesis and pathogenicity have been identified previously in the fungus Ustilago maydis. One of these, the Ubc2 adaptor protein, possesses a basidiomycete-specific structure. In addition to containing SAM and RA domains typical of...

  18. Optimized Adaptor Polymerase Chain Reaction Method for Efficient Genomic Walking

    Institute of Scientific and Technical Information of China (English)

    Peng XU; Rui-Ying HU; Xiao-Yan DING

    2006-01-01

    Genomic walking is one of the most useful approaches in genome-related research. Three kinds of PCR-based methods are available for this purpose. However, none of them has been generally applied because they are either insensitive or inefficient. Here we present an efficient PCR protocol, an optimized adaptor PCR method for genomic walking. Using a combination of a touchdown PCR program and a special adaptor, the optimized adaptor PCR protocol achieves high sensitivity with low background noise. By applying this protocol, the insertion sites of a gene trap mouse line and two gene promoters from the incompletely sequenced Xenopus laevis genome were successfully identified with high efficiency. The general application of this protocol in genomic walking was promising.

  19. ATP binding to p97/VCP D1 domain regulates selective recruitment of adaptors to its proximal N-domain.

    Directory of Open Access Journals (Sweden)

    Wei Sheng Chia

    Full Text Available p97/Valosin-containing protein (VCP is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD. It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

  20. Proteasome inhibition, the pursuit of new cancer therapeutics, and the adaptor molecule p130Cas

    Directory of Open Access Journals (Sweden)

    Anderson Kenneth C

    2011-10-01

    Full Text Available Abstract Current interest in proteasome inhibitors for cancer therapy has stimulated considerable research efforts to identify the molecular pathway to their cytotoxicity with a view to identifying the mechanisms of sensitivity and resistance as well as informing the development of new drugs. Zhao and Vuori describe this month in BMC Biology experiments indicating a novel role of the adaptor protein p130Cas in sensitivity to apoptosis induced not only by proteasome inhibitors but also by the unrelated drug doxorubicin. See research article: http:// http://www.biomedcentral.com/1741-7007/9/73

  1. DMPD: The SAP family of adaptors in immune regulation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15541655 The SAP family of adaptors in immune regulation. Latour S, Veillette A. Se...min Immunol. 2004 Dec;16(6):409-19. (.png) (.svg) (.html) (.csml) Show The SAP family of adaptors in immune ...regulation. PubmedID 15541655 Title The SAP family of adaptors in immune regulation. Authors Latour S, Veill

  2. Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σS by ClpXP.

    Directory of Open Access Journals (Sweden)

    Dimce eMicevski

    2015-04-01

    Full Text Available In Escherichia coli, σS is the master regulator of the general stress response. The level of σS changes in response to multiple stress conditions and it is regulated at many levels including protein turnover. In the absence of stress, σS is rapidly degraded by the AAA+ protease, ClpXP in a regulated manner that depends on the adaptor protein RssB. This two-component response regulator mediates the recognition of σS and its delivery to ClpXP. The turnover of σS however, can be inhibited in a stress specific manner, by one of three anti-adaptor proteins. Each anti-adaptor binds to RssB and inhibits its activity, but how this is achieved is not fully understood at a molecular level. Here we describe details of the interaction between each anti-adaptor and RssB that leads to the stabilization of σS. By defining the domains of RssB using partial proteolysis we demonstrate that each anti-adaptor uses a distinct mode of binding to inhibit RssB activity. IraD docks specifically to the N-terminal domain of RssB, IraP interacts primarily with the C-terminal domain, while IraM interacts with both domains. Despite these differences in binding, we propose that docking of each anti-adaptor induces a conformational change in RssB, which resembles the inactive dimer of RssB. This dimer-like state of RssB not only prevents substrate binding but also triggers substrate release from a pre-bound complex.

  3. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

    Science.gov (United States)

    Hautbergue, Guillaume M; Hung, Ming-Lung; Walsh, Matthew J; Snijders, Ambrosius P L; Chang, Chung-Te; Jones, Rachel; Ponting, Chris P; Dickman, Mark J; Wilson, Stuart A

    2009-12-01

    Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

  4. Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals.

    Science.gov (United States)

    Smith, Stephen C; Joshi, Kamal K; Zik, Justin J; Trinh, Katherine; Kamajaya, Aron; Chien, Peter; Ryan, Kathleen R

    2014-09-30

    The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor.

  5. Spiral biasing adaptor for use in Si drift detectors and Si drift detector arrays

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zheng; Chen, Wei

    2016-07-05

    A drift detector array, preferably a silicon drift detector (SDD) array, that uses a low current biasing adaptor is disclosed. The biasing adaptor is customizable for any desired geometry of the drift detector single cell with minimum drift time of carriers. The biasing adaptor has spiral shaped ion-implants that generate the desired voltage profile. The biasing adaptor can be processed on the same wafer as the drift detector array and only one biasing adaptor chip/side is needed for one drift detector array to generate the voltage profiles on the front side and back side of the detector array.

  6. The Hypoxic Regulator of Sterol Synthesis Nro1 Is a Nuclear Import Adaptor

    Energy Technology Data Exchange (ETDEWEB)

    T Yeh; C Lee; L Amzel; P Espenshade; M Bianchet

    2011-12-31

    Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 {angstrom} resolution shows an all-{alpha}-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.

  7. Adaptor-tagged competitive PCR: a novel method for measuring relative gene expression.

    OpenAIRE

    Kato, K.

    1997-01-01

    A simple and reliable PCR-based method to quantitate gene expression is described. Following the digestion of double-stranded cDNA by a restriction enzyme, an adaptor is ligated to a cDNA from a first RNA sample, and another adaptor to a second RNA sample. The two adaptors share a common sequence at the outer region, but differ in size. Equal amounts of the ligated samples are mixed, and amplified by an adaptor-primer and a primer specific to the gene of interest. Products derived from the tw...

  8. Analysis of Arf1 GTPase-dependent membrane binding and remodeling using the exomer secretory vesicle cargo adaptor

    Science.gov (United States)

    Paczkowski, Jon E.; Fromme, J. Christopher

    2016-01-01

    Summary Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest. PMID:27632000

  9. A Big-Five Personality Profile of the Adaptor and Innovator.

    Science.gov (United States)

    Kwang, Ng Aik; Rodrigues, Daphne

    2002-01-01

    A study explored the relationship between two creative types (adaptor and innovator) and the Big Five personality traits (extraversion, agreeableness, conscientiousness, neuroticism, and openness to experience), in 164 teachers in Singapore. Adaptors were significantly more conscientious than innovators, while innovators were significantly more…

  10. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    Science.gov (United States)

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-01

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

  11. Kit- and Fc epsilonRI-induced differential phosphorylation of the transmembrane adaptor molecule NTAL/LAB/LAT2 allows flexibility in its scaffolding function in mast cells

    DEFF Research Database (Denmark)

    Iwaki, Shoko; Spicka, Jiri; Tkaczyk, Christine;

    2008-01-01

    The transmembrane adaptor protein (TRAP), NTAL, is phosphorylated in mast cells following FcvarepsilonRI aggregation whereby it cooperates with LAT to induce degranulation. The Kit ligand, stem cell factor (SCF), enhances antigen-induced degranulation and this also appears to be NTAL......-knock down-human mast cells. The observations reported herein support the conclusion that NTAL may be differentially utilized by specific receptors for relaying alternative signals and this suggests a flexibility in the function of TRAPs not previously appreciated....

  12. ATM-Dependent Phosphorylation of All Three Members of the MRN Complex: From Sensor to Adaptor.

    Science.gov (United States)

    Lavin, Martin F; Kozlov, Sergei; Gatei, Magtouf; Kijas, Amanda W

    2015-10-23

    The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes.

  13. Study on the isothermal forging process of MB26 magnesium alloy adaptor

    Directory of Open Access Journals (Sweden)

    Xu Wenchen

    2015-01-01

    Full Text Available The isothermal forging process is an effective method to manufacture complex-shaped components of hard-to-work materials, such as magnesium alloys. This study investigates the isothermal forging process of an MB26 magnesium alloy adaptor with three branches. The results show that two-step forging process is appropriate to form the adaptor forging, which not only improves the filling quality but also reduces the forging load compared with one-step forging process. Moreover, the flow line is distributed along the contour of the complex-shaped adaptor forging.

  14. The AP-3 adaptor complex is required for vacuolar function in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Maria Zwiewka; Elena Feraru; Barbara M(o)ller; Inhwan Hwang; Mugurel I Feraru; Jürgen Kleine-Vehn; Dolf Weijers; Ji(n) Friml

    2011-01-01

    Subcellular trafficking is required for a multitude of functions in eukaryotic cells.It involves regulation of cargo sorting,vesicle formation,trafficking and fusion processes at multiple levels.Adaptor protein (AP) complexes are key regulators of cargo sorting into vesicles in yeast and mammals but their existence and function in plants have not been demonstrated.Here we report the identification of the protein-affected trafficking 4 (pat4) mutant defective in the putative δ subunit of the AP-3 complex.pat4 and pat2,a mutant isolated from the same GFP imaging-based forward genetic screen that lacks a functional putative AP-3 β,as well as dominant negative AP-3 μ transgenic lines display undistinguishable phenotypes characterized by largely normal morphology and development,but strong intracellular accumulation of membrane proteins in aberrant vacuolar structures.All mutants are defective in morphology and function of lytic and protein storage vacuoles (PSVs) but show normal sorting of reserve proteins to PSVs.Immunoprecipitation experiments and genetic studies revealed tight functional and physical associations of putative AP-3 β and AP-3 δ subunits.Furthermore,both proteins are closely linked with putative AP-3 μ and σ subunits and several components of the clathrin and dynamin machineries.Taken together,these results demonstrate that AP complexes,similar to those in other eukaryotes,exist in plants,and that AP-3 plays a specific role in the regulation of biogenesis and function of vacuoles in plant cells.

  15. The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

    Directory of Open Access Journals (Sweden)

    Fubito Nakatsu

    2014-11-01

    Full Text Available The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis. Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells.

  16. The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

    Science.gov (United States)

    Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

    2013-04-25

    The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

  17. The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo.

    Science.gov (United States)

    Carvajal-Gonzalez, Jose Maria; Balmer, Sophie; Mendoza, Meg; Dussert, Aurore; Collu, Giovanna; Roman, Angel-Carlos; Weber, Ursula; Ciruna, Brian; Mlodzik, Marek

    2015-04-07

    A key step in generating planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. We describe the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo-distal axis, producing severe PCP phenotypes. Using novel tools, we demonstrate a direct and specific Arf1 involvement in Fz trafficking in vivo. Moreover, we uncover a conserved Arf1 PCP function in vertebrates. Our data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo.

  18. Sub-cellular distribution of UNC-104(KIF1A) upon binding to adaptors as UNC-16(JIP3), DNC-1(DCTN1/Glued) and SYD-2(Liprin-α) in C. elegans neurons.

    Science.gov (United States)

    Hsu, C-C; Moncaleano, J D; Wagner, O I

    2011-03-10

    The accumulation of cargo (tau, amyloid precursor protein, neurofilaments etc.) in neurons is a hallmark of various neurodegenerative diseases while we have only little knowledge how axonal transport is regulated. Kinesin-3 UNC-104(KIF1A) is the major transporter of synaptic vesicles and recent reports suggest that a cargo itself can affect the motor's activity. Inspecting an interactome map, we identify three putative UNC-104 interactors, namely UNC-16(JIP3), DNC-1(DCTN1/Glued) and SYD-2(Liprin-α), known to be adaptors in essential neuronal protein complexes. We then employed the novel method bimolecular fluorescence complementation (BiFC) assay to visualize motor-adaptor complexes in the nervous system of living C. elegans. Interestingly, the binding of UNC-104 to each adaptor protein results in different sub-cellular distributions and has distinctive effects on the motor's motility. Specifically, if UNC-104 bound to UNC-16, the motor is primarily localized in the soma of neurons while bound to DNC-1, the motor is basically found in axonal termini. On the other hand, if UNC-104 is bound to SYD-2 we identify motor populations mostly along axons. Therefore, these three adaptors inherit different functions in steering the motor to specific sub-cellular locations in the neuron.

  19. Increasing the efficiency of SAGE adaptor ligation by directed ligation chemistry

    Science.gov (United States)

    So, Austin P.; Turner, Robin F. B.; Haynes, Charles A.

    2004-01-01

    The ability of Serial Analysis of Gene Expression (SAGE) to provide a quantitative picture of global gene expression relies not only on the depth and accuracy of sequencing into the SAGE library, but also on the efficiency of each step required to generate the SAGE library from the starting mRNA material. The first critical step is the ligation of adaptors containing a Type IIS recognition sequence to the anchored 3′ end cDNA population that permits the release of short sequence tags (SSTs) from defined sites within the 3′ end of each transcript. Using an in vitro transcript as a template, we observed that only a small fraction of anchored 3′ end cDNA are successfully ligated with added SAGE adaptors under typical reaction conditions currently used in the SAGE protocol. Although the introduction of ∼500-fold molar excess of adaptor or the inclusion of 15% (w/v) PEG-8000 increased the yield of the adaptor-modified product, complete conversion to the desired adaptor:cDNA hetero-ligation product is not achieved. An alternative method of ligation, termed as directed ligation, is described which exploits a favourable mass-action condition created by the presence of NlaIII during ligation in combination with a novel SAGE adaptor containing a methylated base within the ligation site. Using this strategy, we were able to achieve near complete conversion of the anchored 3′ end cDNA into the desired adaptor-modified product. This new protocol therefore greatly increases the probability that a SST will be generated from every transcript, greatly enhancing the fidelity of SAGE. Directed ligation also provides a powerful means to achieve near-complete ligation of any appropriately designed adaptor to its respective target. PMID:15247329

  20. A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling.

    Science.gov (United States)

    Ní Cheallaigh, Clíona; Sheedy, Frederick J; Harris, James; Muñoz-Wolf, Natalia; Lee, Jinhee; West, Kim; McDermott, Eva Palsson; Smyth, Alicia; Gleeson, Laura E; Coleman, Michelle; Martinez, Nuria; Hearnden, Claire H A; Tynan, Graham A; Carroll, Elizabeth C; Jones, Sarah A; Corr, Sinéad C; Bernard, Nicholas J; Hughes, Mark M; Corcoran, Sarah E; O'Sullivan, Mary; Fallon, Ciara M; Kornfeld, Hardy; Golenbock, Douglas; Gordon, Stephen V; O'Neill, Luke A J; Lavelle, Ed C; Keane, Joseph

    2016-02-16

    Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.

  1. Ascent Heating Thermal Analysis on Spacecraft Adaptor Fairings

    Science.gov (United States)

    Wang, Xiao Yen; Yuko, James; Motil, Brian

    2011-01-01

    When the Crew Exploration Vehicle (CEV) is launched, the spacecraft adaptor (SA) fairings that cover the CEV service module (SM) are exposed to aero heating. Thermal analysis is performed to compute the fairing temperatures and to investigate whether the temperatures are within the material limits for nominal ascent aeroheating case. The ascent heating is analyzed by using computational fluid dynamics (CFD) and engineering codes at Marshall Space Flight Center. The aeroheating environment data used for this work is known as Thermal Environment 3 (TE3) heating data. One of the major concerns is with the SA fairings covering the CEV SM and the SM/crew launch vehicle (CLV) flange interface. The TE3 heating rate is a function of time, wall temperature, and the spatial locations. The implementation of the TE3 heating rate as boundary conditions in the thermal analysis becomes challenging. The ascent heating thermal analysis on SA fairings and SM/CLV flange interface are performed using two commercial software packages: Cullimore & Ring (C&R) Thermal Desktop (TD) 5.1 and MSC Patran 2007r1 b. TD is the pre-and post-processor for SINDA, which is a finite-difference-based solver. In TD, the geometry is built and meshed, the boundary conditions are defined, and then SINDA is used to compute temperatures. MSC Pthermal is a finite-element- based thermal solver. MSC Patran is the pre- and post-processor for Pthermal. Regarding the boundary conditions, the convection, contact resistance, and heat load can be imposed in different ways in both programs. These two software packages are used to build the thermal model for the same analysis to validate each other and show the differences in the modeling details.

  2. Yeast Interacting Proteins Database: YCL032W, YLR423C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YCL032W STE50 Protein involved in mating response, invasive/filamentous growth, and...lved in mating response, invasive/filamentous growth, and osmotolerance, acts as an adaptor that links G pro

  3. Fuel combustion test in constant volume combustion chamber with built-in adaptor

    Institute of Scientific and Technical Information of China (English)

    JEONG; DongSoo; CHO; GyuBack; CHOI; SuJin; LEE; JinSoo

    2010-01-01

    Combustion tests of pre-mixture of methane and air in constant volume combustion chamber(CVCC) have been carried out by means of flame propagation photo and gas pressure measurement,the effects of CVCC body temperature,intake pressure of pre-mixture of methane and air,equivalence ratio and location of the built-in adaptor have been investigated.The whole combustion chamber can be divided into two parts,i.e.the upper combustion chamber and the lower combustion chamber,by the built-in adaptor with through hole.Owing to the built-in adaptor with through hole,jet ignition or compression ignition(auto-ignition) phenomena may occur in the lower combustion chamber,which is helpful to getting higher flame propagation velocity,higher combustion peak pressure,low cycle-to-cycle variation and more stable combustion process.

  4. A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, Arne; Søndergaard, Birgitte P; Ersbøll, Bjarne

    2004-01-01

    Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequ...... that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor....... sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...

  5. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization.

    Science.gov (United States)

    Pérez-Quintero, Luis-Alberto; Roncagalli, Romain; Guo, Huaijian; Latour, Sylvain; Davidson, Dominique; Veillette, André

    2014-04-07

    Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.

  6. Regulation and function of the CD3¿ DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro

    DEFF Research Database (Denmark)

    Dietrich, J; Kastrup, J; Nielsen, B L

    1997-01-01

    Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR...

  7. Hybridization characteristics of biomolecular adaptors, covalent DNA streptavidin conjugates

    NARCIS (Netherlands)

    Niemeyer, CM; Burger, W; Hoedemakers, RMJ

    1998-01-01

    Semisynthetic, covalent streptavidin-DNA adducts are versatile molecular connectors for the fabrication of both nano-and microstructured protein arrays by use of DNA hybridization. In this study, the hybridization characteristics of six adduct species, each containing a different DNA sequence of 21

  8. ER Adaptor SCAP Translocates and Recruits IRF3 to Perinuclear Microsome Induced by Cytosolic Microbial DNAs

    Science.gov (United States)

    Yu, Huansha; Liu, Xing; Huang, Lulu; Wang, Qiang; Liu, Heng; Cui, Ye; Tang, Yijun; Zhang, Peng; Wang, Chen

    2016-01-01

    Stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS) induces the activation of TBK1 kinase and IRF3 transcription factor, upon sensing of microbial DNAs. How IRF3 is recruited onto the STING signalosome remains unknown. We report here that silencing of the ER adaptor SCAP markedly impairs the IRF3-responsive gene expression induced by STING. Scap knockdown mice are more susceptible to HSV-1 infection. Interestingly, SCAP translocates from ER, via Golgi, to perinuclear microsome in a STING-dependent manner. Mechanistically, the N-terminal transmembrane domain of SCAP interacts with STING, and the C-terminal cytosolic domain of SCAP binds to IRF3, thus recruiting IRF3 onto STING signalosome. Mis-localization of SCAP abolishes its antiviral function. Collectively, this study characterizes SCAP as an essential adaptor in the STING signaling pathway, uncovering a critical missing link in DNAs-triggered host antiviral responses. PMID:26900919

  9. ER Adaptor SCAP Translocates and Recruits IRF3 to Perinuclear Microsome Induced by Cytosolic Microbial DNAs.

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2016-02-01

    Full Text Available Stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS induces the activation of TBK1 kinase and IRF3 transcription factor, upon sensing of microbial DNAs. How IRF3 is recruited onto the STING signalosome remains unknown. We report here that silencing of the ER adaptor SCAP markedly impairs the IRF3-responsive gene expression induced by STING. Scap knockdown mice are more susceptible to HSV-1 infection. Interestingly, SCAP translocates from ER, via Golgi, to perinuclear microsome in a STING-dependent manner. Mechanistically, the N-terminal transmembrane domain of SCAP interacts with STING, and the C-terminal cytosolic domain of SCAP binds to IRF3, thus recruiting IRF3 onto STING signalosome. Mis-localization of SCAP abolishes its antiviral function. Collectively, this study characterizes SCAP as an essential adaptor in the STING signaling pathway, uncovering a critical missing link in DNAs-triggered host antiviral responses.

  10. Bcl-XL cooperatively associates with the Bap31 complex in the endoplasmic reticulum, dependent on procaspase-8 and Ced-4 adaptor.

    Science.gov (United States)

    Ng, F W; Shore, G C

    1998-02-06

    Bap31 is a polytopic integral membrane protein of the endoplasmic reticulum and forms a complex with Bcl-2/Bcl-XL and procaspase-8 (Ng, F. W. H., Nguyen, M., Kwan, T., Branton, P. E., Nicholson, W. D., Cromlish, J. A., and Shore, G. C. (1997) J. Cell Biol. 139, 327-338). In co-transfected human cells, procaspase-8 is capable of interacting with Ced-4, an important adaptor molecule in Caenorhabditis elegans that binds to and activates the C. elegans procaspase, proCed-3. Here, we show that the predicted death effector homology domain within the cytosolic region of Bap31 interacts with Ced-4 and contributes to recruitment of procaspase-8. Bcl-XL, which binds directly but weakly to the polytopic transmembrane region of Bap31, indirectly and cooperatively associates with the Bap31 cytosolic domain, dependent on the presence of procaspase-8 and Ced-4. Ced-4Deltac does not interact with Bcl-XL but rather displaces it from Bap31, suggesting that an endogenous Ced-4-like adaptor is a normal constituent of the Bap31 complex and is required for stable association of Bcl-XL with Bap31 in vivo. These findings indicate that Bap31 is capable of recruiting essential components of a core death regulatory machinery.

  11. The immune adaptor molecule SARM modulates tumor necrosis factor alpha production and microglia activation in the brainstem and restricts West Nile Virus pathogenesis.

    Science.gov (United States)

    Szretter, Kristy J; Samuel, Melanie A; Gilfillan, Susan; Fuchs, Anja; Colonna, Marco; Diamond, Michael S

    2009-09-01

    Sterile alpha and HEAT/Armadillo motif (SARM) is a highly conserved Toll/interleukin-1 receptor (TIR)-containing adaptor protein that is believed to negatively regulate signaling of the pathogen recognition receptors Toll-like receptor 3 (TLR3) and TLR4. To test its physiological function in the context of a microbial infection, we generated SARM(-/-) mice and evaluated the impact of this deficiency on the pathogenesis of West Nile virus (WNV), a neurotropic flavivirus that requires TLR signaling to restrict infection. Although SARM was preferentially expressed in cells of the central nervous system (CNS), studies with primary macrophages, neurons, or astrocytes showed no difference in viral growth kinetics. In contrast, viral replication was increased specifically in the brainstem of SARM(-/-) mice, and this was associated with enhanced mortality after inoculation with a virulent WNV strain. A deficiency of SARM was also linked to reduced levels of tumor necrosis factor alpha (TNF-alpha), decreased microglia activation, and increased neuronal death in the brainstem after WNV infection. Thus, SARM appears to be unique among the TIR adaptor molecules, since it functions to restrict viral infection and neuronal injury in a brain region-specific manner, possibly by modulating the activation of resident CNS inflammatory cells.

  12. Use of Conversion Adaptors to Clone Antigen Genes in Lambda gt11

    Science.gov (United States)

    1987-01-01

    gradients of 19, 30, and 50%. with 4 units ofT 4 DNA ligase for 60 min at Chromosomal DNA was prepared by dode- 16°C. Because the adaptor-insert...0.75 M and 6.5%. respectively. After chill- Biotec. Madison. WI) and 0.5 unit of T4 ing on ice for I h. the mixture was centri- DNA ligase , in 5ul of

  13. Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure.

    Directory of Open Access Journals (Sweden)

    Ryan T Fuchs

    Full Text Available High-throughput sequencing (HTS has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA and other small RNAs (sRNA. Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.

  14. DEAD/H BOX 3 (DDX3) helicase binds the RIG-I adaptor IPS-1 to up-regulate IFN-beta-inducing potential.

    Science.gov (United States)

    Oshiumi, Hiroyuki; Sakai, Keisuke; Matsumoto, Misako; Seya, Tsukasa

    2010-04-01

    Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLR) are members of the DEAD box helicases, and recognize viral RNA in the cytoplasm, leading to IFN-beta induction through the adaptor IFN-beta promoter stimulator-1 (IPS-1) (also known as Cardif, mitochondrial antiviral signaling protein or virus-induced signaling adaptor). Since uninfected cells usually harbor a trace of RIG-I, other RNA-binding proteins may participate in assembling viral RNA into the IPS-1 pathway during the initial response to infection. We searched for proteins coupling with human IPS-1 by yeast two-hybrid and identified another DEAD (Asp-Glu-Ala-Asp) box helicase, DDX3 (DEAD/H BOX 3). DDX3 can bind viral RNA to join it in the IPS-1 complex. Unlike RIG-I, DDX3 was constitutively expressed in cells, and some fraction of DDX3 is colocalized with IPS-1 around mitochondria. The 622-662 a.a DDX3 C-terminal region (DDX3-C) directly bound to the IPS-1 CARD-like domain, and the whole DDX3 protein also associated with RLR. By reporter assay, DDX3 helped IPS-1 up-regulate IFN-beta promoter activation and knockdown of DDX3 by siRNA resulted in reduced IFN-beta induction. This activity was conserved on the DDX3-C fragment. DDX3 only marginally enhanced IFN-beta promoter activation induced by transfected TANK-binding kinase 1 (TBK1) or I-kappa-B kinase-epsilon (IKKepsilon). Forced expression of DDX3 augmented virus-mediated IFN-beta induction and host cell protection against virus infection. Hence, DDX3 is an antiviral IPS-1 enhancer.

  15. Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Zawawi, M.S.F. [Universiti Sains Malaysia (USM) (Malaysia); Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); Dharmapatni, A.A.S.S.K.; Cantley, M.D. [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); McHugh, K.P. [University of Florida, College of Dentistry, Fl (United States); Haynes, D.R. [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); Crotti, T.N., E-mail: tania.crotti@adelaide.edu.au [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real

  16. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects.

    Science.gov (United States)

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A

    2016-09-19

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes.

  17. Yeast Endocytic Adaptor AP-2 Binds the Stress Sensor Mid2 and Functions in Polarized Cell Responses

    Science.gov (United States)

    Chapa-y-Lazo, Bernardo; Allwood, Ellen G; Smaczynska-de Rooij, Iwona I; Snape, Mary L; Ayscough, Kathryn R

    2014-01-01

    The AP-2 complex is a heterotetrameric endocytic cargo-binding adaptor that facilitates uptake of membrane proteins during mammalian clathrin-mediated endocytosis. While budding yeast has clear homologues of all four AP-2 subunits which form a complex and localize to endocytic sites in vivo, the function of yeast AP-2 has remained enigmatic. Here, we demonstrate that AP-2 is required for hyphal growth in Candida albicans and polarized cell responses in Saccharomyces cerevisiae. Deletion of APM4, the cargo-binding mu subunit of AP-2, causes defects in pseudohyphal growth, generation of a mating projection and the cell wall damage response. In an apm4 null mutant, the cell wall stress sensor Mid2 is unable to relocalize to the tip of a mating projection following pheromone addition, or to the mother bud neck in response to cell wall damage. A direct binding interaction between Mid2 and the mu homology domain of Apm4 further supports a model in which AP-2 binds Mid2 to facilitate its internalization and relocalization in response to specific signals. Thus, Mid2 is the first cargo for AP-2 identified in yeast. We propose that endocytic recycling of Mid2 and other components is required for polarized cell responses ensuring cell wall deposition and is tightly monitored during cell growth. PMID:24460703

  18. DMPD: Adaptor usage and Toll-like receptor signaling specificity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 05 Jun 13;579(15):3330-5. Epub 2005 Apr 26. Pathway - PNG File (.png) SVG File (.svg) HTML File (.html) CSML File (.csml) Open .csm...ll LA. FEBS Lett. 2005 Jun 13;579(15):3330-5. Epub 2005 Apr 26. (.png) (.svg) (.html) (.csml) Show Adaptor u...l file with CIOPlayer Open .csml file with CIOPlayer - ※CIO Playerのご利用上の注意 Open .csml file with CIO Open .csml file with CIO - ※CIOのご利用上の注意 ...

  19. Role of Crk Adaptor Proteins in Cellular Migration and Invasion in Human Breast Cancer

    Science.gov (United States)

    2008-03-01

    mice and cell lines have been established. One CrkII tumor has been identified as a fibroadenoma . The tumor is positive for CrkII, CK8, CK14, PCNA...mice were of a diverse phenotype, 7 including fibroadenomas and squamous adenocarcinoma (Figure 5). No metastatic lesions were found in any...animals. The fibroadenomas consisted of several differentiated epithelial cells (CK8, CK14) interspersed within regions of mesenchymal-like cell

  20. Bicaudal d family adaptor proteins control the velocity of Dynein-based movements

    NARCIS (Netherlands)

    Schlager, M.A.; Serra-Marquez, A.; Grigoriev, Ilya; Gumy, Laura; Estevez da Silva, M.; Wulf, Phebe; Akhmanova, Anna; Hoogenraad, Casper

    2014-01-01

    Cargo transport along microtubules is driven by the collective function of microtubule plus- and minus-end-directed motors (kinesins and dyneins). How the velocity of cargo transport is driven by opposing teams of motors is still poorly understood. Here, we combined inducible recruitment of motors a

  1. The Role of Crk Adaptor Proteins in Breast Tumorigenesis and Bone Metastasis

    Science.gov (United States)

    2012-09-01

    metabolomics confirmed changes in metabolite levels, including decreased glucose and lactate levels with Crk knockdown. These findings support a role for Crk...software for analysis of data) - Attended numerous seminars from visiting lecturers in oncology and other areas of biochemistry (Seminars given by job

  2. The adaptor protein soc-1/Gab1 modifies growth factor receptor output in Caenorhabditis elegans.

    Science.gov (United States)

    Hopper, Neil A

    2006-05-01

    Previous genetic analysis has shown that dos/soc-1/Gab1 functions positively in receptor tyrosine kinase (RTK)-stimulated Ras/Map kinase signaling through the recruitment of csw/ptp-2/Shp2. Using sensitized assays in Caenorhabditis elegans for let-23/Egfr and daf-2/InsR (insulin receptor-like) signaling, it is shown that soc-1/Gab1 inhibits phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3'-kinase (PI3K)-mediated signaling. Furthermore, as well as stimulating Ras/Map kinase signaling, soc-1/Gab1 stimulates a poorly defined signaling pathway that represses class 2 daf-2 phenotypes. In addition, it is shown that SOC-1 binds the C-terminal SH3 domain of SEM-5. This binding is likely to be functional as the sem-5(n2195)G201R mutation, which disrupts SOC-1 binding, behaves in a qualitatively similar manner to a soc-1 null allele in all assays for let-23/Egfr and daf-2/InsR signaling that were examined. Further genetic analysis suggests that ptp-2/Shp2 mediates the negative function of soc-1/Gab1 in PI3K-mediated signaling, as well as the positive function in Ras/Map kinase signaling. Other effectors of soc-1/Gab1 are likely to inhibit PLCgamma-mediated signaling and stimulate the poorly defined signaling pathway that represses class 2 daf-2 phenotypes. Thus, the recruitment of soc-1/Gab1, and its effectors, into the RTK-signaling complex modifies the cellular response by enhancing Ras/Map kinase signaling while inhibiting PI3K and PLCgamma-mediated signaling.

  3. CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis.

    Science.gov (United States)

    Bai, Baoyan; Moore, Henna M; Laiho, Marikki

    2013-01-01

    CRM1 is an export factor that together with its adaptor NMD3 transports numerous cargo molecules from the nucleus to cytoplasm through the nuclear pore. Previous studies have suggested that CRM1 and NMD3 are detected in the nucleolus. However, their localization with subnucleolar domains or participation in the activities of the nucleolus are unclear. We demonstrate here biochemically and using imaging analyses that CRM1 and NMD3 co-localize with nucleolar marker proteins in the nucleolus. In particular, their nucleolar localization is markedly increased by inhibition of RNA polymerase I (Pol I) transcription by actinomycin D or by silencing Pol I catalytic subunit, RPA194. We show that CRM1 nucleolar localization is dependent on its activity and the expression of NMD3, whereas NMD3 nucleolar localization is independent of CRM1. This suggests that NMD3 provides nucleolar tethering of CRM1. While inhibition of CRM1 by leptomycin B inhibited processing of 28S ribosomal (r) RNA, depletion of NMD3 did not, suggesting that their effects on 28S rRNA processing are distinct. Markedly, depletion of NMD3 and inhibition of CRM1 reduced the rate of pre-47S rRNA synthesis. However, their inactivation did not lead to nucleolar disintegration, a hallmark of Pol I transcription stress, suggesting that they do not directly regulate transcription. These results indicate that CRM1 and NMD3 have complex functions in pathways that couple rRNA synthetic and processing engines and that the rRNA synthesis rate may be adjusted according to proficiency in rRNA processing and export.

  4. The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.

    Directory of Open Access Journals (Sweden)

    Stefan Pils

    Full Text Available BACKGROUND: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. PRINCIPAL FINDINGS: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. CONCLUSIONS: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.

  5. The late endosomal adaptor p14 is a macrophage host-defense factor against Salmonella infection.

    Science.gov (United States)

    Taub, Nicole; Nairz, Manfred; Hilber, Diana; Hess, Michael W; Weiss, Günter; Huber, Lukas A

    2012-06-01

    The outcome of an infection depends on the balance between host resistance and bacterial virulence. Here, we show that the late endosomal adaptor p14 (also known as LAMTOR2) is one of the components for cellular host defense against the intracellular pathogen Salmonella enterica serovar Typhimurium. During Salmonella infection, the complex of p14 and MP1 is required for the accurately timed transport of Salmonella through the endolysosomal system. Loss of p14 opens a time window that allows Salmonella to populate a replication niche, in which early and late antimicrobial effector systems, comprising NADPH phagocytic oxidase and inducible nitric oxide synthase, respectively, are inappropriately activated. Thus, p14 supports the accurate transport of Salmonella through the endolysosomal system, thereby limiting bacterial replication in both, professional phagocytes and in non-phagocytic cells in vitro, and helps mice to successfully battle Salmonella infection in vivo.

  6. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71

    Science.gov (United States)

    Wang, Xiangxi; Peng, Wei; Ren, Jingshan; Hu, Zhongyu; Xu, Jiwei; Lou, Zhiyong; Li, Xumei; Yin, Weidong; Shen, Xinliang; Porta, Claudine; Walter, Thomas S.; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Rowlands, David J.; Wang, Junzhi; Stuart, David I.; Fry, Elizabeth E.; Rao, Zihe

    2012-01-01

    Enterovirus 71 (EV71), a major agent of hand-foot-and-mouth disease in children, can cause severe central nervous system disease and mortality. At present no vaccine or antiviral therapy is available. We have determined high-resolution structures for the mature virus and natural empty particles. The structure of the mature virus is similar to that of other enteroviruses, whilst the empty particles are dramatically expanded, with notable fissures, resembling elusive enterovirus uncoating intermediates not previously characterized in atomic detail. Hydrophobic capsid pockets within the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. The results provide a paradigm for enterovirus uncoating, in which the VP1 GH loop acts as an adaptor-sensor for the attachment of cellular receptors, converting heterologous inputs to a generic uncoating mechanism, spotlighting novel points for therapeutic intervention. PMID:22388738

  7. The Influences of Connectors and Adaptors to Fiber-To-The-Home Network Performance

    Directory of Open Access Journals (Sweden)

    Mohammad S. Ab-Rahman

    2012-01-01

    Full Text Available Problem statement: The reliability of the entire communications network was dependent on the reliability of each single element. Connector was important devices that can affect the performance of the fiber communication. There were a large number of issues that affect the performance of fiber optic connectors in todays networks. These factors were increasingly as data rates, the number of wavelengths and transmission distances continue to escalate. Approach: Therefore this study was carried out to test on the influence of connectors and adapters to the performance of the optical network. Initially the actual attenuation of connector and adaptor were tested by using multifunction loss tester. The first two 1 m corning optical fibers with a connector at each end are measured. Then, both the 1 m corning optical fibers were joined together by an adaptor and connected to the Multifunction loss tester. Three types of wavelength are used as the source to test the attenuation of the fiber which is 1310, 1490-1550 nm. In order to measure the Bit Error Rate (BER and the power loss in optical fiber communication, a simple simulation was carried out by using software opti sys. Results: The attenuation on the connector was caused mainly by existence of impurities in the connector, less perfect connection, scattering of beam and others. These causes the parameter such as power received, Q-factor, minimum BER and also the eye-height to change. Changes in these parameters also affect the performance at the user end. It was very critical that causes of attenuation to be eliminated. Conclusion/Recommendations: From the result it can be concluded that, the greater the attenuation, the greater the decrease in power received. It also affects the Q-factor of the system where as the attenuation increase, the maximum Q-factor decreases. As for the minimum BER, minimum BER changes as the attenuation increase initially, after a maximum value it decreases as the

  8. Tailed pooled suppression subtractive hybridization (PSSH) adaptors do not alter efficiency.

    Science.gov (United States)

    Gerrish, Robert S; Gill, Steven R

    2010-11-01

    Suppression Subtractive Hybridization (SSH) and its derivative, Pooled Suppression Subtractive hybridization (PSSH), are powerful tools used to study variances larger than ~100 bp in prokaryotic genome structure. The initial steps involve ligating an oligonucleotide of known sequence (the "adaptor") to a fragmented genome to facilitate amplification, subtraction and downstream sequencing. SSH results in the creation of a library of unique DNA fragments which have been traditionally analyzed via Sanger sequencing. Numerous next generation sequencing technologies have entered the market yet SSH is incompatible with these platforms. This is due to the high level of sequence conservation of the oligonucleotide used for SSH. This rigid adherence is partly because it has yet to be determined if alteration of this oligonucleotide will have a deleterious impact on subtraction efficiency. The subtraction occurs when non-unique fragments are inhibited by a secondary self-pairing structure which requires exact nucleotide sequence. We determine if appending custom sequence to the 5' terminal ends of these oligonucleotides during the nested PCR stages of PSSH will reduce subtraction efficiency. We compare a pool of ten S. aureus clinical isolates with a standard PSSH and custom tailed-PSSH. We detected no statistically significant difference between their subtraction efficiencies. Our observations suggest that the adaptor's terminal ends may be labeled during the nested PCR step. This produces libraries labeled with custom sequence. This does not lead to loss of subtraction efficiency and would be invaluable for groups wishing to combine SSH or PSSH with their own downstream applications, such as a high throughput sequencing platform.

  9. Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

    Science.gov (United States)

    Caruccio, Nicholas

    2011-01-01

    DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

  10. Identification of CMS as a cytosolic adaptor of the human pTalpha chain involved in pre-TCR function.

    Science.gov (United States)

    Navarro, María N; Nusspaumer, Gretel; Fuentes, Patricia; González-García, Sara; Alcain, Juan; Toribio, María L

    2007-12-15

    The T-cell receptor beta (TCRbeta)/pre-TCRalpha (pTalpha) pre-TCR complex (pre-TCR) signals the expansion and differentiation of de-veloping thymocytes. Functional pro-perties of the pre-TCR rely on its unique pTalpha chain, which suggests the participation of specific intracellular adaptors. However, pTalpha-interacting molecules remain unknown. Here, we identified a polyproline-arginine sequence in the human pTalpha cytoplasmic tail that interacted in vitro with SH3 domains of the CIN85/CMS family of adaptors, and mediated the recruitment of multiprotein complexes involving all (CMS, CIN85, and CD2BP3) members. Supporting the physiologic relevance of this interaction, we found that 1 such adaptor, CMS, interacted in vivo with human pTalpha, and its expression was selectively up-regulated during human thymopoiesis in pre-TCR-activated thymocytes. Upon activation, pre-TCR clustering was induced, and CMS and polymerized actin were simultaneously recruited to the pre-TCR activation site. CMS also associated via its C-terminal region to the actin cytoskeleton in the endocytic compartment, where it colocalized with internalized pTalpha in traffic to lysosomal degradation. Notably, deletion of the pTalpha CIN85/CMS-binding motif impaired pre-TCR-mediated Ca(2+) mobilization and NFAT transcriptional activity, and precluded activation induced by overexpression of a CMS-SH3 N-terminal mutant. These results provide the first molecular evidence for a pTalpha intracellular adaptor involved in pre-TCR function.

  11. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  12. Lymphocytes and the Dap12 adaptor are key regulators of osteoclast activation associated with gonadal failure.

    Directory of Open Access Journals (Sweden)

    Adrienne Anginot

    Full Text Available Bone resorption by osteoclasts is necessary to maintain bone homeostasis. Osteoclast differentiation from hematopoietic progenitors and their activation depend on M-CSF and RANKL, but also requires co-stimulatory signals acting through receptors associated with DAP12 and FcRgamma adaptors. Dap12 mutant mice (KDelta75 are osteopetrotic due to inactive osteoclasts but, surprisingly, these mice are more sensitive than WT mice to bone loss following an ovariectomy. Because estrogen withdrawal is known to disturb bone mass, at least in part, through lymphocyte interaction, we looked at the role of mature lymphocytes on osteoclastogenesis and bone mass in the absence of functional DAP12. Lymphocytes were found to stimulate an early osteoclast differentiation response from Dap12-deficient progenitors in vitro. In vivo, Rag1-/- mice lacking mature lymphocytes did not exhibit any bone phenotype, but lost their bone mass after ovariectomy like KDelta75 mice. KDelta75;Rag1-/- double mutant female mice exhibited a more severe osteopetrosis than Dap12-deficient animals but lost their bone mass after ovariectomy, like single mutants. These results suggest that both DAP12 and mature lymphocytes act synergistically to maintain bone mass under physiological conditions, while playing similar but not synergistic co-stimulatory roles in protecting bone loss after gonadal failure. Thus, our data support a role for lymphocytes during osteoclast differentiation and suggest that they may function as accessory cells when regular osteoclast function is compromised.

  13. IL-1β-Induced Downregulation of the Multifunctional PDZ Adaptor PDZK1 Is Attenuated by ERK Inhibition, RXRα, or PPARα Stimulation in Enterocytes

    Science.gov (United States)

    Luo, Min; Yeruva, Sunil; Liu, Yongjian; Chodisetti, Giriprakash; Riederer, Brigitte; Menon, Manoj B.; Tachibana, Keisuke; Doi, Takefumi; Seidler, Ursula E.

    2017-01-01

    Background: The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients. Aim and Methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-α, IFN-γ, and IL-1β, and analysing PDZK1 promotor activity, mRNA and protein expression. Results: IL-1β was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1β-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-β downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1β mediated downregulation of PDZK1, while NF-κB, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXRα and PPARα was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1β-treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPARα-agonist GW7647 stimulated PDZK1 mRNA and protein expression and attenuated IL-1β-mediated inhibition. Conclusions: The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1β-mediated RXRα and PPARα repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy.

  14. Fission yeast arrestin-related trafficking adaptor, Arn1/Any1, is ubiquitinated by Pub1 E3 ligase and regulates endocytosis of Cat1 amino acid transporter

    Directory of Open Access Journals (Sweden)

    Akio Nakashima

    2014-05-01

    Full Text Available The Tsc1–Tsc2 complex homologous to human tuberous sclerosis complex proteins governs amino acid uptake by regulating the expression and intracellular distribution of amino acid transporters in Schizosaccharomyces pombe. Here, we performed a genetic screening for molecules that are involved in amino acid uptake and found Arn1 (also known as Any1. Arn1 is homologous to ART1, an arrestin-related trafficking adaptor (ART in Saccharomyces cerevisiae, and contains a conserved arrestin motif, a ubiquitination site, and two PY motifs. Overexpression of arn1+ confers canavanine resistance on cells, whereas its disruption causes hypersensitivity to canavanine. We also show that Arn1 regulates endocytosis of the Cat1 amino acid transporter. Furthermore, deletion of arn1+ suppresses a defect of amino acid uptake and the aberrant Cat1 localization in tsc2Δ. Arn1 interacts with and is ubiquitinated by the Pub1 ubiquitin ligase, which is necessary to regulate Cat1 endocytosis. Cat1 undergoes ubiquitinations on lysine residues within the N-terminus, which are mediated, in part, by Arn1 to determine Cat1 localization. Correctively, Arn1 is an ART in S. pombe and contributes to amino acid uptake through regulating Cat1 endocytosis in which Tsc2 is involved.

  15. Hydrological Modeling Reproducibility Through Data Management and Adaptors for Model Interoperability

    Science.gov (United States)

    Turner, M. A.

    2015-12-01

    Because of a lack of centralized planning and no widely-adopted standards among hydrological modeling research groups, research communities, and the data management teams meant to support research, there is chaos when it comes to data formats, spatio-temporal resolutions, ontologies, and data availability. All this makes true scientific reproducibility and collaborative integrated modeling impossible without some glue to piece it all together. Our Virtual Watershed Integrated Modeling System provides the tools and modeling framework hydrologists need to accelerate and fortify new scientific investigations by tracking provenance and providing adaptors for integrated, collaborative hydrologic modeling and data management. Under global warming trends where water resources are under increasing stress, reproducible hydrological modeling will be increasingly important to improve transparency and understanding of the scientific facts revealed through modeling. The Virtual Watershed Data Engine is capable of ingesting a wide variety of heterogeneous model inputs, outputs, model configurations, and metadata. We will demonstrate one example, starting from real-time raw weather station data packaged with station metadata. Our integrated modeling system will then create gridded input data via geostatistical methods along with error and uncertainty estimates. These gridded data are then used as input to hydrological models, all of which are available as web services wherever feasible. Models may be integrated in a data-centric way where the outputs too are tracked and used as inputs to "downstream" models. This work is part of an ongoing collaborative Tri-state (New Mexico, Nevada, Idaho) NSF EPSCoR Project, WC-WAVE, comprised of researchers from multiple universities in each of the three states. The tools produced and presented here have been developed collaboratively alongside watershed scientists to address specific modeling problems with an eye on the bigger picture of

  16. The Toll-Like receptor adaptor TRIF contributes to otitis media pathogenesis and recovery

    Directory of Open Access Journals (Sweden)

    Pak Kwang

    2009-08-01

    Full Text Available Abstract Background Toll-like receptor (TLR signalling is crucial for innate immune responses to infection. The involvement of TLRs in otitis media (OM, the most prevalent childhood disease in developed countries, has been implicated by studies in middle ear cell lines, by association studies of TLR-related gene polymorphisms, and by altered OM in mice bearing mutations in TLR genes. Activated TLRs signal via two alternative intracellular signaling molecules with differing effects; MyD88 (Myeloid differentiation primary response gene 88 inducing primarily interleukin expression and TRIF (Tir-domain-containing adaptor inducing interferon β mediating type I interferon (IFN expression. We tested the hypothesis that TRIF and type I IFN signaling play a role in OM, using a murine model of OM induced by non-typeable Haemophilus influenzae (NTHi. The ME inflammatory response to NTHi was examined in wild-type (WT and TRIF-/- mice by qPCR, gene microarray, histopathology and bacterial culture. Results Expression of TRIF mRNA was only modesty enhanced during OM, but both type I IFN signalling genes and type I IFN-inducible genes were significantly up-regulated in WT mice. TRIF-deficient mice showed reduced but more persistent mucosal hyperplasia and less leukocyte infiltration into the ME in response to NTHi infection than did WT animals. Viable bacteria could be cultured from MEs of TRIF-/- mice for much longer in the course of disease than was the case for middle ears of WT mice. Conclusion Our results demonstrate that activation of TRIF/type I IFN responses is important in both the pathogenesis and resolution of NTHi-induced OM.

  17. The TIR-domain containing adaptor TRAM is required for TLR7 mediated RANTES production.

    Directory of Open Access Journals (Sweden)

    Enda Shevlin

    Full Text Available Toll-like receptor 7 (TLR7 plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM(-/- murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-β, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNβ, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNβ and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM(-/- cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity.

  18. Negative regulation of the endocytic adaptor disabled-2 (Dab2) in mitosis.

    Science.gov (United States)

    Chetrit, David; Barzilay, Lior; Horn, Galit; Bielik, Tom; Smorodinsky, Nechama I; Ehrlich, Marcelo

    2011-02-18

    Mitotic cells undergo extensive changes in shape and size through the altered regulation and function of their membrane trafficking machinery. Disabled 2 (Dab2), a multidomain cargo-specific endocytic adaptor and a mediator of signal transduction, is a potential integrator of trafficking and signaling. Dab2 binds effectors of signaling and trafficking that localize to different intracellular compartments. Thus, differential localization is a putative regulatory mechanism of Dab2 function. Furthermore, Dab2 is phosphorylated in mitosis and is thus regulated in the cell cycle. However, a detailed description of the intracellular localization of Dab2 in the different phases of mitosis and an understanding of the functional consequences of its phosphorylation are lacking. Here, we show that Dab2 is progressively displaced from the membrane in mitosis. This phenomenon is paralleled by a loss of co-localization with clathrin. Both phenomena culminate in metaphase/anaphase and undergo partial recovery in cytokinesis. Treatment with 2-methoxyestradiol, which arrests cells at the spindle assembly checkpoint, induces the same effects observed in metaphase cells. Moreover, 2-methoxyestradiol also induced Dab2 phosphorylation and reduced Dab2/clathrin interactions, endocytic vesicle motility, clathrin exchange dynamics, and the internalization of a receptor endowed with an NPXY endocytic signal. Serine/threonine to alanine mutations, of residues localized to the central region of Dab2, attenuated its phosphorylation, reduced its membrane displacement, and maintained its endocytic abilities in mitosis. We propose that the negative regulation of Dab2 is part of an accommodation of the cell to the altered physicochemical conditions prevalent in mitosis, aimed at allowing endocytic activity throughout the cell cycle.

  19. A library of 7TM receptor C-terminal tails - Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, A.; Sondergaard, B.P.; Ersbøll, Bjarne Kjær

    2004-01-01

    -coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed......Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...

  20. Functional analyses of Src-like adaptor (SLA), a glucocorticoid-regulated gene in acute lymphoblastic leukemia.

    Science.gov (United States)

    Mansha, Muhammad; Carlet, Michela; Ploner, Christian; Gruber, Georg; Wasim, Muhammad; Wiegers, Gerrit Jan; Rainer, Johannes; Geley, Stephan; Kofler, Reinhard

    2010-04-01

    Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and are used in the therapy of lymphoid malignancies. SLA (Src-like-adaptor), an inhibitor of T- and B-cell receptor signaling, is a promising candidate derived from expression profiling analyses in children with acute lymphoblastic leukemia (ALL). Over-expression and knock-down experiments in ALL in vitro model revealed that transgenic SLA alone had no effect on survival or cell cycle progression, nor did it affect sensitivity to, or kinetics of, GC-induced apoptosis. Although SLA is a prominent GC response gene, it does not seem to contribute to the anti-leukemic effects of GC.

  1. Downstream Toll-like receptor signaling mediates adaptor-specific cytokine expression following focal cerebral ischemia

    Directory of Open Access Journals (Sweden)

    Bolanle Famakin

    2012-07-01

    Full Text Available Abstract Background Deletion of some Toll-like receptors (TLRs affords protection against cerebral ischemia, but disruption of their known major downstream adaptors does not. To determine whether compensation in the production of downstream effectors by one pathway when the other is disrupted can explain these findings, we examined cytokine/chemokine expression and inflammatory infiltrates in wild-type (WT, MyD88−/− and TRIF-mutant mice following permanent middle cerebral artery occlusion (pMCAO. Methods Cytokine/chemokine expression was measured with a 25-plex bead array in the serum and brains of all three groups of mice at baseline (no surgery/naïve and at 3 hours and 24 hours following pMCAO. Brain inflammatory and neutrophil infiltrates were examined 24 hours following pMCAO. Results IL-6, keratinocyte chemoattractant (KC, granulocyte colony-stimulating factor (G-CSF and IL-10 were significantly decreased in MyD88−/− mice compared to WT mice following pMCAO. Significantly, decreased levels of the neutrophil chemoattractants KC and G-CSF corresponded with a trend toward fewer neutrophils in the brains of MyD88−/− mice. IP-10 was significantly decreased when either pathway was disrupted. MIP-1α was significantly decreased in TRIF-mutant mice, consistent with TRIF-dependent production. MyD88−/− mice showed elevations of a number of Th2 cytokines, such as IL-13, at baseline, which became significantly decreased following pMCAO. Conclusions Both MyD88 and TRIF mediate pathway-specific cytokine production following focal cerebral ischemia. Our results also suggest a compensatory Th2-type skew at baseline in MyD88−/− mice and a paradoxical switch to a Th1 phenotype following focal cerebral ischemia. The MyD88 pathway directs the expression of neutrophil chemoattractants following cerebral ischemia.

  2. Advances on the research of adaptor SARM of Toll like receptor%TLR接头蛋白SARM研究进展

    Institute of Scientific and Technical Information of China (English)

    李涛; 徐元宏; 熊自忠

    2012-01-01

    SARM是最后一个发现的TLR接头蛋白,也是唯一具有抑制作用的接头蛋白,SARM的功能在不同物种和不同研究体系中结果差异较大,本文综述了人和小鼠中SARM的基因定位、基因结构和表达情况,并综述了线虫、鲎、文昌鱼、斑马鱼、小鼠和人等不同物种中SARM功能研究的最新进展.%SARM is the last adaptor of Toll like receptor to be found and a unique adaptor to possess inhibitory action. The function of SARM is largely difference in disparate species and research system. This paper reviews the gene location, gene structure and gene expression of SARM in human beings and mice and the advancement of SARM function research in Caenorhabditis elegans, horseshoe crab, amphioxus, zebra fish, mouse and human beings.

  3. Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-GMP binding.

    Science.gov (United States)

    Ouyang, Songying; Song, Xianqiang; Wang, Yaya; Ru, Heng; Shaw, Neil; Jiang, Yan; Niu, Fengfeng; Zhu, Yanping; Qiu, Weicheng; Parvatiyar, Kislay; Li, Yang; Zhang, Rongguang; Cheng, Genhong; Liu, Zhi-Jie

    2012-06-29

    STING is an essential signaling molecule for DNA and cyclic di-GMP (c-di-GMP)-mediated type I interferon (IFN) production via TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) pathway. It contains an N-terminal transmembrane region and a cytosolic C-terminal domain (CTD). Here, we describe crystal structures of STING CTD alone and complexed with c-di-GMP in a unique binding mode. The strictly conserved aa 153-173 region was shown to be cytosolic and participated in dimerization via hydrophobic interactions. The STING CTD functions as a dimer and the dimerization was independent of posttranslational modifications. Binding of c-di-GMP enhanced interaction of a shorter construct of STING CTD (residues 139-344) with TBK1. This suggests an extra TBK1 binding site, other than serine 358. This study provides a glimpse into the unique architecture of STING and sheds light on the mechanism of c-di-GMP-mediated TBK1 signaling.

  4. MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Larsen, Morten K; Tuck, Simon; Færgeman, Nils J.

    2006-01-01

    The budding and fission of vesicles during membrane trafficking requires many proteins, including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydro...

  5. Homer proteins in Ca²⁺ entry.

    Science.gov (United States)

    Jardin, Isaac; López, José J; Berna-Erro, Alejandro; Salido, Ginés M; Rosado, Juan A

    2013-06-01

    The Homer family of proteins consists of three adaptor proteins, Homer1, Homer2 and Homer3, each with various isoforms. Homer1 family presents an EVH1 domain, a coiled coil domain and two leucine zipper domains. Homer proteins regulate a number of Ca2+-handling proteins, including transient receptor potential channels and other Ca2+-permeable channels, ionotropic and metabotropic glutamate receptors, shank scaffolding proteins or endoplasmic reticulum Ca2+ release channels. This review article focuses on the association of Homer 1 proteins with Ca2+-handling proteins and their role on intracellular Ca2+-homeostasis.

  6. Ascent Heating Thermal Analysis on the Spacecraft Adaptor (SA) Fairings and the Interface with the Crew Launch Vehicle (CLV)

    Science.gov (United States)

    Wang, Xiao-Yen; Yuko, James; Motil, Brian

    2009-01-01

    When the crew exploration vehicle (CEV) is launched, the spacecraft adaptor (SA) fairings that cover the CEV service module (SM) are exposed to aero heating. Thermal analysis is performed to compute the fairing temperatures and to investigate whether the temperatures are within the material limits for nominal ascent aero heating case. Heating rates from Thermal Environment (TE) 3 aero heating analysis computed by engineers at Marshall Space Flight Center (MSFC) are used in the thermal analysis. Both MSC Patran 2007r1b/Pthermal and C&R Thermal Desktop 5.1/Sinda models are built to validate each other. The numerical results are also compared with those reported by Lockheed Martin (LM) and show a reasonably good agreement.

  7. A Patient-Controlled Analgesia Adaptor to Mitigate Postsurgical Pain for Combat Casualties With Multiple Limb Amputation: A Case Series.

    Science.gov (United States)

    Pasquina, Paul F; Isaacson, Brad M; Johnson, Elizabeth; Rhoades, Daniel S; Lindholm, Mark P; Grindle, Garrett G; Cooper, Rory A

    2016-08-01

    The use of explosive armaments during Operation Iraqi Freedom, Operation Enduring Freedom, and Operation New Dawn has resulted in a significant number of injured U.S. service members. These weapons often generate substantial extremity trauma requiring multiple surgical procedures to preserve life, limb, and restore function. For those individuals who require multiple surgeries, the use of patient-controlled analgesia (PCA) devices can be an effective way to achieve adequate pain management and promote successful rehabilitation and recovery during inpatient treatment. A subpopulation of patients are unable to independently control a PCA device because of severe multiple limb dysfunction and/or loss. In response to the needs of these patients, our team designed and developed a custom adaptor to assist service members who would otherwise not be able to use a PCA. Patient feedback of the device indicated a positive response, improved independence, and overall satisfaction during inpatient hospitalization.

  8. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

    Science.gov (United States)

    Bondage, Devanand D; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-07-05

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity.

  9. Lipid raft-dependent FcepsilonRI ubiquitination regulates receptor endocytosis through the action of ubiquitin binding adaptors.

    Directory of Open Access Journals (Sweden)

    Rosa Molfetta

    Full Text Available The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcepsilonRI expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcepsilonRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcepsilonRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.

  10. Association of Dishevelled with the clathrin AP-2 adaptor is required for Frizzled endocytosis and planar cell polarity signaling.

    Science.gov (United States)

    Yu, Anan; Rual, Jean-François; Tamai, Keiko; Harada, Yuko; Vidal, Marc; He, Xi; Kirchhausen, Tomas

    2007-01-01

    Upon activation by Wnt, the Frizzled receptor is internalized in a process that requires the recruitment of Dishevelled. We describe a novel interaction between Dishevelled2 (Dvl2) and micro2-adaptin, a subunit of the clathrin adaptor AP-2; this interaction is required to engage activated Frizzled4 with the endocytic machinery and for its internalization. The interaction of Dvl2 with AP-2 requires simultaneous association of the DEP domain and a peptide YHEL motif within Dvl2 with the C terminus of micro2. Dvl2 mutants in the YHEL motif fail to associate with micro2 and AP-2, and prevent Frizzled4 internalization. Corresponding Xenopus Dishevelled mutants show compromised ability to interfere with gastrulation mediated by the planar cell polarity (PCP) pathway. Conversely, a Dvl2 mutant in its DEP domain impaired in PCP signaling exhibits defective AP-2 interaction and prevents the internalization of Frizzled4. We suggest that the direct interaction of Dvl2 with AP-2 is important for Frizzled internalization and Frizzled/PCP signaling.

  11. Adapting for endocytosis: Roles for endocytic sorting adaptors in directing neural development.

    Directory of Open Access Journals (Sweden)

    Chan Choo eYap

    2015-04-01

    Full Text Available Proper cortical development depends on the orchestrated actions of a multitude of guidance receptors and adhesion molecules and their downstream signaling. The levels of these receptors on the surface and their precise locations can greatly affect guidance outcomes. Trafficking of receptors to a particular surface locale and removal by endocytosis thus feed crucially into the final guidance outcomes. In addition, endocytosis of receptors can affect downstream signaling (both quantitatively and qualitatively and regulated endocytosis of guidance receptors is thus an important component of ensuring proper neural development. We will discuss the cell biology of regulated endocytosis and the impact on neural development. We focus our discussion on endocytic accessory proteins (such as numb and disabled and how they regulate endocytosis and subsequent post-endocytic trafficking of their cognate receptors (such as Notch, TrkB, β-APP, VLDLR, and ApoER2.

  12. Adapting for endocytosis: roles for endocytic sorting adaptors in directing neural development.

    Science.gov (United States)

    Yap, Chan Choo; Winckler, Bettina

    2015-01-01

    Proper cortical development depends on the orchestrated actions of a multitude of guidance receptors and adhesion molecules and their downstream signaling. The levels of these receptors on the surface and their precise locations can greatly affect guidance outcomes. Trafficking of receptors to a particular surface locale and removal by endocytosis thus feed crucially into the final guidance outcomes. In addition, endocytosis of receptors can affect downstream signaling (both quantitatively and qualitatively) and regulated endocytosis of guidance receptors is thus an important component of ensuring proper neural development. We will discuss the cell biology of regulated endocytosis and the impact on neural development. We focus our discussion on endocytic accessory proteins (EAPs) (such as numb and disabled) and how they regulate endocytosis and subsequent post-endocytic trafficking of their cognate receptors (such as Notch, TrkB, β-APP, VLDLR, and ApoER2).

  13. DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes.

    Science.gov (United States)

    Hoang, Ha Thi; Schlager, Max A; Carter, Andrew P; Bullock, Simon L

    2017-02-28

    Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein's core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

  14. Interplay of Polarity Proteins and GTPases in T-Lymphocyte Function

    OpenAIRE

    Ivan Fung; Russell, Sarah M.; Jane Oliaro

    2012-01-01

    Polarity refers to the asymmetric distribution of different cellular components within a cell and is central to many cell functions. In T-cells, polarity regulates the activation, migration, and effector function of cytotoxic T-cells (CTLs) during an immune response. The regulation of asymmetric cell division by polarity proteins may also dictate CTL effector and memory differentiation following antigen presentation. Small GTPases, along with their associated polarity and adaptor proteins, ar...

  15. Development of novel on-chip, customer-design spiral biasing adaptor on for Si drift detectors and detector arrays for X-ray and nuclear physics experiments

    Science.gov (United States)

    Li, Zheng; Chen, Wei

    2014-11-01

    A novel on-chip, customer-design spiral biasing adaptor (SBA) has been developed. A single SBA is used for biasing a Si drift detector (SDD) and SDD array. The use of an SBA reduces the biasing current. This paper shows the calculation of the geometry of an SBA and an SDD to get the best drift field in the SDD and SDD array. Prototype SBAs have been fabricated to verify the concept. Electrical measurements on these SBAs are in agreement with the expectations. The new SDD array with an SBA can be used for X-ray detection and in nuclear physics experiments.

  16. Development of novel on-chip, customer-design spiral biasing adaptor on for Si drift detectors and detector arrays for X-ray and nuclear physics experiments

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zheng, E-mail: lizheng@xtu.edu.cn [School of Materials, Optoelectronics and Physics, Xiangtan University, Xiangtan, Hunan 411105 (China); Chen, Wei [Brookhaven National Laboratory, Upton, NY 11973 (United States)

    2014-11-21

    A novel on-chip, customer-design spiral biasing adaptor (SBA) has been developed. A single SBA is used for biasing a Si drift detector (SDD) and SDD array. The use of an SBA reduces the biasing current. This paper shows the calculation of the geometry of an SBA and an SDD to get the best drift field in the SDD and SDD array. Prototype SBAs have been fabricated to verify the concept. Electrical measurements on these SBAs are in agreement with the expectations. The new SDD array with an SBA can be used for X-ray detection and in nuclear physics experiments.

  17. Expression of Chicken Toll-Like Receptors and Signal Adaptors in Spleen and Cecum of Young Chickens Infected with Eimeria tenella

    Institute of Scientific and Technical Information of China (English)

    ZHOU Zuo-yong; HU Shi-jun; WANG Zhi-ying; GUO Zhi-li; QIN Bo; NIE Kui

    2014-01-01

    Toll-like receptors (TLRs) are a group of highly conserved molecules which initiate the innate immune response to pathogens by recognizing structural motifs of microbes. Understanding the changes in chicken Toll-like receptors (ChTLRs) and signal adaptors expression that occur with Eimeria tenella infection will help to elucidate the molecular basis of immune control of coccidiosis caused by Eimeria. The present study detected the dynamic changes in the expression of ChTLRs and associated signal adaptors in the spleen and cecum of E. tenella-infected chickens during the early stage of infection. The results showed that the expression peak for ChTLRs, MyD88 and TRIF occurred at 12 h post-infection (hpi), ChTLR3, ChTLR15 and MyD88 mRNA expression in the spleen of E. tenella infected chickens were signiifcantly higher (P<0.05) than that of negative control chickens, and there were similar tendencies of these molecules expression in the cecum and spleen of E. tenella-infected chickens. The expression of MyD88 was upregulated at four time points in the cecum of E. tenella-infected chickens. The results of this study indicate that ChTLR3, ChTLR15 and MyD88 play a role in young chickens infected with E. tenella.

  18. Ubiquitin in the peroxisomal protein import pathway.

    Science.gov (United States)

    Francisco, Tânia; Rodrigues, Tony A; Pinto, Manuel P; Carvalho, Andreia F; Azevedo, Jorge E; Grou, Cláudia P

    2014-03-01

    PEX5 is the shuttling receptor for newly synthesized peroxisomal matrix proteins. Alone, or with the help of an adaptor protein, this receptor binds peroxisomal matrix proteins in the cytosol and transports them to the peroxisomal membrane docking/translocation module (DTM). The interaction between cargo-loaded PEX5 and the DTM ultimately results in its insertion into the DTM with the concomitant translocation of the cargo protein across the organelle membrane. PEX5 is not consumed in this event; rather it is dislocated back into the cytosol so that it can promote additional rounds of protein transportation. Remarkably, the data collected in recent years indicate that dislocation is preceded by monoubiquitination of PEX5 at a conserved cysteine residue. This mandatory modification is not the only type of ubiquitination occurring at the DTM. Indeed, several findings suggest that defective receptors jamming the DTM are polyubiquitinated and targeted to the proteasome for degradation.

  19. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shi, T.; Niepel, M.; McDermott, J. E.; Gao, Y.; Nicora, C. D.; Chrisler, W. B.; Markillie, L. M.; Petyuk, V. A.; Smith, R. D.; Rodland, K. D.; Sorger, P. K.; Qian, W. -J.; Wiley, H. S.

    2016-07-12

    It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundance of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.

  20. Intrinsically disordered proteins drive membrane curvature

    Science.gov (United States)

    Busch, David J.; Houser, Justin R.; Hayden, Carl C.; Sherman, Michael B.; Lafer, Eileen M.; Stachowiak, Jeanne C.

    2015-07-01

    Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.

  1. The origin of polynucleotide-directed protein synthesis

    Science.gov (United States)

    Orgel, Leslie E.

    1989-01-01

    If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that at first the simple attachment of amino acids to the 2'(3') termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

  2. ITSN protein family: regulation of diversity, role in signalling and pathology

    OpenAIRE

    Tsyba L. O.; Dergai M. V.; Skrypkina I. Ya.; Nikolaienko O. V.; Dergai O. V.; Kropyvko S. V.; Novokhatska O. V.; Morderer D. Ye.; Gryaznova T. A.; Gubar O. S.; Rynditch A. V.

    2013-01-01

    Adaptor/scaffold proteins of the intersectin (ITSN) family are important components of endocytic and signalling complexes. They coordinate trafficking events with actin cytoskeleton rearrangements and modulate the activity of a variety of signalling pathways. In this review, we present our results as a part of recent findings on the function of ITSNs, the role of alternative splicing in the generation of ITSN1 diversity and the potential relevance of ITSNs for neurodegenerative diseases, cancer.

  3. ITSN protein family: regulation of diversity, role in signalling and pathology

    Directory of Open Access Journals (Sweden)

    Tsyba L. O.

    2013-05-01

    Full Text Available Adaptor/scaffold proteins of the intersectin (ITSN family are important components of endocytic and signalling complexes. They coordinate trafficking events with actin cytoskeleton rearrangements and modulate the activity of a variety of signalling pathways. In this review, we present our results as a part of recent findings on the function of ITSNs, the role of alternative splicing in the generation of ITSN1 diversity and the potential relevance of ITSNs for neurodegenerative diseases, cancer.

  4. Alix serves as an adaptor that allows human parainfluenza virus type 1 to interact with the host cell ESCRT system.

    Directory of Open Access Journals (Sweden)

    Jim Boonyaratanakornkit

    Full Text Available The cellular ESCRT (endosomal sorting complex required for transport system functions in cargo-sorting, in the formation of intraluminal vesicles that comprise multivesicular bodies (MVB, and in cytokinesis, and this system can be hijacked by a number of enveloped viruses to promote budding. The respiratory pathogen human parainfluenza virus type I (HPIV1 encodes a nested set of accessory C proteins that play important roles in down-regulating viral transcription and replication, in suppressing the type I interferon (IFN response, and in suppressing apoptosis. Deletion or mutation of the C proteins attenuates HPIV1 in vivo, and such mutants are being evaluated preclinically and clinically as vaccines. We show here that the C proteins interact and co-localize with the cellular protein Alix, which is a member of the class E vacuolar protein sorting (Vps proteins that assemble at endosomal membranes into ESCRT complexes. The HPIV1 C proteins interact with the Bro1 domain of Alix at a site that is also required for the interaction between Alix and Chmp4b, a subunit of ESCRT-III. The C proteins are ubiquitinated and subjected to proteasome-mediated degradation, but the interaction with AlixBro1 protects the C proteins from degradation. Neither over-expression nor knock-down of Alix expression had an effect on HPIV1 replication, although this might be due to the large redundancy of Alix-like proteins. In contrast, knocking down the expression of Chmp4 led to an approximately 100-fold reduction in viral titer during infection with wild-type (WT HPIV1. This level of reduction was similar to that observed for the viral mutant, P(C- HPIV1, in which expression of the C proteins were knocked out. Chmp4 is capable of out-competing the HPIV1 C proteins for binding Alix. Together, this suggests a possible model in which Chmp4, through Alix, recruits the C proteins to a common site on intracellular membranes and facilitates budding.

  5. The cullin protein family.

    Science.gov (United States)

    Sarikas, Antonio; Hartmann, Thomas; Pan, Zhen-Qiang

    2011-01-01

    Cullin proteins are molecular scaffolds that have crucial roles in the post-translational modification of cellular proteins involving ubiquitin. The mammalian cullin protein family comprises eight members (CUL1 to CUL7 and PARC), which are characterized by a cullin homology domain. CUL1 to CUL7 assemble multi-subunit Cullin-RING E3 ubiquitin ligase (CRL) complexes, the largest family of E3 ligases with more than 200 members. Although CUL7 and PARC are present only in chordates, other members of the cullin protein family are found in Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and yeast. A cullin protein tethers both a substrate-targeting unit, often through an adaptor protein, and the RING finger component in a CRL. The cullin-organized CRL thus positions a substrate close to the RING-bound E2 ubiquitin-conjugating enzyme, which catalyzes the transfer of ubiquitin to the substrate. In addition, conjugation of cullins with the ubiquitin-like molecule Nedd8 modulates activation of the corresponding CRL complex, probably through conformational regulation of the interactions between cullin's carboxy-terminal tail and CRL's RING subunit. Genetic studies in several model organisms have helped to unravel a multitude of physiological functions associated with cullin proteins and their respective CRLs. CRLs target numerous substrates and thus have an impact on a range of biological processes, including cell growth, development, signal transduction, transcriptional control, genomic integrity and tumor suppression. Moreover, mutations in CUL7 and CUL4B genes have been linked to hereditary human diseases.

  6. Translocator protein-mediated pharmacology of cholesterol transport and steroidogenesis.

    Science.gov (United States)

    Papadopoulos, Vassilios; Aghazadeh, Yasaman; Fan, Jinjiang; Campioli, Enrico; Zirkin, Barry; Midzak, Andrew

    2015-06-15

    Steroidogenesis begins with cholesterol transfer into mitochondria through the transduceosome, a complex composed of cytosolic proteins that include steroidogenesis acute regulatory protein (STAR), 14-3-3 adaptor proteins, and the outer mitochondrial membrane proteins Translocator Protein (TSPO) and Voltage-Dependent Anion Channel (VDAC). TSPO is a drug- and cholesterol-binding protein found at particularly high levels in steroid synthesizing cells. Its aberrant expression has been linked to cancer, neurodegeneration, neuropsychiatric disorders and primary hypogonadism. Brain steroids serve as local regulators of neural development and excitability. Reduced levels of these steroids have been linked to depression, anxiety and neurodegeneration. Reduced serum testosterone is common among subfertile young men and aging men, and is associated with depression, metabolic syndrome and reduced sexual function. Although testosterone-replacement therapy is available, there are undesired side-effects. TSPO drug ligands have been proposed as therapeutic agents to regulate steroid levels in the brain and testis.

  7. How anchoring proteins shape pain.

    Science.gov (United States)

    Fischer, Michael J M; McNaughton, Peter A

    2014-09-01

    Cellular responsiveness to external stimuli can be altered by extracellular mediators which activate membrane receptors, in turn signalling to the intracellular space via calcium, cyclic nucleotides, membrane lipids or enzyme activity. These signalling events trigger a cascade leading to an effector which can be a channel, an enzyme or a transcription factor. The effectiveness of these intracellular events is enhanced when they are maintained in close proximity by anchoring proteins, which assemble complexes of signalling molecules such as kinases together with their targets, and in this way enhance both the speed and the precision of intracellular signalling. The A kinase anchoring protein (AKAP) family are adaptor proteins originally named for their ability to associate Protein Kinase A and its targets, but several other enzymes bound by AKAPs have now been found and a wide variety of target structures has been described. This review provides an overview of anchoring proteins involved in pain signalling. The key anchoring proteins and their ion channel targets in primary sensory neurons responding to painful stimuli (nociceptors) are discussed.

  8. Novel binding partners and differentially regulated phosphorylation sites clarify Eps8 as a multi-functional adaptor.

    Directory of Open Access Journals (Sweden)

    Debbie L Cunningham

    Full Text Available Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2, proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2, and proteins involved in receptor signalling (IRS4 and Shp2. Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.

  9. Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA

    DEFF Research Database (Denmark)

    Nielsen, Morten S; Gustafsen, Camilla; Madsen, Peder

    2007-01-01

    -formation with the amyloid precursor protein it downregulates generation of Alzheimer's disease-associated Abeta-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLA's trafficking using full-length and chimeric receptors and find that its...... established that the AP-1 adaptor complex is essential to SorLA's transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged...

  10. Suppressor of Cytokine Signaling (SOCS 5 utilises distinct domains for regulation of JAK1 and interaction with the adaptor protein Shc-1.

    Directory of Open Access Journals (Sweden)

    Edmond M Linossi

    Full Text Available Suppressor of Cytokine Signaling (SOCS5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR. Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2 autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.

  11. TLR signaling adaptor protein MyD88 in primary sensory neurons contributes to persistent inflammatory and neuropathic pain and neuroinflammation

    Science.gov (United States)

    Liu, Xing-Jun; Liu, Tong; Chen, Gang; Wang, Bing; Yu, Xiao-Lu; Yin, Cui; Ji, Ru-Rong

    2016-01-01

    Increasing evidence suggests that neuro-immune and neuro-glial interactions are critically involved in chronic pain sensitization. It is well studied how immune/glial mediators sensitize pain, but how sensory neurons control neuroinflammation remains unclear. We employed Myd88 conditional knockout (CKO) mice, in which Myd88 was deleted in sodium channel subunit Nav1.8-expressing primary sensory neurons, to examine the unique role of neuronal MyD88 in regulating acute and chronic pain, and possible underlying mechanisms. We found that baseline pain and the formalin induced acute inflammatory pain were intact in CKO mice. However, the late phase inflammatory pain following complete Freund’s adjuvant injection and the late phase neuropathic pain following chronic constriction injury (CCI), were reduced in CKO mice. CCI induced up-regulation of MyD88 and chemokine C-C motif ligand 2 expression in DRG neurons and macrophage infiltration into DRGs, and microglia activation in spinal dorsal horns in wild-type mice, but all these changes were compromised in CKO mice. Finally, the pain hypersensitivity induced by intraplantar IL-1β was reduced in CKO mice. Our findings suggest that MyD88 in primary sensory neurons plays an active role in regulating IL-1β signaling and neuroinflammation in the peripheral and the central nervous systems, and contributes to the maintenance of persistent pain. PMID:27312666

  12. Ubiquitination and degradation of CFTR by the E3 ubiquitin ligase MARCH2 through its association with adaptor proteins CAL and STX6.

    Science.gov (United States)

    Cheng, Jie; Guggino, William

    2013-01-01

    Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.

  13. Ubiquitin-Mediated Regulation of Endocytosis by Proteins of the Arrestin Family

    Directory of Open Access Journals (Sweden)

    Michel Becuwe

    2012-01-01

    Full Text Available In metazoans, proteins of the arrestin family are key players of G-protein-coupled receptors (GPCRS signaling and trafficking. Following stimulation, activated receptors are phosphorylated, thus allowing the binding of arrestins and hence an “arrest” of receptor signaling. Arrestins act by uncoupling receptors from G proteins and contribute to the recruitment of endocytic proteins, such as clathrin, to direct receptor trafficking into the endocytic pathway. Arrestins also serve as adaptor proteins by promoting the recruitment of ubiquitin ligases and participate in the agonist-induced ubiquitylation of receptors, known to have impact on their subcellular localization and stability. Recently, the arrestin family has expanded following the discovery of arrestin-related proteins in other eukaryotes such as yeasts or fungi. Surprisingly, most of these proteins are also involved in the ubiquitylation and endocytosis of plasma membrane proteins, thus suggesting that the role of arrestins as ubiquitin ligase adaptors is at the core of these proteins' functions. Importantly, arrestins are themselves ubiquitylated, and this modification is crucial for their function. In this paper, we discuss recent data on the intricate connections between arrestins and the ubiquitin pathway in the control of endocytosis.

  14. Assembling Fe/S-clusters and modifying tRNAs: ancient co-factors meet ancient adaptors.

    Science.gov (United States)

    Alfonzo, Juan D; Lukeš, Julius

    2011-06-01

    Trypanosoma brucei undergoes two clearly distinct develomental stages: in the insect vector (procyclic stage) the cells generate the bulk of their energy through respiration, whereas in the bloodstream of the mammalian host (bloodstream stage) they grow mostly glycolytically. Several mitochondrial respiratory proteins require iron-sulfur clusters for activity, and their activation coincides with developmental changes. Likewise some tRNA modification enzymes either require iron-sulfur clusters or use components of the iron-sulfur cluster assembly pathway for activity. These enzymes affect the anticodon loop of various tRNAs and can impact protein synthesis. Herein, the possibility of these pathways being integrated and exploited by T. brucei to carefully coordinate energy demands to translational rates in response to enviromental changes is examined.

  15. Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy.

    Science.gov (United States)

    Haack, Tobias B; Ignatius, Erika; Calvo-Garrido, Javier; Iuso, Arcangela; Isohanni, Pirjo; Maffezzini, Camilla; Lönnqvist, Tuula; Suomalainen, Anu; Gorza, Matteo; Kremer, Laura S; Graf, Elisabeth; Hartig, Monika; Berutti, Riccardo; Paucar, Martin; Svenningsson, Per; Stranneheim, Henrik; Brandberg, Göran; Wedell, Anna; Kurian, Manju A; Hayflick, Susan A; Venco, Paola; Tiranti, Valeria; Strom, Tim M; Dichgans, Martin; Horvath, Rita; Holinski-Feder, Elke; Freyer, Christoph; Meitinger, Thomas; Prokisch, Holger; Senderek, Jan; Wredenberg, Anna; Carroll, Christopher J; Klopstock, Thomas

    2016-09-01

    SQSTM1 (sequestosome 1; also known as p62) encodes a multidomain scaffolding protein involved in various key cellular processes, including the removal of damaged mitochondria by its function as a selective autophagy receptor. Heterozygous variants in SQSTM1 have been associated with Paget disease of the bone and might contribute to neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Using exome sequencing, we identified three different biallelic loss-of-function variants in SQSTM1 in nine affected individuals from four families with a childhood- or adolescence-onset neurodegenerative disorder characterized by gait abnormalities, ataxia, dysarthria, dystonia, vertical gaze palsy, and cognitive decline. We confirmed absence of the SQSTM1/p62 protein in affected individuals' fibroblasts and found evidence of a defect in the early response to mitochondrial depolarization and autophagosome formation. Our findings expand the SQSTM1-associated phenotypic spectrum and lend further support to the concept of disturbed selective autophagy pathways in neurodegenerative diseases.

  16. Protein-protein interactions within late pre-40S ribosomes.

    Directory of Open Access Journals (Sweden)

    Melody G Campbell

    Full Text Available Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps.

  17. Regulation of lifespan, metabolism, and stress responses by the Drosophila SH2B protein, Lnk.

    Directory of Open Access Journals (Sweden)

    Cathy Slack

    2010-03-01

    Full Text Available Drosophila Lnk is the single ancestral orthologue of a highly conserved family of structurally-related intracellular adaptor proteins, the SH2B proteins. As adaptors, they lack catalytic activity but contain several protein-protein interaction domains, thus playing a critical role in signal transduction from receptor tyrosine kinases to form protein networks. Physiological studies of SH2B function in mammals have produced conflicting data. However, a recent study in Drosophila has shown that Lnk is an important regulator of the insulin/insulin-like growth factor (IGF-1 signaling (IIS pathway during growth, functioning in parallel to the insulin receptor substrate, Chico. As this pathway also has an evolutionary conserved role in the determination of organism lifespan, we investigated whether Lnk is required for normal lifespan in Drosophila. Phenotypic analysis of mutants for Lnk revealed that loss of Lnk function results in increased lifespan and improved survival under conditions of oxidative stress and starvation. Starvation resistance was found to be associated with increased metabolic stores of carbohydrates and lipids indicative of impaired metabolism. Biochemical and genetic data suggest that Lnk functions in both the IIS and Ras/Mitogen activated protein Kinase (MapK signaling pathways. Microarray studies support this model, showing transcriptional feedback onto genes in both pathways as well as indicating global changes in both lipid and carbohydrate metabolism. Finally, our data also suggest that Lnk itself may be a direct target of the IIS responsive transcription factor, dFoxo, and that dFoxo may repress Lnk expression. We therefore describe novel functions for a member of the SH2B protein family and provide the first evidence for potential mechanisms of SH2B regulation. Our findings suggest that IIS signaling in Drosophila may require the activity of a second intracellular adaptor, thereby yielding fundamental new insights into the

  18. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway.

    Science.gov (United States)

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-07-12

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling.

  19. Differential splicing of the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC regulates inflammasomes

    Directory of Open Access Journals (Sweden)

    Rojanasakul Yon

    2010-05-01

    Full Text Available Abstract Background The apoptotic speck-like protein containing a caspase recruitment domain (ASC is the essential adaptor protein for caspase 1 mediated interleukin (IL-1β and IL-18 processing in inflammasomes. It bridges activated Nod like receptors (NLRs, which are a family of cytosolic pattern recognition receptors of the innate immune system, with caspase 1, resulting in caspase 1 activation and subsequent processing of caspase 1 substrates. Hence, macrophages from ASC deficient mice are impaired in their ability to produce bioactive IL-1β. Furthermore, we recently showed that ASC translocates from the nucleus to the cytosol in response to inflammatory stimulation in order to promote an inflammasome response, which triggers IL-1β processing and secretion. However, the precise regulation of inflammasomes at the level of ASC is still not completely understood. In this study we identified and characterized three novel ASC isoforms for their ability to function as an inflammasome adaptor. Methods To establish the ability of ASC and ASC isoforms as functional inflammasome adaptors, IL-1β processing and secretion was investigated by ELISA in inflammasome reconstitution assays, stable expression in THP-1 and J774A1 cells, and by restoring the lack of endogenous ASC in mouse RAW264.7 macrophages. In addition, the localization of ASC and ASC isoforms was determined by immunofluorescence staining. Results The three novel ASC isoforms, ASC-b, ASC-c and ASC-d display unique and distinct capabilities to each other and to full length ASC in respect to their function as an inflammasome adaptor, with one of the isoforms even showing an inhibitory effect. Consistently, only the activating isoforms of ASC, ASC and ASC-b, co-localized with NLRP3 and caspase 1, while the inhibitory isoform ASC-c, co-localized only with caspase 1, but not with NLRP3. ASC-d did not co-localize with NLRP3 or with caspase 1 and consistently lacked the ability to function as an

  20. CRACC-targeting Fc-fusion protein induces activation of NK cells and DCs and improves T cell immune responses to antigenic targets.

    Science.gov (United States)

    Aldhamen, Yasser A; Rastall, David P W; Chen, Weimin; Seregin, Sergey S; Pereira-Hicks, Cristiane; Godbehere, Sarah; Kaminski, Norbert E; Amalfitano, Andrea

    2016-06-08

    The CD2-like receptor activating cytotoxic cell (CRACC) receptor is a member of the SLAM family of receptors that are found on several types of immune cells. We previously demonstrated that increasing the abundance of the adaptor protein EAT-2 during vaccination enhanced innate and adaptive immune responses to vaccine antigens. Engagement of the CRACC receptor in the presence of the EAT-2 adaptor generally results in immune cell activation, while activating CRACC signaling in cells that lack EAT-2 adaptor inhibits their effector and regulatory functions. As EAT-2 is the only SAP adaptor that interacts with the CRACC receptor, we hypothesized that technologies that specifically modulate CRACC signaling during vaccination may also improve antigen specific adaptive immune responses. To test this hypothesis, we constructed a CRACC-targeting Fc fusion protein and included it in vaccination attempts. Indeed, mice co-vaccinated with the CRACC-Fc fusion protein and an adenovirus vaccine expressing the HIV-Gag protein had improved Gag-specific T cell responses, as compared to control mice. These responses are characterized by increased numbers of Gag-specific tetramer+ CD8+ T cells and increases in production of IFNγ, TNFα, and IL2, by Gag-specific CD8+ T cells. Moreover, our results revealed that use of the CRACC-Fc fusion protein enhances vaccine-elicited innate immune responses, as characterized by increased dendritic cells (DCs) maturation and IFNγ production from NK cells. This study highlights the importance of CRACC signaling during the induction of an immune response generally, and during vaccinations specifically, and also lends insight into the mechanisms underlying our prior results noting EAT-2-dependent improvements in vaccine efficacy.

  1. The MRL proteins: adapting cell adhesion, migration and growth.

    Science.gov (United States)

    Coló, Georgina P; Lafuente, Esther M; Teixidó, Joaquin

    2012-01-01

    MIG-10, RIAM and Lamellipodin (Lpd) are the founding members of the MRL family of multi-adaptor molecules. These proteins have common domain structures but display distinct functions in cell migration and adhesion, signaling, and in cell growth. The binding of RIAM with active Rap1 and with talin provides these MRL molecules with important regulatory roles on integrin-mediated cell adhesion and migration. Furthermore, RIAM and Lpd can regulate actin dynamics through their binding to actin regulatory Ena/VASP proteins. Recent data generated with the Drosophila MRL ortholog called Pico and with RIAM in melanoma cells indicate that these proteins can also regulate cell growth. As MRL proteins represent a relatively new family, many questions on their structure-function relationships remain unanswered, including regulation of their expression, post-translational modifications, new interactions, involvement in signaling and their knockout mice phenotype.

  2. Identification and isolation of stimulator of interferon genes (STING): an innate immune sensory and adaptor gene from camelids.

    Science.gov (United States)

    Premraj, A; Aleyas, A G; Nautiyal, B; Rasool, T J

    2013-10-01

    The mechanism by which type I interferon-mediated antiviral response is mounted by hosts against invading pathogen is an intriguing one. Of late, an endoplasmic reticulum transmembrane protein encoded by a gene called stimulator of interferon genes (STING) is implicated in the innate signalling pathways and has been identified and cloned in few mammalian species including human, mouse and pig. In this article, we report the identification of STING from three different species of a highly conserved family of mammals - the camelids. cDNAs encoding the STING of Old World camels - dromedary camel (Camelus dromedarius) and bactrian camel (Camelus bactrianus) and a New World camel - llama (Llama glama) were amplified using conserved primers and RACE. The complete STING cDNA of dromedary camel is 2171 bp long with a 706-bp 5' untranslated regions (UTR), an 1137-bp open reading frame (ORF) and a 328-bp 3' UTR. Sequence and phylogenetic analysis of the ORF of STING from these three camelids indicate high level of similarity among camelids and conservation of critical amino acid residues across different species. Quantitative real-time PCR analysis revealed high levels of STING mRNA expression in blood, spleen, lymph node and lung. The identification of camelid STING will help in better understanding of the role of this molecule in the innate immunity of the camelids and other mammals.

  3. Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

    OpenAIRE

    Tsytlonok, Maksym; Ibrahim, Shehu M.; Rowling, Pamela J.E.; Xu, Wenshu; Ruedas-Rama, Maria J.; Orte, Angel; Klenerman, David; Itzhaki, Laura S.

    2015-01-01

    Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unrav...

  4. Integration of the beta-catenin-dependent Wnt pathway with integrin signaling through the adaptor molecule Grb2.

    Directory of Open Access Journals (Sweden)

    Steve P Crampton

    Full Text Available BACKGROUND: THE COMPLEXITY OF WNT SIGNALING LIKELY STEMS FROM TWO SOURCES: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of beta-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA LRP6, Dvl2 or CA-beta-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of beta-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal Adhesion Kinase (FAK, and this is blocked by DN-Grb2. CONCLUSIONS/SIGNIFICANCE: These data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling.

  5. Improved cytotoxic T-lymphocyte immune responses to a tumor antigen by vaccines co-expressing the SLAM-associated adaptor EAT-2.

    Science.gov (United States)

    Aldhamen, Y A; Seregin, S S; Kousa, Y A; Rastall, D P W; Appledorn, D M; Godbehere, S; Schutte, B C; Amalfitano, A

    2013-10-01

    The signaling lymphocytic activation molecule-associated adaptor Ewing's sarcoma's-activated transcript 2 (EAT-2) is primarily expressed in dendritic cells, macrophages and natural killer cells. Including EAT-2 in a vaccination regimen enhanced innate and adaptive immune responses toward pathogen-derived antigens, even in the face of pre-existing vaccine immunity. Herein, we investigate whether co-vaccinations with two recombinant Ad5 (rAd5) vectors, one expressing the carcinoembryonic antigen (CEA) and one expressing EAT-2, can induce more potent CEA-specific cytotoxic T lymphocyte (CTL) and antitumor activity in the therapeutic CEA-expressing MC-38 tumor model. Our results suggest that inclusion of EAT-2 significantly alters the kinetics of Th1-biasing proinflammatory cytokine and chemokine responses, and enhances anti-CEA-specific CTL responses. As a result, rAd5-EAT2-augmented rAd5-CEA vaccinations are more efficient in eliminating CEA-expressing target cells as measured by an in vivo CTL assay. Administration of rAd5-EAT2 vaccines also reduced the rate of growth of MC-38 tumor growth in vivo. Also, an increase in MC-38 tumor cell apoptosis (as measured by hematoxylin and eosin staining, active caspase-3 and granzyme B levels within the tumors) was observed. These data provide evidence that more efficient, CEA-specific effector T cells are generated by rAd5 vaccines expressing CEA, when augmented by rAd5 vaccines expressing EAT-2, and this regimen may be a promising approach for cancer immunotherapy in general.

  6. Fit-to-Flow (F2F) interconnects: universal reversible adhesive-free microfluidic adaptors for lab-on-a-chip systems.

    Science.gov (United States)

    Chen, Arnold; Pan, Tingrui

    2011-02-21

    World-to-chip (macro-to-micro) interface continues to be one of the most complicated, ineffective, and unreliable components in the development of emerging lab-on-a-chip systems involving integrated microfluidic operations. A number of irreversible (e.g., adhesive gluing) and reversible techniques (e.g., press fitting) have attempted to provide dedicated fluidic passage from standard tubing to miniature on-chip devices, none of which completely addresses the above concerns. In this paper, we present standardized adhesive-free microfluidic adaptors, referred to as Fit-to-Flow (F2F) Interconnects, to achieve reliable hermetic seal, high-density tube packing, self-aligned plug-in, reworkable connectivity, straightforward scalability and expandability, and applicability to broad lab-on-a-chip platforms; analogous to the modular plug-and-play USB architecture employed in modern electronics. Specifically, two distinct physical packaging mechanisms are applied, with one utilizing induced tensile stress in elastomeric socket to establish reversible seal and the other using negative pressure to provide on demand vacuum shield, both of which can be adapted to a variety of experimental configurations. The non-leaking performance (up to 336 kPa) along with high tube-packing density (of 1 tube/mm(2)) and accurate self-guided alignment (of 10 μm) have been characterized. In addition, a 3D microfluidic mixer and a 6-level chemical gradient generator paired with the corresponding F2F Interconnects have been devised to illustrate the applicability of the universal fluidic connections to classic lab-on-a-chip operations.

  7. ShcC proteins: brain aging and beyond.

    Science.gov (United States)

    Sagi, Orli; Budovsky, Arie; Wolfson, Marina; Fraifeld, Vadim E

    2015-01-01

    To date, most studies of Shc family of signaling adaptor proteins have been focused on the near-ubiquitously expressed ShcA, indicating its relevance to age-related diseases and longevity. Although the role of the neuronal ShcC protein is much less investigated, accumulated evidence suggests its importance for neuroprotection against such aging-associated conditions as brain ischemia and oxidative stress. Here, we summarize more than decade of studies on the ShcC expression and function in normal brain, age-related brain pathologies and immune disorders with a focus on the interactions of ShcC with signaling proteins/pathways, and the possible implications of these interactions for changes associated with aging.

  8. AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

    OpenAIRE

    Neveu, Gregory; Ziv-Av, Amotz; Barouch-Bentov, Rina; Berkerman, Elena; Mulholland, Jon; Einav, Shirit

    2015-01-01

    Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the μ subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved ant...

  9. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-02

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  10. Prolongamento da vida pós-colheita de bananas-maçã submetidas ao 1-metilciclopropeno (1-MCP - qualidade sensorial e física Extension of postharvest life of 'Apple1 banana subjected to1-methylcyclopropene (1-MCP - sensory and physical quality

    Directory of Open Access Journals (Sweden)

    Ana Carla Marques Pinheiro

    2010-03-01

    Full Text Available Objetivou-se avaliar o efeito do 1-metilciclopropeno (1-MCP sobre a qualidade e extensão da vida pós-colheita de bananas-maçã. Frutas no grau 2 de coloração da casca foram tratadas com 50 nL.L-1 de 1-MCP durante 0 (controle, 6, 9, 12 e 24 horas. Posteriormente, foram armazenadas sob condição ambiente: 22± 2 ºC 85± 5% UR. Avaliaram-se as características sensoriais: aparência dos frutos no grau 4 (frutos mais verdes que amarelos e grau 7 (frutos amarelos com pontuações marrons de coloração da casca; descasque, aroma, sabor, firmeza da polpa e aspecto global nos frutos no grau 7, utilizando-se um teste de aceitabilidade através de escala hedônica de 9 pontos; e ainda a intenção de compra nos frutos nos graus 4 e 7, através de escala de 5 pontos. Avaliou-se também, visualmente, a extensão da vida pós-colheita, e, instrumentalmente, a coloração da casca e firmeza da polpa. Conclui-se que a aplicação de 50 nL.L-1 por um período de 12 horas é a mais adequada, pois promove extensão na vida pós-colheita de bananas-maçã, armazenadas em temperatura ambiente, em aproximadamente 11 dias, sem alterar a qualidade sensorial, a intenção de compra, coloração da casca e firmeza dos frutos quando maduros, comparados ao controle.The objective of this work was to evaluate the effect of 1-methylcyclopropene (1-MCP on the quality and extension of postharvest life of 'Apple1 banana. The fruit, at degree 2 of peel color was treated with 50 nL.L-1 of 1-MCP for 0 (control, 6, 9, 12 and 24 hours. Next, It was stored at room temperature: 22± 2 ºC 85± 5 per cent RH. The following sensory characteristics were evaluated: appearance of the fruit at degree 4 (greener than yellow and degree 7 (yellow with brown spots of peel color; peeling, aroma, flavor, firmness of pulp and global aspect of the fruit at degree 7, using a test of acceptability using the 9-point hedonic scale and also the intention of buying the fruit at degrees 4 and 7

  11. CB1 Cannabinoid Receptors and their Associated Proteins

    Science.gov (United States)

    Howlett, Allyn C.; Blume, Lawrence C.; Dalton, George D.

    2011-01-01

    CB1 receptors are G-protein coupled receptors (GPCRs) abundant in neurons, in which they modulate neurotransmission. The CB1 receptor influence on memory and learning is well recognized, and disease states associated with CB1 receptors are observed in addiction disorders, motor dysfunction, schizophrenia, and in bipolar, depression, and anxiety disorders. Beyond the brain, CB1 receptors also function in liver and adipose tissues, vascular as well as cardiac tissue, reproductive tissues and bone. Signal transduction by CB1 receptors occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPK), inhibit voltage-gated Ca2+ channels, activate K+ currents (Kir), and influence Nitric Oxide (NO) signaling. CB1 receptors are observed in internal organelles as well as plasma membrane. β-Arrestins, adaptor protein AP-3, and G-protein receptor-associated sorting protein 1 (GASP1) modulate cellular trafficking. Cannabinoid Receptor Interacting Protein 1a (CRIP1a) is an accessory protein whose function has not been delineated. Factor Associated with Neutral sphingomyelinase (FAN) regulates ceramide signaling. Such diversity in cellular signaling and modulation by interacting proteins suggests that agonists and allosteric modulators could be developed to specifically regulate unique, cell type-specific responses. PMID:20166926

  12. A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones

    Science.gov (United States)

    Siebert, Matthias; Böhme, Mathias A; Driller, Jan H; Babikir, Husam; Mampell, Malou M; Rey, Ulises; Ramesh, Niraja; Matkovic, Tanja; Holton, Nicole; Reddy-Alla, Suneel; Göttfert, Fabian; Kamin, Dirk; Quentin, Christine; Klinedinst, Susan; Andlauer, Till FM; Hell, Stefan W; Collins, Catherine A; Wahl, Markus C; Loll, Bernhard; Sigrist, Stephan J

    2015-01-01

    Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes. DOI: http://dx.doi.org/10.7554/eLife.06935.001 PMID:26274777

  13. Structure–function–folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights

    Indian Academy of Sciences (India)

    P M Krishna Mohan; Ramakrishna V Hosur

    2009-09-01

    The detailed characterization of the structure, dynamics and folding process of a protein is crucial for understanding the biological functions it performs. Modern biophysical and nuclear magnetic resonance (NMR) techniques have provided a way to obtain accurate structural and thermodynamic information on various species populated on the energy landscape of a given protein. In this context, we review here the structure–function–folding relationship of an important protein, namely, dynein light chain protein (DLC8). DLC8, the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions and dissociates into a pure monomer below pH 4. Cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface are anticipated to be playing crucial roles in the variety of functions the protein performs. NMR investigations have provided great insights into these aspects of DLC8 in recent years.

  14. The ulcerative colitis marker protein WAFL interacts with accessory proteins in endocytosis

    Directory of Open Access Journals (Sweden)

    You Fu Pan, Ing-Marie Viklund, Heng Hang Tsai, Sven Pettersson, Ichiro N. Maruyama

    2010-01-01

    Full Text Available Ulcerative colitis (UC is one of the major forms of inflammatory bowel disease with unknown cause. A molecular marker, WAFL, has recently been found to be up-regulated in the inflamed colonic mucosa of UC patients. Towards understanding biological function of WAFL, we analyzed proteins interacting with WAFL in HEK-293 cells by immunoprecipitation and mass spectrometry. Among four proteins found to specifically interact with WAFL, both KIAA0196 and KIAA1033 bind to α-appendage of the adaptor protein complex 2 (AP2, which acts as an interaction hub for accessory proteins in endocytosis mediated by clathrin-coated vesicle (CCV. The specific interaction between WAFL and KIAA0196 was also confirmed in human colorectal carcinoma HCT-116 cells by co-immunoprecipitation with specific antibodies. Meta-analyses of the databases of expressed genes suggest that the three genes are co-expressed in many tissues and cell types, and that their molecular function may be classified in the category of 'membrane traffic protein'. Therefore, these results suggest that WAFL may play an important role in endocytosis and subsequent membrane trafficking by interacting with AP2 through KIAA0196 and KIAA1033.

  15. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.

    Science.gov (United States)

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

  16. The tight junction protein ZO-2 blocks cell cycle progression and inhibits cyclin D1 expression.

    Science.gov (United States)

    Gonzalez-Mariscal, Lorenza; Tapia, Rocio; Huerta, Miriam; Lopez-Bayghen, Esther

    2009-05-01

    ZO-2 is an adaptor protein of the tight junction that belongs to the MAGUK protein family. ZO-2 is a dual localization protein that in sparse cultures is present at the cell borders and the nuclei, whereas in confluent cultures it is concentrated at the cell boundaries. Here we have studied whether ZO-2 is able to regulate the expression of cyclin D1 (CD1) and cell proliferation. We have demonstrated that ZO-2 negatively regulates CD1 transcription by interacting with c-Myc at an E box present in CD1 promoter. We have further found that ZO-2 transfection into epithelial MDCK cells triggers a diminished expression of CD1 protein and decreases the rate of cell proliferation in a wound-healing assay.

  17. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  18. Vaccine platforms combining circumsporozoite protein and potent immune modulators, rEA or EAT-2, paradoxically result in opposing immune responses.

    Directory of Open Access Journals (Sweden)

    Nathaniel J Schuldt

    Full Text Available BACKGROUND: Malaria greatly impacts the health and wellbeing of over half of the world's population. Promising malaria vaccine candidates have attempted to induce adaptive immune responses to Circumsporozoite (CS protein. Despite the inclusion of potent adjuvants, these vaccines have limited protective efficacy. Conventional recombinant adenovirus (rAd based vaccines expressing CS protein can induce CS protein specific immune responses, but these are essentially equivalent to those generated after use of the CS protein subunit based vaccines. In this study we combined the use of rAds expressing CS protein along with rAds expressing novel innate immune response modulating proteins in an attempt to significantly improve the induction of CS protein specific cell mediated immune (CMI responses. METHODS AND FINDINGS: BALB/cJ mice were co-vaccinated with a rAd vectors expressing CS protein simultaneous with a rAd expressing either TLR agonist (rEA or SLAM receptors adaptor protein (EAT-2. Paradoxically, expression of the TLR agonist uncovered a potent immunosuppressive activity inherent to the combined expression of the CS protein and rEA. Fortunately, use of the rAd vaccine expressing EAT-2 circumvented CS protein's suppressive activity, and generated a fivefold increase in the number of CS protein responsive, IFNγ secreting splenocytes, as well as increased the breadth of T cells responsive to peptides present in the CS protein. These improvements were positively correlated with the induction of a fourfold improvement in CS protein specific CTL functional activity in vivo. CONCLUSION: Our results emphasize the need for caution when incorporating CS protein into malaria vaccine platforms expressing or containing other immunostimulatory compounds, as the immunological outcomes may be unanticipated and/or counter-productive. However, expressing the SLAM receptors derived signaling adaptor EAT-2 at the same time of vaccination with CS protein can

  19. Identification of AOSC-binding proteins in neurons

    Institute of Scientific and Technical Information of China (English)

    LIU Ming; NIE Qin; XIN Xianliang; GENG Meiyu

    2008-01-01

    Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer's Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

  20. Regulation of cargo-selective endocytosis by dynamin 2 GTPase-activating protein girdin.

    Science.gov (United States)

    Weng, Liang; Enomoto, Atsushi; Miyoshi, Hiroshi; Takahashi, Kiyofumi; Asai, Naoya; Morone, Nobuhiro; Jiang, Ping; An, Jian; Kato, Takuya; Kuroda, Keisuke; Watanabe, Takashi; Asai, Masato; Ishida-Takagishi, Maki; Murakumo, Yoshiki; Nakashima, Hideki; Kaibuchi, Kozo; Takahashi, Masahide

    2014-09-17

    In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.

  1. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E;

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane......The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  2. mRNA and protein dataset of autophagy markers (LC3 and p62 in several cell lines

    Directory of Open Access Journals (Sweden)

    Rubén Gómez-Sánchez

    2016-06-01

    Full Text Available We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I to the autophagosomal membrane where it is lipidated (LC3-II and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates, (Klionsky et al., 2016 [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR. To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1 to block fusion of autophagosomes with lysosomes.

  3. mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines

    Science.gov (United States)

    Gómez-Sánchez, Rubén; Yakhine-Diop, Sokhna M.S.; Rodríguez-Arribas, Mario; Bravo-San Pedro, José M.; Martínez-Chacón, Guadalupe; Uribe-Carretero, Elisabet; Pinheiro de Castro, Diana C.J.; Pizarro-Estrella, Elisa; Fuentes, José M.; González-Polo, Rosa A.

    2016-01-01

    We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes. PMID:27054171

  4. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

    Science.gov (United States)

    Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

    2009-02-01

    Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

  5. Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1.

    Directory of Open Access Journals (Sweden)

    Kosuke Tsukamoto

    Full Text Available BACKGROUND: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293 for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. CONCLUSIONS/SIGNIFICANCE: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

  6. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers......, are reported. The aim is to depict how the elucidation of the interplay of structures requires the interplay of methods....

  7. The WW-HECT protein Smurf2 interacts with the Docking Protein NEDD9/HEF1 for Aurora A activation

    Directory of Open Access Journals (Sweden)

    Moore Finola E

    2010-09-01

    Full Text Available Abstract The multi-functional adaptor protein NEDD9/HEF1/Cas-L regulates cell motility, invasion and cell cycle progression, and plays key roles in cancer progression and metastasis. NEDD9 is localized to the centrosome and is required for activation of Aurora A kinase in mitosis. Here we demonstrate that the HECT-WW protein Smurf2 physically associates with NEDD9 and is required for the stability of NEDD9 protein. Smurf2 depletion results in a marked decrease in NEDD9 protein levels, by facilitating polyubiquitination and proteasomal degradation of NEDD9. Conversely, forced overexpression of Smurf2 results in upregulation of endogenous NEDD9 protein, confirming the role for Smurf2 in NEDD9 stability. Cells with Smurf2 depletion fail to activate Aurora A at the G2/M boundary, leading to a marked delay in mitotic entry. These observations suggest that the stable complex of Smurf2 and NEDD9 is required for timely entry into mitosis via Aurora A activation.

  8. Characterisation of the Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop).

    Science.gov (United States)

    Gitau, Grace W; Mandal, Pradipta; Blatch, Gregory L; Przyborski, Jude; Shonhai, Addmore

    2012-03-01

    Malaria is caused by Plasmodium species, whose transmission to vertebrate hosts is facilitated by mosquito vectors. The transition from the cold blooded mosquito vector to the host represents physiological stress to the parasite, and additionally malaria blood stage infection is characterised by intense fever periods. In recent years, it has become clear that heat shock proteins play an essential role during the parasite's life cycle. Plasmodium falciparum expresses two prominent heat shock proteins: heat shock protein 70 (PfHsp70) and heat shock protein 90 (PfHsp90). Both of these proteins have been implicated in the development and pathogenesis of malaria. In eukaryotes, Hsp70 and Hsp90 proteins are functionally linked by an essential adaptor protein known as the Hsp70-Hsp90 organising protein (Hop). In this study, recombinant P. falciparum Hop (PfHop) was heterologously produced in E. coli and purified by nickel affinity chromatography. Using specific anti-PfHop antisera, the expression and localisation of PfHop in P. falciparum was investigated. PfHop was shown to co-localise with PfHsp70 and PfHsp90 in parasites at the trophozoite stage. Gel filtration and co-immunoprecipitation experiments suggested that PfHop was present in a complex together with PfHsp70 and PfHsp90. The association of PfHop with both PfHsp70 and PfHsp90 suggests that this protein may mediate the functional interaction between the two chaperones.

  9. Screening of BRD7 interacting proteins by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    余鹰; 朱诗国; 张必成; 周鸣; 李小玲; 李桂源

    2002-01-01

    BRD7 gene is low or undetectably expressed in nasopharyngeal carcinoma tissue (NPC) and can obviously suppress the growth of NPC cell line HNE1. In this study, the proteins that interacted with BRD7 coding protein were screened by yeast two-hybrid system. The complete reading frame of BRD7 gene was subcloned into pAS2 vector (BRD7-BD). Then the human embryo brain cDNA library was screened by BRD7-BD bait. Eleven positive clones were obtained from 4.8×106 transformed clones. By sequencing directly, 6 interacting proteins of BRD7 coding protein were isolated (Bromodomain-containing 3 protein (BRD3), Bromodomain-containing 2 protein (BRD2), IκB kinase-beta, KIAA1375 protein, interleukin 7 and adaptor-related protein complex 3δ-1 subunit). These results suggest BRD7 protein might form heterogenous dimer or triplex polymer with BRD2 and/or BRD3 to participate in transcriptional regulation.

  10. Stress conditions promote yeast Gap1 permease ubiquitylation and down-regulation via the arrestin-like Bul and Aly proteins.

    Science.gov (United States)

    Crapeau, Myriam; Merhi, Ahmad; André, Bruno

    2014-08-01

    Gap1, the yeast general amino acid permease, is a convenient model for studying how the intracellular traffic of membrane transporters is regulated. Present at the plasma membrane under poor nitrogen supply conditions, it undergoes ubiquitylation, endocytosis, and degradation upon activation of the TORC1 kinase complex in response to an increase in internal amino acids. This down-regulation is stimulated by TORC1-dependent phosphoinhibition of the Npr1 kinase, resulting in activation by dephosphorylation of the arrestin-like Bul1 and Bul2 adaptors recruiting the Rsp5 ubiquitin ligase to Gap1. We report here that Gap1 is also down-regulated when cells are treated with the TORC1 inhibitor rapamycin or subjected to various stresses and that a lack of the Tco89 subunit of TORC1 causes constitutive Gap1 down-regulation. Both the Bul1 and Bul2 and the Aly1 and Aly2 arrestin-like adaptors of Rsp5 promote this down-regulation without undergoing dephosphorylation. Furthermore, they act via the C-terminal regions of Gap1 not involved in ubiquitylation in response to internal amino acids, whereas a Gap1 mutant altered in the N-terminal tail and resistant to ubiquitylation by internal amino acids is efficiently down-regulated under stress via the Bul and Aly adaptors. Although the Bul proteins mediate Gap1 ubiquitylation of two possible lysines, Lys-9 and Lys-16, the Aly proteins promote ubiquitylation of the Lys-16 residue only. This stress-induced pathway of Gap1 down-regulation targets other permeases as well, and it likely allows cells facing adverse conditions to retrieve amino acids from permease degradation.

  11. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.

    Science.gov (United States)

    Bashline, Logan; Li, Shundai; Zhu, Xiaoyu; Gu, Ying

    2015-10-13

    Cellulose biosynthesis is performed exclusively by plasma membrane-localized cellulose synthases (CESAs). Therefore, the trafficking of CESAs to and from the plasma membrane is an important mechanism for regulating cellulose biosynthesis. CESAs were recently identified as cargo proteins of the classic adaptor protein 2 (AP2) complex of the clathrin-mediated endocytosis (CME) pathway. The AP2 complex of the CME pathway is conserved in yeast, animals, and plants, and has been well-characterized in many systems. In contrast, the recently discovered TPLATE complex (TPC), which is proposed to function as a CME adaptor complex, is only conserved in plants and a few other eukaryotes. In this study, we discovered that the TWD40-2 protein, a putative member of the TPC, is also important for the endocytosis of CESAs. Genetic analysis between TWD40-2 and AP2M of the AP2 complex revealed that the roles of TWD40-2 in CME are both distinct from and cooperative with the AP2 complex. Loss of efficient CME in twd40-2-3 resulted in the unregulated overaccumulation of CESAs at the plasma membrane. In seedlings of twd40-2-3 and other CME-deficient mutants, a direct correlation was revealed between endocytic deficiency and cellulose content deficiency, highlighting the importance of controlled CESA endocytosis in regulating cellulose biosynthesis.

  12. Collapsin Response Mediator Protein-2 (CRMP2) is a Plausible Etiological Factor and Potential Therapeutic Target in Alzheimer's Disease: Comparison and Contrast with Microtubule-Associated Protein Tau.

    Science.gov (United States)

    Hensley, Kenneth; Kursula, Petri

    2016-04-15

    Alzheimer's disease (AD) has long been viewed as a pathology that must be caused either by aberrant amyloid-β protein precursor (AβPP) processing, dysfunctional tau protein processing, or a combination of these two factors. This is a reasonable assumption because amyloid-β peptide (Aβ) accumulation and tau hyperphosphorylation are the defining histological features in AD, and because AβPP and tau mutations can cause AD in humans or AD-like features in animal models. Nonetheless, other protein players are emerging that one can argue are significant etiological players in subsets of AD and potentially novel, druggable targets. In particular, the microtubule-associated protein CRMP2 (collapsin response mediator protein-2) bears striking analogies to tau and is similarly relevant to AD. Like tau, CRMP2 dynamically regulates microtubule stability; it is acted upon by the same kinases; collects similarly in neurofibrillary tangles (NFTs); and when sequestered in NFTs, complexes with critical synapse-stabilizing factors. Additionally, CRMP2 is becoming recognized as an important adaptor protein involved in vesicle trafficking, amyloidogenesis and autophagy, in ways that tau is not. This review systematically compares the biology of CRMP2 to that of tau in the context of AD and explores the hypothesis that CRMP2 is an etiologically significant protein in AD and participates in pathways that can be rationally engaged for therapeutic benefit.

  13. Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates.

    Science.gov (United States)

    Martin, Andreas; Baker, Tania A; Sauer, Robert T

    2008-02-29

    ClpX, an archetypal proteolytic AAA+ unfoldase, must engage the ssrA tags of appropriate substrates prior to ATP-dependent unfolding and translocation of the denatured polypeptide into ClpP for degradation. Here, specificity-transplant and disulfide-crosslinking experiments reveal that the ssrA tag interacts with different loops that form the top, middle, and lower portions of the central channel of the ClpX hexamer. Our results support a two-step binding mechanism, in which the top loop serves as a specificity filter and the remaining loops form a binding site for the peptide tag relatively deep within the pore. Crosslinking experiments suggest a staggered arrangement of pore loops in the hexamer and nucleotide-dependent changes in pore-loop conformations. This mechanism of initial tag binding would allow ATP-dependent conformational changes in one or more pore loops to drive peptide translocation, force unfolding, and mediate threading of the denatured protein through the ClpX pore.

  14. Nuclear import of the thyroid hormone receptor α1 is mediated by importin 7, importin β1, and adaptor importin α1.

    Science.gov (United States)

    Roggero, Vincent R; Zhang, Jibo; Parente, Laura E; Doshi, Yazdi; Dziedzic, Rose C; McGregor, Emma L; Varjabedian, Arev D; Schad, Sara E; Bondzi, Cornelius; Allison, Lizabeth A

    2016-01-01

    The thyroid hormone receptor α1 (TRα1) is a nuclear receptor for thyroid hormone that shuttles rapidly between the nucleus and cytoplasm. Our prior studies showed that nuclear import of TRα1 is directed by two nuclear localization signals, one in the N-terminal A/B domain and the other in the hinge domain. Here, we showed using in vitro nuclear import assays that TRα1 nuclear localization is temperature and energy-dependent and can be reconstituted by the addition of cytosol. In HeLa cells expressing green fluorescent protein (GFP)-tagged TRα1, knockdown of importin 7, importin β1 and importin α1 by RNA interference, or treatment with an importin β1-specific inhibitor, significantly reduced nuclear localization of TRα1, while knockdown of other importins had no effect. Coimmunoprecipitation assays confirmed that TRα1 interacts with importin 7, as well as importin β1 and the adapter importin α1, suggesting that TRα1 trafficking into the nucleus is mediated by two distinct pathways.

  15. Traffic of p24 Proteins and COPII Coat Composition Mutually Influence Membrane Scaffolding.

    Science.gov (United States)

    D'Arcangelo, Jennifer G; Crissman, Jonathan; Pagant, Silvere; Čopič, Alenka; Latham, Catherine F; Snapp, Erik L; Miller, Elizabeth A

    2015-05-18

    Eukaryotic protein secretion requires efficient and accurate delivery of diverse secretory and membrane proteins. This process initiates in the ER, where vesicles are sculpted by the essential COPII coat. The Sec13p subunit of the COPII coat contributes to membrane scaffolding, which enforces curvature on the nascent vesicle. A requirement for Sec13p can be bypassed when traffic of lumenally oriented membrane proteins is abrogated. Here we sought to further explore the impact of cargo proteins on vesicle formation. We show that efficient ER export of the p24 family of proteins is a major driver of the requirement for Sec13p. The scaffolding burden presented by the p24 complex is met in part by the cargo adaptor Lst1p, which binds to a subset of cargo, including the p24 proteins. We propose that the scaffolding function of Lst1p is required to generate vesicles that can accommodate difficult cargo proteins that include large oligomeric assemblies and asymmetrically distributed membrane proteins. Vesicles that contain such cargoes are also more dependent on scaffolding by Sec13p, and may serve as a model for large carrier formation in other systems.

  16. Rab Proteins and the Secretory Pathway: The Case of Rab18 in Neuroendocrine Cells

    Directory of Open Access Journals (Sweden)

    RAFAEL eVAZQUEZ-MARTINEZ

    2011-01-01

    Full Text Available The secretory pathway is a process characteristic of cells specialized in secretion such as endocrine cells and neurons. It consists of different stages that are dependent on specific transport of proteins in vesicular-tubular carriers. Biochemical analyses have unveiled a number of protein families that confer identity to carrier vesicles and specificity to their transport. Among them is the family of Rab proteins, Ras-like small GTPases that anchor to the surface of transport vesicles and participate in vesicle formation from the donor compartment, transport along cytoskeletal tracks, and docking and fusion with the acceptor compartment. All of these functions are accomplished through the recruitment of effector proteins, such as sorting adaptors, tethering factors, kinases, phosphatases and motors. The numerous Rab proteins have distinct subcellular distributions throughout the endomembrane system, which ensures efficient cargo transfer. Rab proteins act as molecular switches that alternate between a cytosolic GDP-bound, inactive form and a membrane-associated GTP-bound, active conformation. Cycling between inactive and active states is a highly regulated process that enables Rabs to confer spatio-temporal precision to the different stages through which a vesicle passes during its lifespan. This review focuses on our current knowledge on Rab functioning, from their structural features to the multiple regulatory proteins and effectors that control Rab activity and translate Rab function. Furthermore, we also summarize the information available on a particular Rab protein, Rab18, which has been linked to the control of secretory granule traffic in neuroendocrine cells.

  17. Estrogen effects on actin cytoskeletal and endocytic proteins associated with tubulobulbar complex disruption in rat testes.

    Science.gov (United States)

    Upadhyay, Rahul D; Kumar, Anita V; Sonawane, Shobha; Gaonkar, Reshma; Balasinor, Nafisa H

    2013-10-01

    Tubulobulbar complexes (TBCs), evaginations of mature spermatids, penetrate into the surrounding Sertoli cell cytoplasm of testis seminiferous epithelium during rat spermatogenesis. These structures prepare mature spermatids for their release into the seminiferous tubular lumen via a process called spermiation. Based on their functions of transient attachment and endocytosis, many actin-regulatory and endocytic proteins are associated with TBCs. Previously, exogenous 17β-estradiol administration to adult male rats showed spermiation failure that was attributed to TBC disruption. To determine the molecular basis of estrogen-induced TBC disruption, we examined the expressions and localizations of actin-regulatory proteins, endocytic proteins, Rho-GTPases, and phosphorylation in TBCs during sperm release. Results demonstrated absence of neural Wiscott Aldrich syndrome protein, cortactin, adaptor-related protein complex 2 sigma-1 subunit, dynamin 2, cell division control protein 42, and phosphocortactin in the concavity of spermatid head where TBCs are present without change in their protein expression levels. Absence of these proteins could have led to collapse of the TBC structure which is involved in its formation and function.

  18. Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways†

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A.; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-01-01

    Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome. PMID:16973564

  19. Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-10-01

    Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

  20. Protein Condensation

    Science.gov (United States)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  1. Growth-arrest-specific 7C protein inhibits tumor metastasis via the N-WASP/FAK/F-actin and hnRNP U/β-TrCP/β-catenin pathways in lung cancer

    OpenAIRE

    Tseng, Ruo-Chia; Chang, Jer-Wei; Mao, Jiou-Shan; Tsai, Charng-Dar; Wu, Pei-Chen; Lin, Cuei-Jyuan; Lu, Yi-Lin; Liao, Sheng-You; Cheng, Hung-Chi; Hsu, Han-Shui; Wang, Yi-Ching

    2015-01-01

    Growth-arrest-specific 7 (GAS7) belongs to a group of adaptor proteins that coordinate the actin cytoskeleton. Among human GAS7 isoforms, only GAS7C possesses a Src homology 3 domain. We report here that GAS7C acts as a migration suppressor and can serve as a prognostic biomarker in lung cancer. GAS7C overexpression reduces lung cancer migration, whereas GAS7C knockdown enhances cancer cell migration. Importantly, ectopically overexpressed GAS7C binds tightly with N-WASP thus inactivates the ...

  2. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

    Science.gov (United States)

    Jin, Takashi; Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M

    2010-11-01

    To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.

  3. Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum.

    Science.gov (United States)

    Mund, Thomas; Gewies, Andreas; Schoenfeld, Nicole; Bauer, Manuel K A; Grimm, Stefan

    2003-04-01

    We have isolated Spike, a novel and evolutionary conserved BH3-only protein. BH3-only proteins constitute a family of apoptosis inducers that mediate proapoptotic signals. In contrast to most proteins of this family, Spike was not found to be associated with mitochondria. Furthermore, unlike the known BH3-only proteins, Spike could not interact with all tested Bcl-2 family members, despite its BH3 domain being necessary for cell killing. Our findings indicate that Spike is localized to the endoplasmic reticulum. The endoplasmic reticulum is an organelle that has only recently been implicated in regulation of apoptosis. At this locale, Spike interacts with Bap31, an adaptor protein for pro-caspase-8 and Bcl-XL. In doing so, Spike is able to inhibit the formation of a complex between Bap31 and the antiapoptotic Bcl-XL protein. Furthermore, Spike transmits the signal of specific death receptors. Its down-regulation in certain tumors suggests that Spike may also play a role in tumorigenesis. Our findings add new insight for how BH3-only and antiapoptotic Bcl-2 proteins regulate cell death.

  4. Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Snakenborg, Detlef; Nielsen, Søren Skou

    2011-01-01

    A new microfluidic sample-preparation system is presented for the structural investigation of proteins using small-angle X-ray scattering (SAXS) at synchrotrons. The system includes hardware and software features for precise fluidic control, sample mixing by diffusion, automated X-ray exposure...... control, UV absorbance measurements and automated data analysis. As little as 15 l of sample is required to perform a complete analysis cycle, including sample mixing, SAXS measurement, continuous UV absorbance measurements, and cleaning of the channels and X-ray cell with buffer. The complete analysis...... cycle can be performed in less than 3 min. Bovine serum albumin was used as a model protein to characterize the mixing efficiency and sample consumption of the system. The N2 fragment of an adaptor protein (p120-RasGAP) was used to demonstrate how the device can be used to survey the structural space...

  5. MoVrp1, a putative verprolin protein, is required for asexual development and infection in the rice blast fungus Magnaporthe oryzae

    Science.gov (United States)

    Huang, Lin; Zhang, Shengpei; Yin, Ziyi; Liu, Muxing; Li, Bing; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2017-01-01

    Endocytosis is a crucial cellular process in eukaryotic cells which involves clathrin and/or adaptor proteins, lipid kinases, phosphatases and the actin cytoskeleton. Verprolin proteins, such as Vrp1 in Saccharomyces cerevisiae, are conserved family proteins that regulate actin binding and endocytosis. Here, we identified and characterized MoVrp1 as the yeast Vrp1 homolog in Magnaporthe oryzae. Deletion of the MoVRP1 gene resulted in defects in vegetative growth, asexual development, and infection of the host plant. The ∆Movrp1 mutants also exhibited decreased extracellular peroxidase and laccase activities and showed defects in colony pigmentation, hyphal surface hydrophobicity, cell wall integrity, autophagy, endocytosis, and secretion of avirulent effector. Our studies provided new evidences that MoVrp1 involved in actin cytoskeleton is important for growth, morphogenesis, cellular trafficking, and fungal pathogenesis. PMID:28117435

  6. In vitro nuclear interactome of the HIV-1 Tat protein.

    LENUS (Irish Health Repository)

    Gautier, Virginie W

    2009-01-01

    BACKGROUND: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. RESULTS: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. CONCLUSION: We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will

  7. Functions of Kinesin Superfamily Proteins in Neuroreceptor Trafficking

    Directory of Open Access Journals (Sweden)

    Na Wang

    2015-01-01

    Full Text Available Synaptic plasticity is widely regarded as the cellular basis of learning and memory. Understanding the molecular mechanism of synaptic plasticity has been one of center pieces of neuroscience research for more than three decades. It has been well known that the trafficking of α-amino-3-hydroxy-5-methylisoxazoloe-4-propionic acid- (AMPA- type, N-methyl-D-aspartate- (NMDA- type glutamate receptors to and from synapses is a key molecular event underlying many forms of synaptic plasticity. Kainate receptors are another type of glutamate receptors playing important roles in synaptic transmission. In addition, GABA receptors also play important roles in modulating the synaptic plasticity. Kinesin superfamily proteins (also known as KIFs transport various cargos in both anterograde and retrograde directions through the interaction with different adaptor proteins. Recent studies indicate that KIFs regulate the trafficking of NMDA receptors, AMPA receptors, kainate receptors, and GABA receptors and thus play important roles in neuronal activity. Here we review the essential functions of KIFs in the trafficking of neuroreceptor and synaptic plasticity.

  8. Asymmetric Inheritance of Aggregated Proteins and Age Reset in Yeast Are Regulated by Vac17-Dependent Vacuolar Functions

    Directory of Open Access Journals (Sweden)

    Sandra Malmgren Hill

    2016-07-01

    Full Text Available Age can be reset during mitosis in both yeast and stem cells to generate a young daughter cell from an aged and deteriorated one. This phenomenon requires asymmetry-generating genes (AGGs that govern the asymmetrical inheritance of aggregated proteins. Using a genome-wide imaging screen to identify AGGs in Saccharomyces cerevisiae, we discovered a previously unknown role for endocytosis, vacuole fusion, and the myosin-dependent adaptor protein Vac17 in asymmetrical inheritance of misfolded proteins. Overproduction of Vac17 increases deposition of aggregates into cytoprotective vacuole-associated sites, counteracts age-related breakdown of endocytosis and vacuole integrity, and extends replicative lifespan. The link between damage asymmetry and vesicle trafficking can be explained by a direct interaction between aggregates and vesicles. We also show that the protein disaggregase Hsp104 interacts physically with endocytic vesicle-associated proteins, such as the dynamin-like protein, Vps1, which was also shown to be required for Vac17-dependent sequestration of protein aggregates. These data demonstrate that two physiognomies of aging—reduced endocytosis and protein aggregation—are interconnected and regulated by Vac17.

  9. Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia

    Directory of Open Access Journals (Sweden)

    Julie eMcLean

    2015-04-01

    Full Text Available AIMS: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM. Results: One-hundred and fifty-eight proteins were confirmed in three biological replicates. Thirty-three were identified as secreted proteins, including 14-3-3 proteins, which are highly conserved adaptor proteins known to have over 200 binding partners. We confirmed the presence of extracellular 14-3-3 proteins in LCM via western blot and discovered that LCM contained less 14-3-3 content than media conditioned with C2C12 myotubes. Using a neutralizing antibody, we depleted extracellular 14-3-3 proteins in myotube culture medium, which resulted in diminished myosin content. We identified the proposed receptor for 14-3-3 proteins, CD13, in differentiated C2C12 myotubes and found that inhibiting CD13 via Bestatin also resulted in diminished myosin content. Conclusions: Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines and may signal via CD13 to help maintain muscle mass.

  10. Contamination of sequence databases with adaptor sequences

    Energy Technology Data Exchange (ETDEWEB)

    Yoshikawa, Takeo; Sanders, A.R.; Detera-Wadleigh, S.D. [National Institute of Mental Health, Bethesda, MD (United States)

    1997-02-01

    Because of the exponential increase in the amount of DNA sequences being added to the public databases on a daily basis, it has become imperative to identify sources of contamination rapidly. Previously, contaminations of sequence databases have been reported to alert the scientific community to the problem. These contaminations can be divided into two categories. The first category comprises host sequences that have been difficult for submitters to manage or control. Examples include anomalous sequences derived from Escherichia coli, which are inserted into the chromosomes (and plasmids) of the bacterial hosts. Insertion sequences are highly mobile and are capable of transposing themselves into plasmids during cloning manipulation. Another example of the first category is the infection with yeast genomic DNA or with bacterial DNA of some commercially available cDNA libraries from Clontech. The second category of database contamination is due to the inadvertent inclusion of nonhost sequences. This category includes incorporation of cloning-vector sequences and multicloning sites in the database submission. M13-derived artifacts have been common, since M13-based vectors have been widely used for subcloning DNA fragments. Recognizing this problem, the National Center for Biotechnology Information (NCBI) started to screen, in April 1994, all sequences directly submitted to GenBank, against a set of vector data retrieved from GenBank by use of key-word searches, such as {open_quotes}vector.{close_quotes} In this report, we present evidence for another sequence artifact that is widespread but that, to our knowledge, has not yet been reported. 11 refs., 1 tab.

  11. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  12. Expanding the substantial interactome of NEMO using protein microarrays.

    LENUS (Irish Health Repository)

    Fenner, Beau J

    2010-01-01

    Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.

  13. Protein Structure

    Science.gov (United States)

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  14. Degradation of phosphorylated p53 by viral protein-ECS E3 ligase complex.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Sato

    2009-07-01

    Full Text Available p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection.

  15. Phosphorylation by Dyrk1A of clathrin coated vesicle-associated proteins: identification of the substrate proteins and the effects of phosphorylation.

    Directory of Open Access Journals (Sweden)

    Noriko Murakami

    Full Text Available Dyrk1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and β-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [(32P]-labeling and immunological methods. The Dyrk1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, Dyrk1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by Dyrk1A. Surprisingly, the Dyrk1A-mediated phosphorylation of α- and β-adaptins led to dissociation of the AP2 complex, and released only β-adaptin from the CCVs. AP180 was phosphorylated by Dyrk1A also in the membrane-free fractions, but α- and β-adaptins were not. Dyrk1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that Dyrk1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, Dyrk1A promotes the uncoating process of endocytosed CCVs.

  16. IDENTIFICATION AND EXPRESSION ANALYSIS OF TWO 14-3-3 PROTEINS IN THE MOSQUITO Aedes aegypti, AN IMPORTANT ARBOVIRUSES VECTOR.

    Science.gov (United States)

    Trujillo-Ocampo, Abel; Cázares-Raga, Febe Elena; Celestino-Montes, Antonio; Cortés-Martínez, Leticia; Rodríguez, Mario H; Hernández-Hernández, Fidel de la Cruz

    2016-11-01

    The 14-3-3 proteins are evolutionarily conserved acidic proteins that form a family with several isoforms in many cell types of plants and animals. In invertebrates, including dipteran and lepidopteran insects, only two isoforms have been reported. 14-3-3 proteins are scaffold molecules that form homo- or heterodimeric complexes, acting as molecular adaptors mediating phosphorylation-dependent interactions with signaling molecules involved in immunity, cell differentiation, cell cycle, proliferation, apoptosis, and cancer. Here, we describe the presence of two isoforms of 14-3-3 in the mosquito Aedes aegypti, the main vector of dengue, yellow fever, chikungunya, and zika viruses. Both isoforms have the conserved characteristics of the family: two protein signatures (PS1 and PS2), an annexin domain, three serine residues, targets for phosphorylation (positions 58, 184, and 233), necessary for their function, and nine alpha helix-forming segments. By sequence alignment and phylogenetic analysis, we found that the molecules correspond to Ɛ and ζ isoforms (Aeae14-3-3ε and Aeae14-3-3ζ). The messengers and protein products were present in all stages of the mosquito life cycle and all the tissues analyzed, with a small predominance of Aeae14-3-3ζ except in the midgut and ovaries of adult females. The 14-3-3 proteins in female midgut epithelial cells were located in the cytoplasm. Our results may provide insights to further investigate the functions of these proteins in mosquitoes.

  17. Interferon-inducible p200-family protein IFI16, an innate immune sensor for cytosolic and nuclear double-stranded DNA: regulation of subcellular localization.

    Science.gov (United States)

    Veeranki, Sudhakar; Choubey, Divaker

    2012-01-01

    The interferon (IFN)-inducible p200-protein family includes structurally related murine (for example, p202a, p202b, p204, and Aim2) and human (for example, AIM2 and IFI16) proteins. All proteins in the family share a partially conserved repeat of 200-amino acid residues (also called HIN-200 domain) in the C-terminus. Additionally, most proteins (except the p202a and p202b proteins) also share a protein-protein interaction pyrin domain (PYD) in the N-terminus. The HIN-200 domain contains two consecutive oligosaccharide/oligonucleotide binding folds (OB-folds) to bind double stranded DNA (dsDNA). The PYD domain in proteins allows interactions with the family members and an adaptor protein ASC. Upon sensing cytosolic dsDNA, Aim2, p204, and AIM2 proteins recruit ASC protein to form an inflammasome, resulting in increased production of proinflammatory cytokines. However, IFI16 protein can sense cytosolic as well as nuclear dsDNA. Interestingly, the IFI16 protein contains a nuclear localization signal (NLS). Accordingly, the initial studies had indicated that the endogenous IFI16 protein is detected in the nucleus and within the nucleus in the nucleolus. However, several recent reports suggest that subcellular localization of IFI16 protein in nuclear versus cytoplasmic (or both) compartment depends on cell type. Given that the IFI16 protein can sense cytosolic as well as nuclear dsDNA and can initiate different innate immune responses (production of IFN-β versus proinflammatory cytokines), here we evaluate the experimental evidence for the regulation of subcellular localization of IFI16 protein in various cell types. We conclude that further studies are needed to understand the molecular mechanisms that regulate the subcellular localization of IFI16 protein.

  18. APC/C(Cdh1-mediated degradation of the F-box protein NIPA is regulated by its association with Skp1.

    Directory of Open Access Journals (Sweden)

    Christine von Klitzing

    Full Text Available NIPA (Nuclear Interaction Partner of Alk kinase is an F-box like protein that targets nuclear Cyclin B1 for degradation. Integrity and therefore activity of the SCF(NIPA E3 ligase is regulated by cell-cycle-dependent phosphorylation of NIPA, restricting substrate ubiquitination to interphase. Here we show that phosphorylated NIPA is degraded in late mitosis in an APC/C(Cdh1-dependent manner. Binding of the unphosphorylated form of NIPA to Skp1 interferes with binding to the APC/C-adaptor protein Cdh1 and therefore protects unphosphorylated NIPA from degradation in interphase. Our data thus define a novel mode of regulating APC/C-mediated ubiquitination.

  19. VESICULAR TRANSPORT. A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly.

    Science.gov (United States)

    Dodonova, S O; Diestelkoetter-Bachert, P; von Appen, A; Hagen, W J H; Beck, R; Beck, M; Wieland, F; Briggs, J A G

    2015-07-10

    Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.

  20. ATAD3B is a human embryonic stem cell specific mitochondrial protein, re-expressed in cancer cells, that functions as dominant negative for the ubiquitous ATAD3A.

    Science.gov (United States)

    Merle, Nicolas; Féraud, Olivier; Gilquin, Benoit; Hubstenberger, Arnaud; Kieffer-Jacquinot, Sylvie; Assard, Nicole; Bennaceur-Griscelli, Annelise; Honnorat, Jérôme; Baudier, Jacques

    2012-07-01

    Here we report on the identification of a human pluripotent embryonic stem cell (hESC) specific mitochondrial protein that is re-expressed in cancer cells, ATAD3B. ATAD3B belongs to the AAA+ ATPase ATAD3 protein family of mitochondrial proteins specific to multicellular eukaryotes. Using loss- and gain-of-function approaches, we show that ATAD3B associates with the ubiquitous ATAD3A species, negatively regulates the interaction of ATAD3A with matrix nucleoid complexes and contributes to a mitochondria fragmentation phenotype. We conclude that ATAD3B is a negative regulator of ATAD3A and may function as an adaptor of mitochondrial homeostasis and metabolism in hESCs and cancer cells.

  1. Interplay of Polarity Proteins and GTPases in T-Lymphocyte Function

    Directory of Open Access Journals (Sweden)

    Ivan Fung

    2012-01-01

    Full Text Available Polarity refers to the asymmetric distribution of different cellular components within a cell and is central to many cell functions. In T-cells, polarity regulates the activation, migration, and effector function of cytotoxic T-cells (CTLs during an immune response. The regulation of asymmetric cell division by polarity proteins may also dictate CTL effector and memory differentiation following antigen presentation. Small GTPases, along with their associated polarity and adaptor proteins, are critical for mediating the polarity changes necessary for T-cell activation and function, and in turn, are regulated by guanine exchange factors (GEFS and GTPase activating proteins (GAPS. For example, a novel GEF, dedicator of cytokinesis 8 (DOCK8 was recently identified as a regulator of immune cell function and mutations in DOCK8 have been detected in patients with severe combined immunodeficiency. Both B and T-cells from DOCK8 mutant mice form defective immunological synapses and have abnormal functions, in addition to impaired immune memory development. This paper will discuss the interplay between polarity proteins and GTPases, and their role in T-cell function.

  2. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    LENUS (Irish Health Repository)

    Gegenbauer, Kristina

    2013-11-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

  3. Differential expression of speckled POZ protein, SPOP: Putative regulation by miR-145

    Indian Academy of Sciences (India)

    Chiu-Jung Huang; Hsing-Yu Chen; Wan-Yi Lin; Kongbung Choo

    2014-06-01

    The speckle POZ protein, SPOP, is an adaptor of the Cul3-based ubiquitination process, and has been implicated in the carcinogenesis process. Despite recent elucidation of biological functions, regulation of SPOP gene expression has not been reported. In this study, the mRNA levels of the mouse SPOP (mSPOP) gene were first shown to vary noticeably in different tissues. However, the SPOP protein was detected in high abundance only in Purkinje cells of the cerebellum and seminiferous tubule of the testis, echoing previous reports of involvement of ubiquitination in neuron cells and in spermatogenesis. In other mouse tissues and human cancer cell lines analysed, only low SPOP protein levels were detected. The 3′-untranslated regions of both the mSPOP and human SPOP transcripts harbor a conserved putative miR-145 binding site (BS). In some tissues and cell lines, miR-145 and SPOP protein levels were in an inverse relationship suggesting miR-145 regulation. Luciferase assays of deletion and point mutation constructs of the miR-145 BS, and miR-145 induction by serum starvation that resulted in reduced endogenous SPOP levels provided further evidence that miR-145 is likely involved in post-transcriptional regulation of SPOP expression in selected tissues, and possibly with the participation of other miRNA species.

  4. Multiplex assay for live-cell monitoring of cellular fates of amyloid-β precursor protein (APP.

    Directory of Open Access Journals (Sweden)

    Maria Merezhko

    Full Text Available Amyloid-β precursor protein (APP plays a central role in pathogenesis of Alzheimer's disease. APP has a short half-life and undergoes complex proteolytic processing that is highly responsive to various stimuli such as changes in cellular lipid or energy homeostasis. Cellular trafficking of APP is controlled by its large protein interactome, including dozens of cytosolic adaptor proteins, and also by interactions with lipids. Currently, cellular regulation of APP is mostly studied based on appearance of APP-derived proteolytic fragments to conditioned media and cellular extracts. Here, we have developed a novel live-cell assay system based on several indirect measures that reflect altered APP trafficking and processing in cells. Protein-fragment complementation assay technology for detection of APP-BACE1 protein-protein interaction forms the core of the new assay. In a multiplex form, the assay can measure four endpoints: total cellular APP level, total secreted sAPP level in media, APP-BACE1 interaction in cells and in exosomes released by the cells. Functional validation of the assay with pharmacological and genetic tools revealed distinct patterns of cellular fates of APP, with immediate mechanistic implications. This new technology will facilitate functional genomics studies of late-onset Alzheimer's disease, drug discovery efforts targeting APP and characterization of the physiological functions of APP and its proteolytic fragments.

  5. TRIM32 protein modulates type I interferon induction and cellular antiviral response by targeting MITA/STING protein for K63-linked ubiquitination.

    Science.gov (United States)

    Zhang, Jing; Hu, Ming-Ming; Wang, Yan-Yi; Shu, Hong-Bing

    2012-08-17

    Viral infection activates several transcription factors including NF-κB and IRF3, which collaborate to induce type I interferons (IFNs) and innate antiviral response. MITA (also called STING) is a critical adaptor protein that links virus-sensing receptors to IRF3 activation upon infection by both RNA and DNA pathogens. Here we show that the E3 ubiquitin ligase tripartite motif protein 32 (TRIM32) ubiquitinated MITA and dramatically enhanced MITA-mediated induction of IFN-β. Overexpression of TRIM32 potentiated virus-triggered IFNB1 expression and cellular antiviral response. Consistently, knockdown of TRIM32 had opposite effects. TRIM32 interacted with MITA, and was located at the mitochondria and endoplasmic reticulum. TRIM32 targeted MITA for K63-linked ubiquitination at K20/150/224/236 through its E3 ubiquitin ligase activity, which promoted the interaction of MITA with TBK1. These findings suggest that TRIM32 is an important regulatory protein for innate immunity against both RNA and DNA viruses by targeting MITA for K63-linked ubiquitination and downstream activation.

  6. Identification of Max binding protein as a novel binding protein of Nck1 and characterization of its role in inhibiting human liver cancer SK-HEP-1 cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Qi; HUANG Tao; WANG Ya-feng; ZHANG Kun-sun; CHEN Dong; PENG Bao-gang

    2012-01-01

    Background The tendency of tumor cells to disperse throughout the liver is a distinct feature of hepatocellular carcinoma (HCC).Nck family adaptor proteins function to regulate actin cytoskeletal reorganization that leads to cell motility.We previously found that Max binding protein (MNT) was differentially expressed in HCC,and interacted with Nck1 by 2-DE.MNT is a protein member of the Myc/Max/Mad network which plays roles in cell proliferation,differentiation,and death.We investigated the effects of MNT on migration of human liver cancer SK-HEP-1 cells to study the migration regulatory role of MNT in HCC cells.Methods Interaction between MNT and Nck1 was further validated in hepatoma cells by GST-pull down assay and immunoprecipitation,siRNAs specific to MNT (MNT siRNA) were used to knockdown MNT expression.Western blotting,transwell assay were used to determine the migration potential of cells.Results Interaction between MNT and Nck1 was validated in hepatoma cells.MNT knockdown promoted the migration of human liver cancer SK-HEP-1 cells (P <0.01 ).Conclusion The results suggest that MNT,via interaction with Nck1,inhibits hepatoma cell migration.

  7. Gab Adapter Proteins as Therapeutic Targets for Hematologic Disease

    Directory of Open Access Journals (Sweden)

    Sheetal Verma

    2012-01-01

    Full Text Available The Grb-2 associated binder (Gab family of scaffolding/adaptor/docking proteins is a group of three molecules with significant roles in cytokine receptor signaling. Gabs possess structural motifs for phosphorylation-dependent receptor recruitment, Grb2 binding, and activation of downstream signaling pathways through p85 and SHP-2. In addition, Gabs participate in hematopoiesis and regulation of immune response which can be aberrantly activated in cancer and inflammation. The multifunctionality of Gab adapters might suggest that they would be too difficult to consider as candidates for “targeted” therapy. However, the one drug/one target approach is giving way to the concept of one drug/multiple target approach since few cancers are addicted to a single signaling molecule for survival and combination drug therapies can be problematic. In this paper, we cover recent findings on Gab multi-functionality, binding partners, and their role in hematological malignancy and examine the concept of Gab-targeted therapy.

  8. Whey Protein

    Science.gov (United States)

    ... Fraction de Lactosérum, Fraction de Petit-Lait, Goat Milk Whey, Goat Whey, Isolat de Protéine de Lactosérum, Isolat ... Lactosérum de Lait de Chèvre, MBP, Milk Protein, Milk Protein Isolate, Mineral Whey Concentrate, Proteínas del Suero de la Leche, Protéine ...

  9. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  10. Insight into the intermolecular recognition mechanism between Keap1 and IKKβ combining homology modelling, protein-protein docking, molecular dynamics simulations and virtual alanine mutation.

    Directory of Open Access Journals (Sweden)

    Zheng-Yu Jiang

    Full Text Available Degradation of certain proteins through the ubiquitin-proteasome pathway is a common strategy taken by the key modulators responsible for stress responses. Kelch-like ECH-associated protein-1(Keap1, a substrate adaptor component of the Cullin3 (Cul3-based ubiquitin E3 ligase complex, mediates the ubiquitination of two key modulators, NF-E2-related factor 2 (Nrf2 and IκB kinase β (IKKβ, which are involved in the redox control of gene transcription. However, compared to the Keap1-Nrf2 protein-protein interaction (PPI, the intermolecular recognition mechanism of Keap1 and IKKβ has been poorly investigated. In order to explore the binding pattern between Keap1 and IKKβ, the PPI model of Keap1 and IKKβ was investigated. The structure of human IKKβ was constructed by means of the homology modeling method and using reported crystal structure of Xenopus laevis IKKβ as the template. A protein-protein docking method was applied to develop the Keap1-IKKβ complex model. After the refinement and visual analysis of docked proteins, the chosen pose was further optimized through molecular dynamics simulations. The resulting structure was utilized to conduct the virtual alanine mutation for the exploration of hot-spots significant for the intermolecular interaction. Overall, our results provided structural insights into the PPI model of Keap1-IKKβ and suggest that the substrate specificity of Keap1 depend on the interaction with the key tyrosines, namely Tyr525, Tyr574 and Tyr334. The study presented in the current project may be useful to design molecules that selectively modulate Keap1. The selective recognition mechanism of Keap1 with IKKβ or Nrf2 will be helpful to further know the crosstalk between NF-κB and Nrf2 signaling.

  11. Insight into the intermolecular recognition mechanism between Keap1 and IKKβ combining homology modelling, protein-protein docking, molecular dynamics simulations and virtual alanine mutation.

    Science.gov (United States)

    Jiang, Zheng-Yu; Chu, Hong-Xi; Xi, Mei-Yang; Yang, Ting-Ting; Jia, Jian-Min; Huang, Jing-Jie; Guo, Xiao-Ke; Zhang, Xiao-Jin; You, Qi-Dong; Sun, Hao-Peng

    2013-01-01

    Degradation of certain proteins through the ubiquitin-proteasome pathway is a common strategy taken by the key modulators responsible for stress responses. Kelch-like ECH-associated protein-1(Keap1), a substrate adaptor component of the Cullin3 (Cul3)-based ubiquitin E3 ligase complex, mediates the ubiquitination of two key modulators, NF-E2-related factor 2 (Nrf2) and IκB kinase β (IKKβ), which are involved in the redox control of gene transcription. However, compared to the Keap1-Nrf2 protein-protein interaction (PPI), the intermolecular recognition mechanism of Keap1 and IKKβ has been poorly investigated. In order to explore the binding pattern between Keap1 and IKKβ, the PPI model of Keap1 and IKKβ was investigated. The structure of human IKKβ was constructed by means of the homology modeling method and using reported crystal structure of Xenopus laevis IKKβ as the template. A protein-protein docking method was applied to develop the Keap1-IKKβ complex model. After the refinement and visual analysis of docked proteins, the chosen pose was further optimized through molecular dynamics simulations. The resulting structure was utilized to conduct the virtual alanine mutation for the exploration of hot-spots significant for the intermolecular interaction. Overall, our results provided structural insights into the PPI model of Keap1-IKKβ and suggest that the substrate specificity of Keap1 depend on the interaction with the key tyrosines, namely Tyr525, Tyr574 and Tyr334. The study presented in the current project may be useful to design molecules that selectively modulate Keap1. The selective recognition mechanism of Keap1 with IKKβ or Nrf2 will be helpful to further know the crosstalk between NF-κB and Nrf2 signaling.

  12. Structure of a PE–PPE–EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    Science.gov (United States)

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-01-01

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed “type VII.” How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE–PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways. PMID:25275011

  13. p66Shc Aging Protein in Control of Fibroblasts Cell Fate

    Directory of Open Access Journals (Sweden)

    Mariusz R. Wieckowski

    2011-08-01

    Full Text Available Reactive oxygen species (ROS are wieldy accepted as one of the main factors of the aging process. These highly reactive compounds modify nucleic acids, proteins and lipids and affect the functionality of mitochondria in the first case and ultimately of the cell. Any agent or genetic modification that affects ROS production and detoxification can be expected to influence longevity. On the other hand, genetic manipulations leading to increased longevity can be expected to involve cellular changes that affect ROS metabolism. The 66-kDa isoform of the growth factor adaptor Shc (p66Shc has been recognized as a relevant factor to the oxygen radical theory of aging. The most recent data indicate that p66Shc protein regulates life span in mammals and its phosphorylation on serine 36 is important for the initiation of cell death upon oxidative stress. Moreover, there is strong evidence that apart from aging, p66Shc may be implicated in many oxidative stress-associated pathologies, such as diabetes, mitochondrial and neurodegenerative disorders and tumorigenesis. This article summarizes recent knowledge about the role of p66Shc in aging and senescence and how this protein can influence ROS production and detoxification, focusing on studies performed on skin and skin fibroblasts.

  14. Single-molecule FRET reveals hidden complexity in a protein energy landscape.

    Science.gov (United States)

    Tsytlonok, Maksym; Ibrahim, Shehu M; Rowling, Pamela J E; Xu, Wenshu; Ruedas-Rama, Maria J; Orte, Angel; Klenerman, David; Itzhaki, Laura S

    2015-01-06

    Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.

  15. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    Science.gov (United States)

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

  16. The F-BAR protein PSTPIP1 controls extracellular matrix degradation and filopodia formation in macrophages.

    Science.gov (United States)

    Starnes, Taylor W; Bennin, David A; Bing, Xinyu; Eickhoff, Jens C; Grahf, Daniel C; Bellak, Jason M; Seroogy, Christine M; Ferguson, Polly J; Huttenlocher, Anna

    2014-04-24

    PSTPIP1 is a cytoskeletal adaptor and F-BAR protein that has been implicated in autoinflammatory disease, most notably in the PAPA syndrome: pyogenic sterile arthritis, pyoderma gangrenosum, and acne. However, the mechanism by which PSTPIP1 regulates the actin cytoskeleton and contributes to disease pathogenesis remains elusive. Here, we show that endogenous PSTPIP1 negatively regulates macrophage podosome organization and matrix degradation. We identify a novel PSTPIP1-R405C mutation in a patient presenting with aggressive pyoderma gangrenosum. Identification of this mutation reveals that PSTPIP1 regulates the balance of podosomes and filopodia in macrophages. The PSTPIP1-R405C mutation is in the SRC homology 3 (SH3) domain and impairs Wiskott-Aldrich syndrome protein (WASP) binding, but it does not affect interaction with protein-tyrosine phosphatase (PTP)-PEST. Accordingly, WASP inhibition reverses the elevated F-actin content, filopodia formation, and matrix degradation induced by PSTPIP1-R405C. Our results uncover a novel role for PSTPIP1 and WASP in orchestrating different types of actin-based protrusions. Our findings implicate the cytoskeletal regulatory functions of PSTPIP1 in the pathogenesis of pyoderma gangrenosum and suggest that the cytoskeleton is a rational target for therapeutic intervention in autoinflammatory disease.

  17. The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling.

    Directory of Open Access Journals (Sweden)

    Alessia Soldano

    Full Text Available Wnt Planar Cell Polarity (PCP signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs are intensely investigated because of their link to Alzheimer's disease (AD. APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh by Abelson kinase (Abl. Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

  18. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware......Years of meticulous curation of scientific literature and increasingly reliable computational predictions have resulted in creation of vast databases of protein interaction data. Over the years, these repositories have become a basic framework in which experiments are analyzed and new directions...

  19. Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex.

    Directory of Open Access Journals (Sweden)

    Ziyan Wu

    Full Text Available The G protein-coupled receptor kinases (GRKs phosphorylate agonist occupied G protein-coupled receptors (GPCRs and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA-binding protein 1 (DDB1, an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex-dependent proteolysis pathway.

  20. The protein interaction network of a taxis signal transduction system in a Halophilic Archaeon

    Directory of Open Access Journals (Sweden)

    Schlesner Matthias

    2012-11-01

    Full Text Available Abstract Background The taxis signaling system of the extreme halophilic archaeon Halobacterium (Hbt. salinarum differs in several aspects from its model bacterial counterparts Escherichia coli and Bacillus subtilis. We studied the protein interactions in the Hbt. salinarum taxis signaling system to gain an understanding of its structure, to gain knowledge about its known components and to search for new members. Results The interaction analysis revealed that the core signaling proteins are involved in different protein complexes and our data provide evidence for dynamic interchanges between them. Fifteen of the eighteen taxis receptors (halobacterial transducers, Htrs can be assigned to four different groups depending on their interactions with the core signaling proteins. Only one of these groups, which contains six of the eight Htrs with known signals, shows the composition expected for signaling complexes (receptor, kinase CheA, adaptor CheW, response regulator CheY. From the two Hbt. salinarum CheW proteins, only CheW1 is engaged in signaling complexes with Htrs and CheA, whereas CheW2 interacts with Htrs but not with CheA. CheY connects the core signaling structure to a subnetwork consisting of the two CheF proteins (which build a link to the flagellar apparatus, CheD (the hub of the subnetwork, two CheC complexes and the receptor methylesterase CheB. Conclusions Based on our findings, we propose two hypotheses. First, Hbt. salinarum might have the capability to dynamically adjust the impact of certain Htrs or Htr clusters depending on its current needs or environmental conditions. Secondly, we propose a hypothetical feedback loop from the response regulator to Htr methylation made from the CheC proteins, CheD and CheB, which might contribute to adaptation analogous to the CheC/CheD system of B. subtilis.

  1. Protein Crystallization

    Science.gov (United States)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  2. An SH3 binding motif within the nucleocapsid protein of porcine reproductive and respiratory syndrome virus interacts with the host cellular signaling proteins STAMI, TXK, Fyn, Hck, and cortactin.

    Science.gov (United States)

    Kenney, Scott P; Meng, Xiang-Jin

    2015-06-02

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease, and has a complicated virus-host immunomodulation that often leads to a weak Th2 immune response and viral persistence. In this study, we identified a Src homology 3 (SH3) binding motif, PxxPxxP, that is conserved within the N protein of PRRSV strains. Subsequently, we identified five host cellular proteins [signal transducing adaptor molecule (STAM)I, TXK tyrosine kinase (TXK), protein tyrosine kinase fyn (Fyn), hematopoietic cell kinase (Hck), and cortactin] that interact with this SH3 motif. We demonstrated that binding of SH3 proteins with PRRSV N protein depends on at least one intact PxxP motif as disruption of P53 within the motif significantly reduced interaction of each of the 5 proteins. The first PxxP motif appears to be more important for STAMI-N protein interactions whereas the second PxxP motif was more important for Hck interaction. Both STAMI and Hck interactions with PRRSV N protein required an unhindered C-terminal domain as the interaction was only observed with STAMI and Hck proteins with N-terminal but not C-terminal fluorescent tags. We showed that the P56 residue within the SH3 motif is critical for virus lifecycle as mutation resulted in a loss of virus infectivity, however the P50 and P53 mutations did not abolish virus infectivity suggesting that these highly conserved proline residues within the SH3 motif may provide a selective growth advantage through interactions with the host rather than a vital functional element. These results have important implications in understanding PRRSV-host interactions.

  3. Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.

    Science.gov (United States)

    Alvarez-Curto, Elisa; Inoue, Asuka; Jenkins, Laura; Raihan, Sheikh Zahir; Prihandoko, Rudi; Tobin, Andrew B; Milligan, Graeme

    2016-12-30

    G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca(2+) in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signaling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins.

  4. Age-related decline of myelin proteins is highly correlated with activation of astrocytes and microglia in the rat CNS.

    Science.gov (United States)

    Xie, Fang; Zhang, Jiu-Cong; Fu, Han; Chen, Jun

    2013-11-01

    It has been shown that aging can greatly influence the integrity and ultrastructure of white matter and the myelin sheath; however, studies regarding the effects of aging on the expression of myelin proteins are still limited. In the present study, immunohistochemical mapping was used to investigate the overall expression of myelin basic protein (Mbp) and myelin oligodendrocyte glycoprotein (Mog) in the central nervous system (CNS) of rats in postnatal months 2, 5, 18 and 26. Astrocyte and microglia activation was also detected by glial fibrillary acidic protein (GFAP) or ionized calcium-binding adaptor molecule 1 (Iba1) staining and western blotting. A significant decline of Mbp and Mog was identified as a universal alteration in the CNS of aged rats. Aging also induced significant astrocyte and microglial activation. Correlation analysis indicated a negative correlation between the reduction of age‑related myelin proteins and glial activation in aging. This correlation of myelin breakdown and glial activation in aging may reveal new evidence in connecting the inflammation and myelin breakdown mechanism of age‑related neurodegenerative diseases.

  5. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics.

    Science.gov (United States)

    Dai, Jin; Niemi, Antti J; He, Jianfeng

    2016-07-28

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  6. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics

    Science.gov (United States)

    Dai, Jin; Niemi, Antti J.; He, Jianfeng

    2016-07-01

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  7. Structure-function analysis of the retinoblastoma tumor suppressor protein – is the whole a sum of its parts?

    Directory of Open Access Journals (Sweden)

    Dick Frederick A

    2007-09-01

    Full Text Available Abstract Biochemical analysis of the retinoblastoma protein's function has received considerable attention since it was cloned just over 20 years ago. During this time pRB has emerged as a key regulator of the cell division cycle and its ability to block proliferation is disrupted in the vast majority of human cancers. Much has been learned about the regulation of E2F transcription factors by pRB in the cell cycle. However, many questions remain unresolved and researchers continue to explore this multifunctional protein. In particular, understanding how its biochemical functions contribute to its role as a tumor suppressor remains to be determined. Since pRB has been shown to function as an adaptor molecule that links different proteins together, or to particular promoters, analyzing pRB by disrupting individual protein interactions holds tremendous promise in unraveling the intricacies of its function. Recently, crystal structures have reported how pRB interacts with some of its molecular partners. This information has created the possibility of rationally separating pRB functions by studying mutants that disrupt individual binding sites. This review will focus on literature that investigates pRB by isolating functions based on binding sites within the pocket domain. This article will also discuss the prospects for using this approach to further explore the unknown functions of pRB.

  8. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Karin [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden); Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Grawé, Jan [Department of Genetics and Pathology, Uppsala University, Uppsala 75185 (Sweden); McKinney-Freeman, Shannon L. [Department of Hematology, St. Jude Children' s Research Hospital, Memphis, TN 38105 (United States); Daley, George Q. [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Welsh, Michael, E-mail: michael.welsh@mcb.uu.se [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden)

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via

  9. Modeling and simulation of aggregation of membrane protein LAT with molecular variability in the number of binding sites for cytosolic Grb2-SOS1-Grb2.

    Directory of Open Access Journals (Sweden)

    Ambarish Nag

    Full Text Available The linker for activation of T cells (LAT, the linker for activation of B cells (LAB, and the linker for activation of X cells (LAX form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1:1 and 2:1 complexes with the guanine nucleotide exchange factor SOS1. The 2:1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate. We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.

  10. NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses.

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    Jie Zhou

    Full Text Available Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.

  11. The Endocytic Receptor Megalin and its Associated Proteins in Proximal Tubule Epithelial Cells

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    Shankhajit De

    2014-07-01

    Full Text Available Receptor-mediated endocytosis in renal proximal tubule epithelial cells (PTECs is important for the reabsorption and metabolization of proteins and other substances, including carrier-bound vitamins and trace elements, in glomerular filtrates. Impairment of this endocytic process results in the loss of such substances and development of proteinuria, which is an important clinical indicator of kidney diseases and is also a risk marker for cardiovascular disease. Megalin, a member of the low-density lipoprotein receptor gene family, is a multiligand receptor expressed in the apical membrane of PTECs and plays a central role in the endocytic process. Megalin interacts with various intracellular adaptor proteins for intracellular trafficking and cooperatively functions with other membrane molecules, including the cubilin-amnionless complex. Evidence suggests that megalin and the cubilin-amnionless complex are involved in the uptake of toxic substances into PTECs, which leads to the development of kidney disease. Studies of megalin and its associated molecules will be useful for future development of novel strategies for the diagnosis and treatment of kidney diseases.

  12. The adapter protein ADAP is required for selected dendritic cell functions

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    Togni Mauro

    2012-06-01

    Full Text Available Abstract Background The cytosolic adaptor protein ADAP (adhesion and degranulation promoting adapter protein is expressed by T cells, natural killer cells, myeloid cells and platelets. ADAP is involved in T-cell-receptor-mediated inside-out signaling, which leads to integrin activation, adhesion and reorganization of the actin cytoskeleton. However, little is known about the role of ADAP in myeloid cells. In the present study, we analyzed the function of ADAP in bone-marrow-derived dendritic cells (BMDCs from ADAP-deficient mice. Results ADAP-deficient BMDCs showed almost normal levels of antigen uptake, adhesion, maturation, migration from the periphery to the draining lymph nodes, antigen-specific T-cell activation, and production of the proinflammatory cytokines IL-6 and TNF-∝. Furthermore, we provide evidence that the activation of signaling pathways after lipopolysaccharide (LPS stimulation are not affected by the loss of ADAP. In contrast, ADAP-deficient BMDCs showed defects in CD11c-mediated cellular responses, with significantly diminished production of IL-6, TNF-∝ and IL-10. Actin polymerization was enhanced after CD11c integrin stimulation. Conclusions In summary, we propose that the adapter molecule ADAP is critical for selected CD11c integrin-mediated functions of dendritic cells.

  13. Poly(C)-binding protein 1 (PCBP1) mediates housekeeping degradation of mitochondrial antiviral signaling (MAVS)

    Institute of Scientific and Technical Information of China (English)

    Xiang Zhou; Fuping You; Huihui Chen; Zhengfan Jiang

    2012-01-01

    Mitochondrial antiviral signaling (MAVS) is a key adaptor in cellular antiviral innate immunity.We previously identified poly(C)-binding protein 2 (PCBP2) as a feedback inhibitor of MAVS that facilitates its degradation after viral infection,but little is known about the regulatory potential of poly(C)-binding protein 1 (PCBP1),which highly resembles PCBP2.Here we report that PCBP1 mediates housekeeping degradation of MAVS using the same mechanism as PCBP2 employs.Overexpression of PCBP1 impairs MAVS-mediated antiviral responses,while knockdown of PCBP1 exerts the opposite effect.The suppression is due to PCBP1-induced MAVS degradation.We observe that PCBP1 and PCBP2 show synergy in MAVS inhibition,but their expression patterns are distinct:PCBP1 is stably and abundantly expressed,while PCBP2 shows low basal expression with rapid induction after infection.Individual knockdown and subcellular fractionation analyses reveal that unlike the postinfection inhibitor PCBP2,PCBP1 continuously eliminates cellular MAVS.Our findings unravel a critical role of PCBP1 in regulating MAVS for both finetuning the antivirai immunity and preventing inflammation.

  14. Role of Toll-Interacting Protein Gene Polymorphisms in Leprosy Mexican Patients

    Science.gov (United States)

    Montoya-Buelna, Margarita; Tovar-Cuevas, Alvaro J.; Alvarado-Navarro, Anabell; Valle, Yeminia; Padilla-Gutierrez, Jorge R.; Muñoz-Valle, Jose F.; Figuera-Villanueva, Luis E.

    2013-01-01

    Background. Leprosy is a debilitating infectious disease of human skin and nerves. Genetics factors of the host play an important role in the disease susceptibility. Toll-interacting protein (TOLLIP) is an inhibitory adaptor protein within the toll-like receptor (TLR) pathway, which recognizes structurally conserved molecular patterns of microbial pathogens, initiating immune responses. The objective of this study was to investigate the association of variants in the TOLLIP gene with susceptibility to leprosy in Mexican patients. Methods. TOLLIP polymorphisms were studied using a case-control design of Mexican patients with lepromatous leprosy (LL). The polymorphisms of TOLLIP at loci −526 C>G (rs5743854), 1309956C>T (rs3750920), 1298430C>A (rs5744015), and 1292831 G>A (rs3750919) were analyzed by PCR, with sequence-specific primers in LL patients and healthy subjects (HS) as controls. Results. Genotype distributions were in Hardy Weinberg equilibrium for all sites except for rs3750920. Neither genotype nor allele frequencies were statistically different between LL patients and controls (P > 0.05). The maximum pairwise D' coefficient reached was 0.44 of linkage (P = 0.01) for all the polymorphisms except for rs5743854. The three loci haplotype comparison yielded no significant differences between groups. Conclusions. Just the individuals with genotype C/C of rs3750920 have a trend of protective effect to developing LL. PMID:24294608

  15. Role of Toll-Interacting Protein Gene Polymorphisms in Leprosy Mexican Patients

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    Margarita Montoya-Buelna

    2013-01-01

    Full Text Available Background. Leprosy is a debilitating infectious disease of human skin and nerves. Genetics factors of the host play an important role in the disease susceptibility. Toll-interacting protein (TOLLIP is an inhibitory adaptor protein within the toll-like receptor (TLR pathway, which recognizes structurally conserved molecular patterns of microbial pathogens, initiating immune responses. The objective of this study was to investigate the association of variants in the TOLLIP gene with susceptibility to leprosy in Mexican patients. Methods. TOLLIP polymorphisms were studied using a case-control design of Mexican patients with lepromatous leprosy (LL. The polymorphisms of TOLLIP at loci −526 C>G (rs5743854, 1309956C>T (rs3750920, 1298430C>A (rs5744015, and 1292831 G>A (rs3750919 were analyzed by PCR, with sequence-specific primers in LL patients and healthy subjects (HS as controls. Results. Genotype distributions were in Hardy Weinberg equilibrium for all sites except for rs3750920. Neither genotype nor allele frequencies were statistically different between LL patients and controls (P>0.05. The maximum pairwise D’ coefficient reached was 0.44 of linkage (P=0.01 for all the polymorphisms except for rs5743854. The three loci haplotype comparison yielded no significant differences between groups. Conclusions. Just the individuals with genotype C/C of rs3750920 have a trend of protective effect to developing LL.

  16. Receptor-mediated sorting of soluble vacuolar proteins: myths, facts, and a new model.

    Science.gov (United States)

    Robinson, David G; Neuhaus, Jean-Marc

    2016-08-01

    To prevent their being released to the cell exterior, acid hydrolases are recognized by receptors at some point in the secretory pathway and diverted towards the lytic compartment of the cell (lysosome or vacuole). In animal cells, the receptor is called the mannosyl 6-phosphate receptor (MPR) and it binds hydrolase ligands in the trans-Golgi network (TGN). These ligands are then sequestered into clathrin-coated vesicles (CCVs) because of motifs in the cytosolic tail of the MPR which interact first with monomeric adaptors (Golgi-localized, Gamma-ear-containing, ARF-binding proteins, GGAs) and then with tetrameric (adaptin) adaptor complexes. The CCVs then fuse with an early endosome, whose more acidic lumen causes the ligands to dissociate. The MPRs are then recycled back to the TGN via retromer-coated carriers. Plants have vacuolar sorting receptors (VSRs) which were originally identified in CCVs isolated from pea (Pisum sativum L.) cotyledons. It was therefore assumed that VSRs would have an analogous function in plants to MPRs in animals. Although this dogma has enjoyed wide support over the last 20 years there are many inconsistencies. Recently, results have been published which are quite contrary to it. It now emerges that VSRs and their ligands can interact very early in the secretory pathway, and dissociate in the TGN, which, in contrast to its mammalian counterpart, has a pH of 5.5. Multivesicular endosomes in plants lack proton pump complexes and consequently have an almost neutral internal pH, which discounts them as organelles of pH-dependent receptor-ligand dissociation. These data force a critical re-evaluation of the role of CCVs at the TGN, especially considering that vacuolar cargo ligands have never been identified in them. We propose that one population of TGN-derived CCVs participate in retrograde transport of VSRs from the TGN. We also present a new model to explain how secretory and vacuolar cargo proteins are effectively separated after

  17. A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2.

    Science.gov (United States)

    Breitkopf, Susanne B; Yang, Xuemei; Begley, Michael J; Kulkarni, Meghana; Chiu, Yu-Hsin; Turke, Alexa B; Lauriol, Jessica; Yuan, Min; Qi, Jie; Engelman, Jeffrey A; Hong, Pengyu; Kontaridis, Maria I; Cantley, Lewis C; Perrimon, Norbert; Asara, John M

    2016-02-03

    Using a series of immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

  18. A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2

    Science.gov (United States)

    Breitkopf, Susanne B.; Yang, Xuemei; Begley, Michael J.; Kulkarni, Meghana; Chiu, Yu-Hsin; Turke, Alexa B.; Lauriol, Jessica; Yuan, Min; Qi, Jie; Engelman, Jeffrey A.; Hong, Pengyu; Kontaridis, Maria I.; Cantley, Lewis C.; Perrimon, Norbert; Asara, John M.

    2016-02-01

    Using a series of immunoprecipitation (IP) - tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

  19. Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

    Directory of Open Access Journals (Sweden)

    Yuan Jian

    2010-06-01

    Full Text Available Abstract Background The downstream of tyrosine kinase/docking protein (Dok adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk receptor family, which has three members (TrkA, TrkB and TrkC, are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST precipitation assays and coimmunoprecipitation (Co-IP experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3 stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

  20. Agrobacterium delivers VirE2 protein into host cells via clathrin-mediated endocytosis

    Science.gov (United States)

    Li, Xiaoyang; Pan, Shen Q.

    2017-01-01

    Agrobacterium tumefaciens can cause crown gall tumors on a wide range of host plants. As a natural genetic engineer, the bacterium can transfer both single-stranded DNA (ssDNA) [transferred DNA (T-DNA)] molecules and bacterial virulence proteins into various recipient cells. Among Agrobacterium-delivered proteins, VirE2 is an ssDNA binding protein that is involved in various steps of the transformation process. However, it is not clear how plant cells receive the T-DNA or protein molecules. Using a split–green fluorescent protein approach, we monitored the VirE2 delivery process inside plant cells in real time. We observed that A. tumefaciens delivered VirE2 from the bacterial lateral sides that were in close contact with plant membranes. VirE2 initially accumulated on plant cytoplasmic membranes at the entry points. VirE2-containing membranes were internalized through clathrin-mediated endocytosis to form endomembrane compartments. VirE2 colocalized with the early endosome marker SYP61 but not with the late endosome marker ARA6, suggesting that VirE2 escaped from early endosomes for subsequent trafficking inside the cells. Dual endocytic motifs at the carboxyl-terminal tail of VirE2 were involved in VirE2 internalization and could interact with the μ subunit of the plant clathrin-associated adaptor AP2 complex (AP2M). Both the VirE2 cargo motifs and AP2M were important for the transformation process. Because AP2-mediated endocytosis is well conserved, our data suggest that the A. tumefaciens pathogen hijacks conserved endocytic pathways to facilitate the delivery of virulence factors. This might be important for Agrobacterium to achieve both a wide host range and a high transformation efficiency.

  1. S100B protein in tissue development,repair and regeneration

    Institute of Scientific and Technical Information of China (English)

    Guglielmo; Sorci; Francesca; Riuzzi; Cataldo; Arcuri; Claudia; Tubaro; Roberta; Bianchi; Ileana; Giambanco; Rosario; Donato

    2013-01-01

    The Ca 2+-binding protein of the EF-hand type,S100B,exerts both intracellular and extracellular regulatory activities.As an intracellular regulator,S100B is involved in the regulation of energy metabolism,transcription,protein phosphorylation,cell proliferation,survival,differentiation and motility,and Ca 2+ homeostasis,by interacting with a wide array of proteins(i.e.,enzymes,enzyme substrates,cytoskeletal subunits,scaffold/adaptor proteins,transcription factors,ubiquitin E3 ligases,ion channels) in a restricted number of cell types.As an extracellular signal,S100B engages the pattern recognition receptor,receptor for advanced glycation end-products(RAGE),on immune cells as well as on neuronal,astrocytic and microglial cells,vascular smooth muscle cells,skeletal myoblasts and cardiomyocytes.However,RAGE may not be the sole receptor activated by S100B,the protein being able to enhance bFGF-FGFR1 signaling by interacting with FGFR1-bound bFGF in particular cell types.Moreover,extracellular effects of S100B vary depending on its local concentration.Increasing evidence suggests that at the concentration found in extracellular fluids in normal physiological conditions and locally upon acute tissue injury,which is up to a few nM levels,S100B exerts trophic effects in the central and peripheral nervous system and in skeletal muscle tissue thus participating in tissue homeostasis.The present commentary summarizes results implicating intracellular and extracellular S100B in tissue development,repair and regeneration.

  2. Protein inference: A protein quantification perspective.

    Science.gov (United States)

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/.

  3. Adiponectin is protective against oxidative stress induced cytotoxicity in amyloid-beta neurotoxicity.

    Directory of Open Access Journals (Sweden)

    Koon-Ho Chan

    Full Text Available Beta-amyloid (Aβ neurotoxicity is important in Alzheimer's disease (AD pathogenesis. Aβ neurotoxicity causes oxidative stress, inflammation and mitochondrial damage resulting in neuronal degeneration and death. Oxidative stress, inflammation and mitochondrial failure are also pathophysiological mechanisms of type 2 diabetes (T(2DM which is characterized by insulin resistance. Interestingly, T(2DM increases risk to develop AD which is associated with reduced neuronal insulin sensitivity (central insulin resistance. We studied the potential protective effect of adiponectin (an adipokine with insulin-sensitizing, anti-inflammatory and anti-oxidant properties against Aβ neurotoxicity in human neuroblastoma cells (SH-SY5Y transfected with the Swedish amyloid precursor protein (Sw-APP mutant, which overproduced Aβ with abnormal intracellular Aβ accumulation. Cytotoxicity was measured by assay for lactate dehydrogenase (LDH released upon cell death and lysis. Our results revealed that Sw-APP transfected SH-SY5Y cells expressed both adiponectin receptor 1 and 2, and had increased AMP-activated protein kinase (AMPK activation and enhanced nuclear factor-kappa B (NF-κB activation compared to control empty-vector transfected SH-SY5Y cells. Importantly, adiponectin at physiological concentration of 10 µg/ml protected Sw-APP transfected SH-SY5Y cells against cytotoxicity under oxidative stress induced by hydrogen peroxide. This neuroprotective action of adiponectin against Aβ neurotoxicity-induced cytotoxicity under oxidative stress involved 1 AMPK activation mediated via the endosomal adaptor protein APPL1 (adaptor protein with phosphotyrosine binding, pleckstrin homology domains and leucine zipper motif and possibly 2 suppression of NF-κB activation. This raises the possibility of novel therapies for AD such as adiponectin receptor agonists.

  4. Transcriptional regulation of human FE65, a ligand of Alzheimer's disease amyloid precursor protein, by Sp1.

    LENUS (Irish Health Repository)

    Yu, Hoi-Tin

    2010-03-01

    FE65 is a neuronal-enriched adaptor protein that binds to the Alzheimer\\'s disease amyloid precursor protein (APP). FE65 forms a transcriptionally active complex with the APP intracellular domain (AICD). The precise gene targets for this complex are unclear but several Alzheimer\\'s disease-linked genes have been proposed. Additionally, evidence suggests that FE65 influences APP metabolism. The mechanism by which FE65 expression is regulated is as yet unknown. To gain insight into the regulatory mechanism, we cloned a 1.6 kb fragment upstream of the human FE65 gene and found that it possesses particularly strong promoter activity in neurones. To delineate essential regions in the human FE65 promoter, a series of deletion mutants were generated. The minimal FE65 promoter was located between -100 and +5, which contains a functional Sp1 site. Overexpression of the transcription factor Sp1 potentiates the FE65 promoter activity. Conversely, suppression of the FE65 promoter was observed in cells either treated with an Sp1 inhibitor or in which Sp1 was knocked down. Furthermore, reduced levels of Sp1 resulted in downregulation of endogenous FE65 mRNA and protein. These findings reveal that Sp1 plays a crucial role in transcriptional control of the human FE65 gene.

  5. Structural Determinants of Human FANCF Protein That Function in the Assembly of a DNA Damage Signaling Complex

    Energy Technology Data Exchange (ETDEWEB)

    Kowal,P.; Gurtan, A.; Stuckert, P.; D' Andrea, A.; Ellenberger, T.

    2007-01-01

    Fanconi anemia (FA) is a rare autosomal recessive and X-linked chromosomal instability disorder. At least eight FA proteins (FANCA, B, C, E, F, G, L, and M) form a nuclear core complex required for monoubiquitination of a downstream protein, FANCD2. The human FANCF protein reportedly functions as a molecular adaptor within the FA nuclear complex, bridging between the subcomplexes A:G and C:E. Our x-ray crystallographic studies of the C-terminal domain of FANCF reveal a helical repeat structure similar to the Cand1 regulator of the Cul1-Rbx1-Skp1-Fbox(Skp2) ubiquitin ligase complex. Two C-terminal loops of FANCF are essential for monoubiquitination of FANCD2 and normal cellular resistance to the DNA cross-linking agent mitomycin C. FANCF mutants bearing amino acid substitutions in this C-terminal surface fail to interact with other components of the FA complex, indicating that this surface is critical for the proper assembly of the FA core complex.

  6. Giardia lamblia low-density lipoprotein receptor-related protein is involved in selective lipoprotein endocytosis and parasite replication.

    Science.gov (United States)

    Rivero, Maria R; Miras, Silvana L; Quiroga, Rodrigo; Rópolo, Andrea S; Touz, Maria C

    2011-03-01

    As Giardia lamblia is unable to synthesize cholesterol de novo, this steroid might be obtained from the host's intestinal milieu by endocytosis of lipoproteins. In this work, we identified a putative Giardia lamblia low-density lipoprotein receptor-related proteins (GlLRP), a type I membrane protein, which shares the substrate N-terminal binding domain and a FXNPXY-type endocytic motif with human LRPs. Expression of tagged GlLRP showed that it was localized predominantly in the endoplasmic reticulum, lysosomal-like peripheral vacuoles and plasma membrane. However, the FXNPXY-deleted GlLRP was retained at the plasma membrane suggesting that it is abnormally transported and processed. The low-density lipoprotein and chylomicrons interacted with GlLRP, with this interaction being necessary for lipoprotein internalization and cell proliferation. Finally, we show that GlLRP binds directly to the medium subunit of Giardia adaptor protein 2, indicating that receptor-mediated internalization occurs through an adaptin mechanism.

  7. A Staphylococcus aureus TIR domain protein virulence factor blocks TLR2-mediated NF-κB signaling.

    Science.gov (United States)

    Askarian, Fatemeh; van Sorge, Nina M; Sangvik, Maria; Beasley, Federico C; Henriksen, Jørn R; Sollid, Johanna U E; van Strijp, Jos A G; Nizet, Victor; Johannessen, Mona

    2014-01-01

    Signaling through Toll-like receptors (TLRs), crucial molecules in the induction of host defense responses, requires adaptor proteins that contain a Toll/interleukin-1 receptor (TIR) domain. The pathogen Staphylococcus aureus produces several innate immune-evasion molecules that interfere with the host's innate immune response. A database search analysis suggested the presence of a gene encoding a homologue of the human TIR domain in S. aureus MSSA476 which was named staphylococcal TIR domain protein (TirS). Ectopic expression of TirS in human embryonic kidney, macrophage and keratinocyte cell lines interfered with signaling through TLR2, including MyD88 and TIRAP, NF-κB and/or mitogen-activated protein kinase pathways. Moreover, the presence of TirS reduced the levels of cytokines MCP-1 and G-CSF secreted in response to S. aureus. The effects on NF-κB pathway were confirmed using S. aureus MSSA476 wild type, an isogenic mutant MSSA476ΔtirS, and complemented MSSA476ΔtirS +pTirS in a Transwell system where bacteria and host cells were physically separated. Finally, in a systematic mouse infection model, TirS promoted bacterial accumulation in several organs 4 days postinfection. The results of this study reveal a new S. aureus virulence factor that can interfere with PAMP-induced innate immune signaling in vitro and bacterial survival in vivo.

  8. Characterization of the interaction between Actinin-Associated LIM Protein (ALP and the rod domain of α-actinin

    Directory of Open Access Journals (Sweden)

    Permi Perttu

    2009-03-01

    Full Text Available Abstract Background The PDZ-LIM proteins are a family of signalling adaptors that interact with the actin cross-linking protein, α-actinin, via their PDZ domains or via internal regions between the PDZ and LIM domains. Three of the PDZ-LIM proteins have a conserved 26-residue ZM motif in the internal region, but the structure of the internal region is unknown. Results In this study, using circular dichroism and nuclear magnetic resonance (NMR, we showed that the ALP internal region (residues 107–273 was largely unfolded in solution, but was able to interact with the α-actinin rod domain in vitro, and to co-localize with α-actinin on stress fibres in vivo. NMR analysis revealed that the titration of ALP with the α-actinin rod domain induces stabilization of ALP. A synthetic peptide (residues 175–196 that contained the N-terminal half of the ZM motif was found to interact directly with the α-actinin rod domain in surface plasmon resonance (SPR measurements. Short deletions at or before the ZM motif abrogated the localization of ALP to actin stress fibres. Conclusion The internal region of ALP appeared to be largely unstructured but functional. The ZM motif defined part of the interaction surface between ALP and the α-actinin rod domain.

  9. Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2.

    Directory of Open Access Journals (Sweden)

    Veronika Mikitova

    Full Text Available BACKGROUND: Two phenylalanines (FF in an acidic tract (FFAT-motifs were originally described as having seven elements: an acidic flanking region followed by 6 residues (EFFDA-E. Such motifs are found in several lipid transfer protein (LTP families, and they interact with a protein on the cytosolic face of the ER called vesicle-associated membrane protein-associated protein (VAP. Mutation of which causes ER stress and motor neuron disease, making it important to determine which proteins bind VAP. Among other proteins that bind VAP, some contain FFAT-like motifs that are missing one or more of the seven elements. Defining how much variation is tolerated in FFAT-like motifs is a preliminary step prior to the identification of the full range of VAP interactors. RESULTS: We used a quantifiable in vivo system that measured ER targeting in a reporter yeast strain that over-expressed VAP to study the effect of substituting different elements of FFAT-like motifs in turn. By defining FFAT-like motifs more widely than before, we found them in novel proteins the functions of which had not previously been directly linked to the ER, including: two PKA anchoring proteins, AKAP220 and AKAP110; a family of plant LTPs; and the glycolipid LTP phosphatidylinositol-four-phosphate adaptor-protein-2 (FAPP-2. CONCLUSION: All of the seven essential elements of a FFAT motif tolerate variation, and weak targeting to the ER via VAP is still detected if two elements are substituted. In addition to the strong FFAT motifs already known, there are additional proteins with weaker FFAT-like motifs, which might be functionally important VAP interactors.

  10. Systematic identification of regulatory proteins critical for T-cell activation

    Directory of Open Access Journals (Sweden)

    Kolbinger Frank

    2003-09-01

    Full Text Available Abstract Background The activation of T cells, mediated by the T-cell receptor (TCR, activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. Results In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1, transmembrane molecules (EDG1, IL-10Rα and integrin α2, cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1, and cytoskeletal molecules (moesin and vimentin. Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. Conclusions This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.

  11. Sec24D-dependent transport of extracellular matrix proteins is required for zebrafish skeletal morphogenesis.

    Directory of Open Access Journals (Sweden)

    Swapnalee Sarmah

    Full Text Available Protein transport from endoplasmic reticulum (ER to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM, but membrane bound receptor beta1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C(6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects.

  12. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  13. The nuclear protein Sam68 is redistributed to the cytoplasm and is involved in PI3K/Akt activation during EV71 infection.

    Science.gov (United States)

    Zhang, Hua; Cong, Haolong; Song, Lei; Tien, Po

    2014-02-13

    Nuclear proteins can be triggered to be redistributed to the cytoplasm to assist with EV71 virus replication. This process is frequently involved in cellular signal transduction upon virus infection. In this study, we have demonstrated that a new nuclear protein, 68-kDa Src-associated in mitosis protein (Sam68), was translocated to the cytoplasm and was co-localized with EV71 during virus infection. Confocal microscopy and subcellular fractionation assay confirmed that virus 3C protease triggered the redistribution of Sam68 to the cytoplasm. Knockdown of Sam68 expression using ShRNA significantly inhibited virus replication, suggesting that Sam68 may be a host factor involved in EV71 life cycle. In addition, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Sam68 is known to be an upstream regulator of PI3K and our immunoprecipitation studies confirmed that Sam68 interacted directly with the p85 regulatory subunit of PI3K and mediated PI3K/Akt activation during EV71 infection. On the contrary, silencing of Sam68 dramatically abrogated Akt phosphorylation. These data, plus the fact that Sam68 is known to be a signaling adaptor protein, indicated that Sam68 is a signal molecule with a functional role in the PI3K/Akt signal pathway during EV71 infection.

  14. A New Role for Annexin A11 in the Early Secretory Pathway via Stabilizing Sec31A Protein at the Endoplasmic Reticulum Exit Sites (ERES)*

    Science.gov (United States)

    Shibata, Hideki; Kanadome, Takashi; Sugiura, Hirofumi; Yokoyama, Takeru; Yamamuro, Minami; Moss, Stephen E.; Maki, Masatoshi

    2015-01-01

    Exit of cargo molecules from the endoplasmic reticulum (ER) for transport to the Golgi is the initial step in intracellular vesicular trafficking. The coat protein complex II (COPII) machinery is recruited to specialized regions of the ER, called ER exit sites (ERES), where it plays a central role in the early secretory pathway. It has been known for more than two decades that calcium is an essential factor in vesicle trafficking from the ER to Golgi apparatus. However, the role of calcium in the early secretory pathway is complicated and poorly understood. We and others previously identified Sec31A, an outer cage component of COPII, as an interacting protein for the penta-EF-hand calcium-binding protein ALG-2. In this study, we show that another calcium-binding protein, annexin A11 (AnxA11), physically associates with Sec31A by the adaptor function of ALG-2. Depletion of AnxA11 or ALG-2 decreases the population of Sec31A that is stably associated with the ERES and causes scattering of juxtanuclear ERES to the cell periphery. The synchronous ER-to-Golgi transport of transmembrane cargoes is accelerated in AnxA11- or ALG-2-knockdown cells. These findings suggest that AnxA11 maintains architectural and functional features of the ERES by coordinating with ALG-2 to stabilize Sec31A at the ERES. PMID:25540196

  15. Membrane Tension Inhibits Deformation by Coat Proteins in Clathrin-Mediated Endocytosis

    Science.gov (United States)

    Hassinger, Julian; Drubin, David; Oster, George; Rangamani, Padmini

    2016-02-01

    In clathrin-mediated endocytosis (CME), clathrin and various adaptor proteins coat a patch of the plasma membrane, which is reshaped to form a budded vesicle. Experimental studies have demonstrated that elevated membrane tension can inhibit bud formation by a clathrin coat. In this study, we investigate the impact of membrane tension on the mechanics of membrane budding by simulating clathrin coats that either grow in area or progressively induce greater curvature. At low membrane tension, progressively increasing the area of a curvature-generating coat causes the membrane to smoothly evolve from a flat to budded morphology, whereas the membrane remains essentially flat at high membrane tensions. Interestingly, at physiologically relevant, intermediate membrane tensions, the shape evolution of the membrane undergoes a snapthrough instability in which increasing coat area causes the membrane to "snap" from an open, U-shaped bud to a closed, $\\Omega$-shaped bud. This instability is accompanied by a large energy barrier, which could cause a developing endocytic pit to stall if the binding energy of additional coat is insufficient to overcome this barrier. Similar results were found for a coat of constant area in which the spontaneous curvature progressively increases. Additionally, a pulling force on the bud, simulating a force from actin polymerization, is sufficient to drive a transition from an open to closed bud, overcoming the energy barrier opposing this transition.

  16. p66(Shc) protein, oxidative stress, and cardiovascular complications of diabetes: the missing link.

    Science.gov (United States)

    Francia, Pietro; Cosentino, Francesco; Schiavoni, Marzia; Huang, Yale; Perna, Enrico; Camici, Giovani G; Lüscher, Thomas F; Volpe, Massimo

    2009-09-01

    Diabetes affects more than 150 million people worldwide, and it is estimated that this would increase to 299 million by the year 2025. The incidence of and mortality from cardiovascular disease are two- to eightfold higher in subjects with diabetes than in those without, coronary artery disease accounting for the large majority of deaths. Among the full spectrum of biochemical effects of high glucose, generation of oxygen-derived free radicals is one of the main pathophysiological mechanisms linking hyperglycemia to atherosclerosis, nephropathy, and cardiomyopathy. The adaptor protein p66(Shc) is implicated in mitochondrial reactive oxygen species (ROS) generation and translation of oxidative signals into apoptosis. Indeed, p66(Shc-/-) mice display prolonged lifespan, reduced production of intracellular oxidants, and increased resistance to oxidative stress-induced apoptosis. Accordingly, a series of studies defined the pathophysiological role of p66(Shc) in cardiovascular disease where ROS represent a substantial triggering component. As p66(Shc) modulates the production of cellular ROS, it represents a proximal node through which high glucose exerts its deleterious effects on different cell types; indeed, several studies tested the hypothesis that deletion of the p66(Shc) gene may confer protection against diabetes-related cardiovascular complications. The present review focuses on the reported evidence linking p66(Shc) signaling pathway to high glucose-associated endothelial dysfunction, atherogenesis, nephropathy, and cardiomyopathy.

  17. EDITORIAL: Precision proteins Precision proteins

    Science.gov (United States)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  18. Scaffolds for blocking protein-protein interactions.

    Science.gov (United States)

    Hershberger, Stefan J; Lee, Song-Gil; Chmielewski, Jean

    2007-01-01

    Due to the pivotal roles that protein-protein interactions play in a plethora of biological processes, the design of therapeutic agents targeting these interactions has become an attractive and important area of research. The development of such agents is faced with a variety of challenges. Nevertheless, considerable progress has been made in the design of proteomimetics capable of disrupting protein-protein interactions. Those inhibitors based on molecular scaffold designs hold considerable interest because of the ease of variation in regard to their displayed functionality. In particular, protein surface mimetics, alpha-helical mimetics, beta-sheet/beta-strand mimetics, as well as beta-turn mimetics have successfully modulated protein-protein interactions involved in such diseases as cancer and HIV. In this review, current progress in the development of molecular scaffolds designed for the disruption of protein-protein interactions will be discussed with an emphasis on those active against biological targets.

  19. Fusion-protein-assisted protein crystallization.

    Science.gov (United States)

    Kobe, Bostjan; Ve, Thomas; Williams, Simon J

    2015-07-01

    Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

  20. Retention of OsNMD3 in the cytoplasm disturbs protein synthesis efficiency and affects plant development in rice.

    Science.gov (United States)

    Shi, Yanyun; Liu, Xiangling; Li, Rui; Gao, Yaping; Xu, Zuopeng; Zhang, Baocai; Zhou, Yihua

    2014-07-01

    The ribosome is the basic machinery for translation, and biogenesis of ribosomes involves many coordinated events. However, knowledge about ribosomal dynamics in higher plants is very limited. This study chose a highly conserved trans-factor, the 60S ribosomal subunit nuclear export adaptor NMD3, to characterize the mechanism of ribosome biogenesis in the monocot plant Oryza sativa (rice). O. sativa NMD3 (OsNMD3) shares all the common motifs and shuttles between the nucleus and cytoplasm via CRM1/XPO1. A dominant negative form of OsNMD3 with a truncated nuclear localization sequence (OsNMD3(ΔNLS)) was retained in the cytoplasm, consequently interfering with the release of OsNMD3 from pre-60S particles and disturbing the assembly of ribosome subunits. Analyses of the transactivation activity and cellulose biosynthesis level revealed low protein synthesis efficiency in the transgenic plants compared with the wild-type plants. Pharmaceutical treatments demonstrated structural alterations in ribosomes in the transgenic plants. Moreover, global expression profiles of the wild-type and transgenic plants were investigated using the Illumina RNA sequencing approach. These expression profiles suggested that overexpression of OsNMD3(ΔNLS) affected ribosome biogenesis and certain basic pathways, leading to pleiotropic abnormalities in plant growth. Taken together, these results strongly suggest that OsNMD3 is important for ribosome assembly and the maintenance of normal protein synthesis efficiency.

  1. HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression.

    Science.gov (United States)

    Diani, Erica; Avesani, Francesca; Bergamo, Elisa; Cremonese, Giorgia; Bertazzoni, Umberto; Romanelli, Maria Grazia

    2015-02-01

    The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression.

  2. Identification and characterization of a novel bacterial virulence factor that shares homology with mammalian Toll/interleukin-1 receptor family proteins.

    Science.gov (United States)

    Newman, Ruchi M; Salunkhe, Prabhakar; Godzik, Adam; Reed, John C

    2006-01-01

    Many important bacterial virulence factors act as mimics of mammalian proteins to subvert normal host cell processes. To identify bacterial protein mimics of components of the innate immune signaling pathway, we searched the bacterial genome database for proteins with homology to the Toll/interleukin-1 receptor (TIR) domain of the mammalian Toll-like receptors (TLRs) and their adaptor proteins. A previously uncharacterized gene, which we have named tlpA (for TIR-like protein A), was identified in the Salmonella enterica serovar Enteritidis genome that is predicted to encode a protein resembling mammalian TIR domains, We show that overexpression of TlpA in mammalian cells suppresses the ability of mammalian TIR-containing proteins TLR4, IL-1 receptor, and MyD88 to induce the transactivation and DNA-binding activities of NF-kappaB, a downstream target of the TIR signaling pathway. In addition, TlpA mimics the previously characterized Salmonella virulence factor SipB in its ability to induce activation of caspase-1 in a mammalian cell transfection model. Disruption of the chromosomal tlpA gene rendered a virulent serovar Enteritidis strain defective in intracellular survival and IL-1beta secretion in a cell culture infection model using human THP1 macrophages. Bacteria with disrupted tlpA also displayed reduced lethality in mice, further confirming an important role for this factor in pathogenesis. Taken together, our findings demonstrate that the bacterial TIR-like protein TlpA is a novel prokaryotic modulator of NF-kappaB activity and IL-1beta secretion that contributes to serovar Enteritidis virulence.

  3. Protein docking prediction using predicted protein-protein interface

    Directory of Open Access Journals (Sweden)

    Li Bin

    2012-01-01

    Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  4. Protein Crystal Based Nanomaterials

    Science.gov (United States)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  5. Shotgun protein sequencing.

    Energy Technology Data Exchange (ETDEWEB)

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  6. Chikungunya virus induces IPS-1-dependent innate immune activation and protein kinase R-independent translational shutoff.

    Science.gov (United States)

    White, Laura K; Sali, Tina; Alvarado, David; Gatti, Evelina; Pierre, Philippe; Streblow, Daniel; Defilippis, Victor R

    2011-01-01

    Chikungunya virus (CHIKV) is an arthritogenic mosquito-transmitted alphavirus that is undergoing reemergence in areas around the Indian Ocean. Despite the current and potential danger posed by this virus, we know surprisingly little about the induction and evasion of CHIKV-associated antiviral immune responses. With this in mind we investigated innate immune reactions to CHIKV in human fibroblasts, a demonstrable in vivo target of virus replication and spread. We show that CHIKV infection leads to activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent transcription of IRF3-dependent antiviral genes, including beta interferon (IFN-β). IRF3 activation occurs by way of a virus-induced innate immune signaling pathway that includes the adaptor molecule interferon promoter stimulator 1 (IPS-1). Despite strong transcriptional upregulation of these genes, however, translation of the corresponding proteins is not observed. We further demonstrate that translation of cellular (but not viral) genes is blocked during infection and that although CHIKV is found to trigger inactivation of the translational molecule eukaryotic initiation factor subunit 2α by way of the double-stranded RNA sensor protein kinase R, this response is not required for the block to protein synthesis. Furthermore, overall diminution of cellular RNA synthesis is also observed in the presence of CHIKV and transcription of IRF3-dependent antiviral genes appears specifically blocked late in infection. We hypothesize that the observed absence of IFN-β and antiviral proteins during infection results from an evasion mechanism exhibited by CHIKV that is dependent on widespread shutoff of cellular protein synthesis and a targeted block to late synthesis of antiviral mRNA transcripts.

  7. Protein folding, protein homeostasis, and cancer

    Institute of Scientific and Technical Information of China (English)

    John H. Van Drie

    2011-01-01

    Proteins fold into their functional 3-dimensional structures from a linear amino acid sequence. In vitro this process is spontaneous; while in vivo it is orchestrated by a specialized set of proteins, called chaperones. Protein folding is an ongoing cellular process, as cellular proteins constantly undergo synthesis and degradation. Here emerging links between this process and cancer are reviewed. This perspective both yields insights into the current struggle to develop novel cancer chemotherapeutics and has implications for future chemotherapy discovery.

  8. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  9. Protein-losing enteropathy

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  10. An Aberrant Phosphorylation of Amyloid Precursor Protein Tyrosine Regulates Its Trafficking and the Binding to the Clathrin Endocytic Complex in Neural Stem Cells of Alzheimer's Disease Patients

    Science.gov (United States)

    Poulsen, Ebbe T.; Iannuzzi, Filomena; Rasmussen, Helle F.; Maier, Thorsten J.; Enghild, Jan J.; Jørgensen, Arne L.; Matrone, Carmela

    2017-01-01

    Alzheimer's disease (AD) is the most common cause of dementia and is likely caused by defective amyloid precursor protein (APP) trafficking and processing in neurons leading to amyloid plaques containing the amyloid-β (Aβ) APP peptide byproducts. Understanding how APP is targeted to selected destinations inside neurons and identifying the mechanisms responsible for the generation of Aβ are thus the keys for the advancement of new therapies. We previously developed a mouse model with a mutation at tyrosine (Tyr) 682 in the C-terminus of APP. This residue is needed for APP to bind to the coating protein Clathrin and to the Clathrin adaptor protein AP2 as well as for the correct APP trafficking and sorting in neurons. By extending these findings to humans, we found that APP binding to Clathrin is decreased in neural stem cells from AD sufferers. Increased APP Tyr phosphorylation alters APP trafficking in AD neurons and it is associated to Fyn Tyr kinase activation. We show that compounds affecting Tyr kinase activity and counteracting defects in AD neurons can control APP location and compartmentalization. APP Tyr phosphorylation is thus a potential therapeutic target for AD.

  11. Retinoids modulate expression of the endocytic partners megalin, cubilin, and disabled-2 and uptake of vitamin D-binding protein in human mammary cells.

    Science.gov (United States)

    Chlon, Timothy M; Taffany, David A; Welsh, Joellen; Rowling, Matthew J

    2008-07-01

    The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells.

  12. Inflammation in mice ectopically expressing human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1 A230T mutant proteins.

    Science.gov (United States)

    Wang, Donghai; Höing, Susanne; Patterson, Heide Christine; Ahmad, Umtul M; Rathinam, Vijay A K; Rajewsky, Klaus; Fitzgerald, Katherine A; Golenbock, Douglas T

    2013-02-15

    Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne Syndrome (PAPA syndrome) is an autoinflammatory disease caused by aberrant production of the proinflammatory cytokine interleukin-1. Mutations in the gene encoding proline serine threonine phosphatase-interacting protein-1 (PSTPIP1) have been linked to PAPA syndrome. PSTPIP1 is an adaptor protein that interacts with PYRIN, the protein encoded by the Mediterranean Fever (MEFV) gene whose mutations cause Familial Mediterranean Fever (FMF). However, the pathophysiological function of PSTPIP1 remains to be elucidated. We have generated mouse strains that either are PSTPIP1 deficient or ectopically express mutant PSTPIP1. Results from analyzing these mice suggested that PSTPIP1 is not an essential regulator of the Nlrp3, Aim2, or Nlrc4 inflammasomes. Although common features of human PAPA syndrome such as pyogenic arthritis and skin inflammation were not recapitulated in the mouse model, ectopic expression of the mutant but not the wild type PSTPIP1 in mice lead to partial embryonic lethality, growth retardation, and elevated level of circulating proinflammatory cytokines.

  13. SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus

    Science.gov (United States)

    Jöhnk, Bastian; Bayram, Özgür; Heinekamp, Thorsten; Mattern, Derek J.; Brakhage, Axel A.; Jacobsen, Ilse D.; Valerius, Oliver; Braus, Gerhard H.

    2016-01-01

    F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus. PMID:27649508

  14. Conductometric monitoring of protein-protein interactions.

    Science.gov (United States)

    Spera, Rosanna; Festa, Fernanda; Bragazzi, Nicola L; Pechkova, Eugenia; LaBaer, Joshua; Nicolini, Claudio

    2013-12-06

    Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.

  15. Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    Dong Ji; Jun Cheng; Guo-Feng Chen; Yan Liu; Lin Wang; Jiang Guo

    2005-01-01

    AIM: To investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH)technique, and to pave the way for elucidating the pathogenesis of HBV infection.METHODS: pcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 (-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ.After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library.Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.RESULTS: The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 (-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences

  16. UNC-16 (JIP3) Acts Through Synapse-Assembly Proteins to Inhibit the Active Transport of Cell Soma Organelles to Caenorhabditis elegans Motor Neuron Axons.

    Science.gov (United States)

    Edwards, Stacey L; Morrison, Logan M; Yorks, Rosalina M; Hoover, Christopher M; Boominathan, Soorajnath; Miller, Kenneth G

    2015-09-01

    The conserved protein UNC-16 (JIP3) inhibits the active transport of some cell soma organelles, such as lysosomes, early endosomes, and Golgi, to the synaptic region of axons. However, little is known about UNC-16's organelle transport regulatory function, which is distinct from its Kinesin-1 adaptor function. We used an unc-16 suppressor screen in Caenorhabditis elegans to discover that UNC-16 acts through CDK-5 (Cdk5) and two conserved synapse assembly proteins: SAD-1 (SAD-A Kinase), and SYD-2 (Liprin-α). Genetic analysis of all combinations of double and triple mutants in unc-16(+) and unc-16(-) backgrounds showed that the three proteins (CDK-5, SAD-1, and SYD-2) are all part of the same organelle transport regulatory system, which we named the CSS system based on its founder proteins. Further genetic analysis revealed roles for SYD-1 (another synapse assembly protein) and STRADα (a SAD-1-interacting protein) in the CSS system. In an unc-16(-) background, loss of the CSS system improved the sluggish locomotion of unc-16 mutants, inhibited axonal lysosome accumulation, and led to the dynein-dependent accumulation of lysosomes in dendrites. Time-lapse imaging of lysosomes in CSS system mutants in unc-16(+) and unc-16(-) backgrounds revealed active transport defects consistent with the steady-state distributions of lysosomes. UNC-16 also uses the CSS system to regulate the distribution of early endosomes in neurons and, to a lesser extent, Golgi. The data reveal a new and unprecedented role for synapse assembly proteins, acting as part of the newly defined CSS system, in mediating UNC-16's organelle transport regulatory function.

  17. Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling.

    Science.gov (United States)

    Chowdhury, Saiful M; Zhu, Xuewei; Aloor, Jim J; Azzam, Kathleen M; Gabor, Kristin A; Ge, William; Addo, Kezia A; Tomer, Kenneth B; Parks, John S; Fessler, Michael B

    2015-07-01

    Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.

  18. A cell-permeable fusion protein based on Clostridium botulinum C2 toxin for delivery of p53 tumorsuppressor into cancer cells.

    Directory of Open Access Journals (Sweden)

    Jörg Fahrer

    Full Text Available Genetically engineered bacterial protein toxins are attractive systems for delivery of exogenous proteins into the cytosol of mammalian cells. The binary C2 toxin from C. botulinum has emerged as powerful delivery vehicle, which rests on its binding/translocation component C2IIa and the genetically modified adaptor domain C2IN that act in concert to trigger cell uptake. The p53 tumor suppressor protein has a crucial function in suppressing carcinogenesis and is frequently inactivated by diverse mechanisms in human tumor cells. Therefore, we constructed a C2IN-p53 fusion protein, which is internalized into cancer cells by C2IIa. To this end, the C2IN-p53 fusion construct was overexpressed in E. coli with good solubility, purified by heparin affinity chromatography and protein identity was confirmed by immunoblotting. We demonstrated that the fusion protein is capable of binding to the p53 consensus-DNA with high affinity in a p53-specific manner in vitro. Next, the internalization of C2IN-p53 was monitored in HeLa cells by cell fractionation and immunoblot analysis, which revealed a C2IIa-mediated translocation of the fusion protein into the cytosol. The uptake was also shown in A549 and Saos-2 cells with similar efficiency. These findings were further corroborated by confocal immunofluorescence analyses of C2IN-p53/C2IIa-treated HeLa and A549 cells, displaying predominantly cytoplasmic localization of the fusion construct.

  19. Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases

    Institute of Scientific and Technical Information of China (English)

    Weihua Zhou; Wenyi Wei; Yi Sun

    2013-01-01

    The SCF (SKP1 (S-phase-kinase-associated protein 1),Cullin-1,F-box protein) E3 ubiquitin ligases,the founding member of Cullin-RING ligases (CRLs),are the largest family of E3 ubiquitin ligases in mammals.Each individual SCF E3 ligase consists of one adaptor protein SKP1,one scaffold protein cullin-1 (the first family member of the eight cullins),one F-box protein out of 69 family members,and one out of two RING (Really Interesting New Gene) family proteins RBX1/ROC1 or RBX2/ROC2/SAG/RNF7.Various combinations of these four components construct a large number of SCF E3s that promote the degradation of many key regulatory proteins in cell-context,temporally,and spatially dependent manners,thus controlling precisely numerous important cellular processes,including cell cycle progression,apoptosis,gene transcription,signal transduction,DNA replication,maintenance of genome integrity,and tumorigenesis.To understand how the SCF E3 ligases regulate these cellular processes and embryonic development under in vivo physiological conditions,a number of mouse models with transgenic (Tg) expression or targeted deletion of components of SCF have been established and characterized.In this review,we will provide a brief introduction to the ubiquitin-proteasome system (UPS) and the SCF E3 ubiquitin ligases,followed by a comprehensive overview on the existing Tg and knockout (KO) mouse models of the SCF E3s,and discuss the role of each component in mouse embryogenesis,cell proliferation,apoptosis,carcinogenesis,as well as other pathogenic processes associated with human diseases.We will end with a brief discussion on the future directions of this research area and the potential applications of the knowledge gained to more effective therapeutic interventions of human diseases.

  20. Protein sequence comparison and protein evolution

    Energy Technology Data Exchange (ETDEWEB)

    Pearson, W.R. [Univ. of Virginia, Charlottesville, VA (United States). Dept. of Biochemistry

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  1. Prediction of Protein-Protein Interactions Using Protein Signature Profiling

    Institute of Scientific and Technical Information of China (English)

    Mahmood A. Mahdavi; Yen-Han Lin

    2007-01-01

    Protein domains are conserved and functionally independent structures that play an important role in interactions among related proteins. Domain-domain inter- actions have been recently used to predict protein-protein interactions (PPI). In general, the interaction probability of a pair of domains is scored using a trained scoring function. Satisfying a threshold, the protein pairs carrying those domains are regarded as "interacting". In this study, the signature contents of proteins were utilized to predict PPI pairs in Saccharomyces cerevisiae, Caenorhabditis ele- gans, and Homo sapiens. Similarity between protein signature patterns was scored and PPI predictions were drawn based on the binary similarity scoring function. Results show that the true positive rate of prediction by the proposed approach is approximately 32% higher than that using the maximum likelihood estimation method when compared with a test set, resulting in 22% increase in the area un- der the receiver operating characteristic (ROC) curve. When proteins containing one or two signatures were removed, the sensitivity of the predicted PPI pairs in- creased significantly. The predicted PPI pairs are on average 11 times more likely to interact than the random selection at a confidence level of 0.95, and on aver- age 4 times better than those predicted by either phylogenetic profiling or gene expression profiling.

  2. Inferring Protein Associations Using Protein Pulldown Assays

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, Julia L.; Anderson, Kevin K.; Daly, Don S.; Auberry, Deanna L.; Borkowski, John J.; Cannon, William R.

    2007-02-01

    Background: One method to infer protein-protein associations is through a “bait-prey pulldown” assay using a protein affinity agent and an LC-MS (liquid chromatography-mass spectrometry)-based protein identification method. False positive and negative protein identifications are not uncommon, however, leading to incorrect inferences. Methods: A pulldown experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. Our method evaluates the presence/absence pattern across a prey protein (row) with a Likelihood Ratio Test (LRT), computing its p-value with simulated LRT test statistic distributions after a check with simulated binomial random variates disqualified the large sample 2 test. A pulldown experiment often involves hundreds of tests so we apply the false discovery rate method to control the false positive rate. Based on the p-value, each prey protein is assigned a category (specific association, non-specific association, or not associated) and appraised with respect to the pulldown experiment’s goal and design. The method is illustrated using a pulldown experiment investigating the protein complexes of Shewanella oneidensis MR-1. Results: The Monte Carlo simulated LRT p-values objectively reveal specific and ubiquitous prey, as well as potential systematic errors. The example analysis shows the results to be biologically sensible and more realistic than the ad hoc screening methods previously utilized. Conclusions: The method presented appears to be informative for screening for protein-protein associations.

  3. Induction of Covalently Crosslinked p62 Oligomers with Reduced Binding to Polyubiquitinated Proteins by the Autophagy Inhibitor Verteporfin.

    Directory of Open Access Journals (Sweden)

    Elizabeth Donohue

    Full Text Available Autophagy is a cellular catabolic process responsible for the degradation of cytoplasmic constituents, including organelles and long-lived proteins, that helps maintain cellular homeostasis and protect against various cellular stresses. Verteporfin is a benzoporphyrin derivative used clinically in photodynamic therapy to treat macular degeneration. Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light. We report that verteporfin directly targets and modifies p62, a scaffold and adaptor protein that binds both polyubiquitinated proteins destined for degradation and LC3 on autophagosomal membranes. Western blotting experiments revealed that exposure of cells or purified p62 to verteporfin causes the formation of covalently crosslinked p62 oligomers by a mechanism involving low-level singlet oxygen production. Rose bengal, a singlet oxygen producer structurally unrelated to verteporfin, also produced crosslinked p62 oligomers and inhibited autophagosome formation. Co-immunoprecipitation experiments demonstrated that crosslinked p62 oligomers retain their ability to bind to LC3 but show defective binding to polyubiquitinated proteins. Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation. Interestingly, small amounts of crosslinked p62 oligomers were detected in untreated cells, and other groups noted the accumulation of p62 forms with reduced SDS-PAGE mobility in cellular and animal models of oxidative stress and aging. These data indicate that p62 is particularly susceptible to oxidative crosslinking and lead us to propose a model whereby oxidized crosslinked p62 oligomers generated rapidly by drugs like verteporfin or over time during the aging process interfere with autophagy.

  4. Induction of Covalently Crosslinked p62 Oligomers with Reduced Binding to Polyubiquitinated Proteins by the Autophagy Inhibitor Verteporfin.

    Science.gov (United States)

    Donohue, Elizabeth; Balgi, Aruna D; Komatsu, Masaaki; Roberge, Michel

    2014-01-01

    Autophagy is a cellular catabolic process responsible for the degradation of cytoplasmic constituents, including organelles and long-lived proteins, that helps maintain cellular homeostasis and protect against various cellular stresses. Verteporfin is a benzoporphyrin derivative used clinically in photodynamic therapy to treat macular degeneration. Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light. We report that verteporfin directly targets and modifies p62, a scaffold and adaptor protein that binds both polyubiquitinated proteins destined for degradation and LC3 on autophagosomal membranes. Western blotting experiments revealed that exposure of cells or purified p62 to verteporfin causes the formation of covalently crosslinked p62 oligomers by a mechanism involving low-level singlet oxygen production. Rose bengal, a singlet oxygen producer structurally unrelated to verteporfin, also produced crosslinked p62 oligomers and inhibited autophagosome formation. Co-immunoprecipitation experiments demonstrated that crosslinked p62 oligomers retain their ability to bind to LC3 but show defective binding to polyubiquitinated proteins. Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation. Interestingly, small amounts of crosslinked p62 oligomers were detected in untreated cells, and other groups noted the accumulation of p62 forms with reduced SDS-PAGE mobility in cellular and animal models of oxidative stress and aging. These data indicate that p62 is particularly susceptible to oxidative crosslinking and lead us to propose a model whereby oxidized crosslinked p62 oligomers generated rapidly by drugs like verteporfin or over time during the aging process interfere with autophagy.

  5. Differential expression analysis of nuclear oligomerization domain proteins NOD1 and NOD2 in orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Hou, Qing-Hua; Yi, Shi-Bai; Ding, Xu; Zhang, Hui-Xian; Sun, Yan; Zhang, Yong; Liu, Xiao-Chun; Lu, Dan-Qi; Lin, Hao-Ran

    2012-11-01

    Nucleotide-binding oligomerization domain-containing proteins-1 and -2 (NOD1 and NOD2) are members of the NOD-like receptors (NLRs) family. They are both cytoplasmic receptors, and sense microbial infections/danger molecules to induce host innate immune response. In this study, the full-length ORF sequences of NOD1 and NOD2 were cloned, and the putative amino acid sequences were identified in orange-spotted grouper (Epinephelus coioides). The complete open reading frame (ORF) of grouper NOD1 contained 2823 bp encoding a 940 amino acid protein. Grouper NOD2 cDNA contained a 2967 bp ORF, encoding a protein of 988 amino acid residues. Both grouper NOD1 and NOD2 had similar domains to human and fish counterparts. Phylogenetic tree analysis showed that grouper NOD1 clustered with grass carp, zebrafish and channel catfish, while NOD2 was most closely related to fugu. Expression patterns of grouper NOD1 and NOD2 were next studied. NOD1 had the highest level of expression in skin while NOD2 in trunk kidney. Post Vibrio alginolyticus (strain EcGS020401), lipopolysaccharide (LPS) or PolyI:C challenges, gene expression of grouper NOD1 and NOD2 was stimulated to different extents. NOD1 showed a significant enhancement after LPS stimulation, but NOD2 increased more significantly after PolyI:C invasion, indicating that NOD1 and NOD2 may exert different effects on the eradication of bacteria and virus. The adaptor protein RIP-like-interacting CLARP kinase (RICK) and downstream molecule interleukin-8 (IL-8) were also induced at different levels after stimulation, which indicated that NOD1 and NOD2 signal transduction was involved in grouper innate immune protection against bacterial and viral infections.

  6. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    2013-01-01

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  7. NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, Masaya [Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Nishihara, Hiroshi, E-mail: hnishihara@med.hokudai.ac.jp [Department of Translational Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Hasegawa, Hideki [Department of Pathology, National Institute of Infectious Diseases, Sinjuku-ku, Tokyo (Japan); Tashiro, Masato [Influenza Virus Research Center, National Institute of Infectious Diseases, Sinjuku-ku, Tokyo (Japan); Wang, Lei [Department of Translational Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Kimura, Taichi; Tanino, Mishie; Tsuda, Masumi [Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Tanaka, Shinya [Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Department of Translational Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan)

    2013-11-29

    Highlights: •NS1 induced excessive phosphorylation of ERK and elevated cell viability. •NS1-BP expression and CRKL knockdown abolished survival effect of NS1. •NS1-BP and NS1 formed the complex through the interaction with CRKL-SH3(N). -- Abstract: The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while the physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated.

  8. Mirror image proteins.

    Science.gov (United States)

    Zhao, Le; Lu, Wuyuan

    2014-10-01

    Proteins composed entirely of unnatural d-amino acids and the achiral amino acid glycine are mirror image forms of their native l-protein counterparts. Recent advances in chemical protein synthesis afford unique and facile synthetic access to domain-sized mirror image d-proteins, enabling protein research to be conducted through 'the looking glass' and in a way previously unattainable. d-Proteins can facilitate structure determination of their native l-forms that are difficult to crystallize (racemic X-ray crystallography); d-proteins can serve as the bait for library screening to ultimately yield pharmacologically superior d-peptide/d-protein therapeutics (mirror-image phage display); d-proteins can also be used as a powerful mechanistic tool for probing molecular events in biology. This review examines recent progress in the application of mirror image proteins to structural biology, drug discovery, and immunology.

  9. Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

    Science.gov (United States)

    Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

    2013-06-01

    Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

  10. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  11. 自噬蛋白NDP52与细菌感染的研究进展%Research Advances in Autophagy Protein NDP52 and Bacterial Infection

    Institute of Scientific and Technical Information of China (English)

    王芬芬; 张吉翔

    2012-01-01

    自噬(autophagy)是哺乳动物清除入侵细菌的主要途径,可保卫宿主细胞免受细菌的损伤.核点蛋白52(nuclear dot protein 52,NDP52)——核点家族成员之一,是除p62/SQSTM1和NBR1 等之外最新发现的自噬关键蛋白.它连接自噬体表面的微管相关蛋白1轻链3(microtubule associated protein l light chain 3,LC3),将披上“泛素大衣”的病原菌(如沙门氏菌和化脓性链球菌)递送至自噬体内加以清除.这一发现有助于人们深入了解自噬抵抗病原微生物感染的具体分子机制,为预防和治疗细菌感染提供了新靶点.%Autophagy is an important pathway to eliminate intracellular bacteria in mammals and defend the host cell from bacteria damage. In addition to p62/SQSTMl and NBR1, NDP52 (nuclear dot protein 52)—one of the members of nuclear dots, is a novel autophagy related protein has been found. NDP52 is an adaptor protein that binds to both ubiquitinated bacteria (such as Salmonella and Streptococcus pyogenes) and LC3 (microtubule associated protein 1 light chain 3), and then delivers these invaders into autophagosomes. This finding helps further understand the specific molecular mechanism of autophagy resisting pathogens infection, and provides a new target for the prevention and treatment of bacteria infection.

  12. Urine Protein and Urine Protein to Creatinine Ratio

    Science.gov (United States)

    ... limited. Home Visit Global Sites Search Help? Urine Protein and Urine Protein to Creatinine Ratio Share this page: Was this page helpful? Also known as: 24-Hour Urine Protein; Urine Total Protein; Urine Protein to Creatinine Ratio; ...

  13. Y682 mutation of amyloid precursor protein promotes endo-lysosomal dysfunction by disrupting APP-SorLA interaction

    Directory of Open Access Journals (Sweden)

    Luca Rosario La Rosa

    2015-04-01

    Full Text Available The intracellular transport and localization of amyloid precursor protein (APP are critical determinants of APP processing and β-amyloid peptide production, thus crucially important for the pathophysiology of Alzheimer’s disease (AD. Notably, the C-terminal Y682ENPTY687 domain of APP binds to specific adaptors controlling APP trafficking and sorting in neurons. Mutation on the Y682 residue to glycine (Y682G leads to altered APP sorting in hippocampal neurons that favors its accumulation in intracellular compartments and the release of soluble APPα. Such alterations induce premature aging and learning and cognitive deficits in APP Y682G mutant mice (APPYG/YG. Here, we report that Y682G mutation affects formation of the APP complex with sortilin-related receptor (SorLA, resulting in endo-lysosomal dysfunctions and neuronal degeneration. Moreover, disruption of the APP/SorLA complex changes the trafficking pathway of SorLA, with its consequent increase in secretion outside neurons. Mutations in the SorLA gene are a prognostic factor in AD, and increases in SorLA levels in cerebrospinal fluid are predictive of AD in humans. These results might open new possibilities in comprehending the role played by SorLA in its interaction with APP and in the progression of neuronal degeneration. In addition, they further underline the crucial role played by Y682 residue in controlling APP trafficking in neurons.

  14. IGSF9 Family Proteins

    DEFF Research Database (Denmark)

    Hansen, Maria; Walmod, Peter Schledermann

    2013-01-01

    The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...... facilitates homophilic cell adhesion. Moreover, IGSF9 family proteins have been implicated in the outgrowth and branching of neurites, axon guidance, synapse maturation, self-avoidance, and tiling. However, despite the few published studies on IGSF9 family proteins, reports on the functions of both Turtle...

  15. Role of cortactin and the adaptor proteins NCK and CRK in pedestal formation by entherpathogenic "Escherichia coli" enteropatógena (EPEC). Papel de cortactina y de las proteínas adaptadoras Nck y Crk en la formación del pedestal por "Escherichia coli" enteropatógena (EPEC)

    OpenAIRE

    Nieto Pelegrín, Elvira

    2013-01-01

    La adherencia de la bacteria Escherichia coli enteropatógena (EPEC) a las células del epitelio intestinal conlleva la formación de estructuras ricas en filamentos de actina denominadas pedestales. El proceso requiere la translocación del efector bacteriano Tir (del inglés translocated intimin receptor) en el citoplasma de la célula hospedadora a través del sistema de secreción tipo III (TTSS). La inserción de Tir en la membrana plasmática de la célula conlleva la fosforilación en residuos de ...

  16. Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

    Science.gov (United States)

    Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

    2015-01-01

    Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

  17. Cellular inhibitors of apoptosis proteins cIAP1 and cIAP2 are required for efficient caspase-1 activation by the inflammasome.

    Science.gov (United States)

    Labbé, Katherine; McIntire, Christian R; Doiron, Karine; Leblanc, Philippe M; Saleh, Maya

    2011-12-23

    Pathogen and danger recognition by the inflammasome activates inflammatory caspases that mediate inflammation and cell death. The cellular inhibitor of apoptosis proteins (cIAPs) function in apoptosis and innate immunity, but their role in modulating the inflammasome and the inflammatory caspases is unknown. Here we report that the cIAPs are critical effectors of the inflammasome and are required for efficient caspase-1 activation. cIAP1, cIAP2, and the adaptor protein TRAF2 interacted with caspase-1-containing complexes and mediated the activating nondegradative K63-linked polyubiquitination of caspase-1. Deficiency in cIAP1 (encoded by Birc2) or cIAP2 (Birc3) impaired caspase-1 activation after spontaneous or agonist-induced inflammasome assembly, and Birc2(-/-) or Birc3(-/-) mice or mice administered with an IAP antagonist had a dampened response to inflammasome agonists and were resistant to peritonitis. Our results describe a role for the cIAPs in innate immunity and further demonstrate the evolutionary conservation between cell death and inflammation mechanisms.

  18. The ER-associated protein ZDHHC1 is a positive regulator of DNA virus-triggered, MITA/STING-dependent innate immune signaling.

    Science.gov (United States)

    Zhou, Qian; Lin, Heng; Wang, Suyun; Wang, Shuai; Ran, Yong; Liu, Ying; Ye, Wen; Xiong, Xiaozhe; Zhong, Bo; Shu, Hong-Bing; Wang, Yan-Yi

    2014-10-01

    Viral DNA sensing within the cytosol of infected cells activates type I interferon (IFN) expression. MITA/STING plays an essential role in this pathway by acting as both a sensor for the second messenger cGAMP and as an adaptor for downstream signaling components. In an expression screen for proteins that can activate the IFNB1 promoter, we identified the ER-associated protein ZDHHC1 as a positive regulator of virus-triggered, MITA/STING-dependent immune signaling. Zdhhc1(-/-) cells failed to effectively produce IFNs and other cytokines in response to infection with DNA but not RNA viruses. Zdhhc1(-/-) mice infected with the neurotropic DNA virus HSV-1 exhibited lower cytokine levels and higher virus titers in the brain, resulting in higher lethality. ZDHHC1 constitutively associated with MITA/STING and mediates dimerization/aggregation of MITA/STING and recruitment of the downstream signaling components TBK1 and IRF3. These findings support a role for ZDHHC1 in mediating MITA/STING-dependent innate immune response against DNA viruses.

  19. Physics of protein motility and motor proteins

    Science.gov (United States)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  20. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  1. Gene expression profiling of TIR-domain-containing adaptor molecule (TICAM)in channel catfish Ictalurus punctatus challenged with different pathogens including bacteria and virus%斑点叉尾(鱼回)TICAM在细菌和病毒感染后的基因表达特征

    Institute of Scientific and Technical Information of China (English)

    王启龙; 李敏; 路飏; 黄爱平; 曾令兵; 王文琪; 陈松林; 沙珍霞

    2012-01-01

    In mammals, Toll-IL-1 receptor (TIR) domain-containing adaptor molecule 1(TICAM-1) is a signaling adaptor for TLR3 and TLR4 that activates the transcription factors IRF-3, NF-kB, and AP-1, leading to the induction of type I interferon and cytokines. TICAM is also identified in some fish species, however, the gene expression profiling of TICAM is largely unknown in teleosts. Because bacteria such as Aeromonas hydrophila , Streptococcus spp. And Edwardsiella tarda and viruses such as channel catfish virus cause a multisystemic disease responsible for severe losses in channel catfish aquaculture in China. In this study, gene expression profiling of TICAM in different immune tissues(iver, headkidney, spleen,and intestine) after infection with these pathogens assayed by quantitative RT-PCR was described. After infection with A. Hydrophila, TICAM was up-regulated approximately 2. 3-fold at 24 h in liver and 1. 9-fold at 12 h in spleen, while expression of this gene was down-regulated in headkidney and intestine, with the lowest expression as 0. 15-fold at 48 h in headkidney, 0. 53-fold at 24 h in intestine, respectively. TICAM was up-regulated drastically in liver, spleen, headkidney and intestine after infection with Streptococcus spp. It reached the highest level with 23-fold in liver at 7 d post infection, and it increased about 10 times in headkidney and spleen after infection. The expression of TICAM increased in all tested tissues after infection with E. Tarda, especially it was up-regulated to the highest (23. 1-fold) at 7d in spleen. After infection with channel catfish virus, the gene TICAM expression was up-regulated in liver, headkidney and intestine moderately, with the highest expression of 3. 7-fold in liver at 72 h, 2. 8-fold in headkidney at 7 d, 1. 5-fold at 24 h in intestine. However, it was down-regulated in spleen,and its lowest expression was 0. 13-fold at 24 h. In conclusion, the results of this study suggest that the TICAM gene may play crucial

  2. Protein and protein hydrolysates in sports nutrition.

    Science.gov (United States)

    van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

    2007-08-01

    With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

  3. Disorders of the calcium-sensing receptor and partner proteins: insights into the molecular basis of calcium homeostasis

    Science.gov (United States)

    Hannan, Fadil M; Babinsky, Valerie N

    2016-01-01

    The extracellular calcium (Ca2+o)-sensing receptor (CaSR) is a family C G protein-coupled receptor, which detects alterations in Ca2+o concentrations and modulates parathyroid hormone secretion and urinary calcium excretion. The central role of the CaSR in Ca2+o homeostasis has been highlighted by the identification of mutations affecting the CASR gene on chromosome 3q21.1. Loss-of-function CASR mutations cause familial hypocalciuric hypercalcaemia (FHH), whereas gain-of-function mutations lead to autosomal dominant hypocalcaemia (ADH). However, CASR mutations are only detected in ≤70% of FHH and ADH cases, referred to as FHH type 1 and ADH type 1, respectively, and studies in other FHH and ADH kindreds have revealed these disorders to be genetically heterogeneous. Thus, loss- and gain-of-function mutations of the GNA11 gene on chromosome 19p13.3, which encodes the G-protein α-11 (Gα11) subunit, lead to FHH type 2 and ADH type 2, respectively; whilst loss-of-function mutations of AP2S1 on chromosome 19q13.3, which encodes the adaptor-related protein complex 2 sigma (AP2σ) subunit, cause FHH type 3. These studies have demonstrated Gα11 to be a key mediator of downstream CaSR signal transduction, and also revealed a role for AP2σ, which is involved in clathrin-mediated endocytosis, in CaSR signalling and trafficking. Moreover, FHH type 3 has been demonstrated to represent a more severe FHH variant that may lead to symptomatic hypercalcaemia, low bone mineral density and cognitive dysfunction. In addition, calcimimetic and calcilytic drugs, which are positive and negative CaSR allosteric modulators, respectively, have been shown to be of potential benefit for these FHH and ADH disorders. PMID:27647839

  4. Protein electrophoresis - serum

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003540.htm Protein electrophoresis - serum To use the sharing features on ... JavaScript. This lab test measures the types of protein in the fluid (serum) part of a blood ...

  5. Protein Data Bank (PDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Protein Data Bank (PDB) archive is the single worldwide repository of information about the 3D structures of large biological molecules, including proteins and...

  6. CARMA3: A novel scaffold protein in regulation of NF-κB activation and diseases

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    CARD recruited membrane associated protein 3 (CARMA3) is a novel scaffold protein. It belongs to the CARMA protein family, and is known to activate nuclear factor (NF)- κB. However, it is still unknown which receptor functions upstream of CARMA3 to trigger NF-κB activation. Recently, several studies have demonstrated that CARMA3 serves as an indispensable adaptor protein in NF-κB signaling under some G protein-coupled receptors (GP- CRs), such as lysophosphatidic acid (LPA) receptor and angiotensin (Ang) Ⅱ receptor. Mechanistically, CARMA3 recruits its essential downstream molecules Bcl10 and MALT1 to form the CBM (CARMA3-Bcl10-MALT1) signalosome whereby it triggers NF-κB activation. GPCRs and NF-κB play pivotal roles in the regulation of various cellular functions, therefore, aberrant regulation of the GPCR/NF-κB signaling axis leads to the development of many types of diseases, such as cancer and atherogenesis. Recently, the GPCR/CARMA3/NF-κB signaling axis has been confirmed in these specific diseases and it plays crucial roles in the pathogenesis of disease progression. In ovarian cancer cell lines, knockdown of CARMA3 abolishes LPA receptor-induced NF-κB activation, and reduces LPA-induced ovarian cancer invasion. In vascular smooth cells, downregulation of CARMA3 substantially impairs Ang-Ⅱ-receptor-induced NF-κB activation, and in vivo studies have confirmed that Bcl10- deficient mice are protected from developing Ang-Ⅱ-receptor-induced atherosclerosis and aortic aneurysms. In this review, we summarize the biology of CARMA3, describe the role of the GPCR/CARMA3/NF-κB signaling axis in ovarian cancer and atherogenesis, and speculate about the potential roles of this signaling axis in other types of cancer and diseases. With a significant increase in the identification of LPA- and Ang-Ⅱ-like ligands, such as endothelin-1, which also activates NF-κB via CARMA3 and contributes to the development of many diseases, CARMA3 is emerging as a novel

  7. Abnormal protein aggregationand neurodegenerativediseases

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Abnormal protein aggregation or amyloid is the major cause ofmany neurodegenerative disorders. The present review focuses on the correlation between sequence and structure features of proteins related to the diseases and abnormal protein aggregation. Recent progress has improved our knowledge on understand-ing the mechanism of amyloid formation. We suggest a nucleation model for ordered protein aggregation, which can also explain pathogenesis mechanisms of these neurodegenerative diseases in vivo.

  8. Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.

    Science.gov (United States)

    Tunnicliffe, Richard B; Hautbergue, Guillaume M; Kalra, Priti; Jackson, Brian R; Whitehouse, Adrian; Wilson, Stuart A; Golovanov, Alexander P

    2011-01-06

    The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104-112 and 103-120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

  9. CSF total protein

    Science.gov (United States)

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  10. Protein domain prediction

    NARCIS (Netherlands)

    Ingolfsson, Helgi; Yona, Golan

    2008-01-01

    Domains are considered to be the building blocks of protein structures. A protein can contain a single domain or multiple domains, each one typically associated with a specific function. The combination of domains determines the function of the protein, its subcellular localization and the interacti

  11. Destabilized bioluminescent proteins

    Science.gov (United States)

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  12. Protein Frustratometer 2

    DEFF Research Database (Denmark)

    Gonzalo Parra, R.; Schafer, Nicholas P.; Radusky, Leandro G.

    2016-01-01

    The protein frustratometer is an energy landscape theory-inspired algorithm that aims at localizing and quantifying the energetic frustration present in protein molecules. Frustration is a useful concept for analyzing proteins' biological behavior. It compares the energy distributions of the nati...... a user-friendly interface. The webserver is freely available at URL: http://frustratometer.qb.fcen.uba.ar....

  13. Kaposi's sarcoma-associated herpesvirus Lana-1 is a major activator of the serum response element and mitogen-activated protein kinase pathways via interactions with the Mediator complex.

    Science.gov (United States)

    Roupelieva, Maria; Griffiths, Samantha J; Kremmer, Elisabeth; Meisterernst, Michael; Viejo-Borbolla, Abel; Schulz, Thomas; Haas, Jürgen

    2010-05-01

    In cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV), the activation of mitogen-activated protein kinase (MAPK) pathways plays a crucial role early after virus infection as well as during reactivation. In order to systematically identify viral proteins activating MAPK pathways in KSHV-infected cells, a clone collection of KSHV open reading frames (ORFs) was screened for induction of the serum response element (SRE), as SRE is induced by MAPKs. The strongest induction of the SRE was found with ORF73 (latency-associated nuclear antigen 1, or Lana-1), although weaker activation was also found with the kaposin B isoform, ORF54 (dUTPase) and ORF74 (G-protein-coupled receptor). The bipartite SRE is bound by a ternary complex consisting of serum response factor (SRF) and ternary complex factor. Lana-1 bound directly to SRF, but also to the MED25 (ARC92/ACID-1), MED15 (PCQAP) and MED23 (Sur-2) subunits of the Mediator complex, a multi-subunit transcriptional co-activator complex for RNA polymerase II. Lana-1-induced SRE activation was inhibited by the dominant-negative N-terminal domain of the MED25 mediator subunit, suggesting that this subunit mediates Lana-1-induced SRE activation. In summary, these data suggest a model in which Lana-1 acts as an adaptor between the transcription factor SRF and the basal transcriptional machinery.

  14. A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.

    Directory of Open Access Journals (Sweden)

    Mustapha Aouida

    Full Text Available Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p. The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA signaling and AMP activated protein kinase (AMPK phosphorylation in S. cerevisiae, which are

  15. A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

    KAUST Repository

    Aouida, Mustapha

    2013-06-07

    Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides\\' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to

  16. Protopia: a protein-protein interaction tool

    Science.gov (United States)

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  17. Protein crystallization with paper

    Science.gov (United States)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  18. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  19. Protein oxidation and peroxidation

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2016-01-01

    and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function....... Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides...

  20. Protein aggregate myopathies

    Directory of Open Access Journals (Sweden)

    Sharma M

    2005-01-01

    Full Text Available Protein aggregate myopathies (PAM are an emerging group of muscle diseases characterized by structural abnormalities. Protein aggregate myopathies are marked by the aggregation of intrinsic proteins within muscle fibers and fall into four major groups or conditions: (1 desmin-related myopathies (DRM that include desminopathies, a-B crystallinopathies, selenoproteinopathies caused by mutations in the, a-B crystallin and selenoprotein N1 genes, (2 hereditary inclusion body myopathies, several of which have been linked to different chromosomal gene loci, but with as yet unidentified protein product, (3 actinopathies marked by mutations in the sarcomeric ACTA1 gene, and (4 myosinopathy marked by a mutation in the MYH-7 gene. While PAM forms 1 and 2 are probably based on impaired extralysosomal protein degradation, resulting in the accumulation of numerous and diverse proteins (in familial types in addition to respective mutant proteins, PAM forms 3 and 4 may represent anabolic or developmental defects because of preservation of sarcomeres outside of the actin and myosin aggregates and dearth or absence of other proteins in these actin or myosin aggregates, respectively. The pathogenetic principles governing protein aggregation within muscle fibers and subsequent structural sarcomeres are still largely unknown in both the putative catabolic and anabolic forms of PAM. Presence of inclusions and their protein composition in other congenital myopathies such as reducing bodies, cylindrical spirals, tubular aggregates and others await clarification. The hitherto described PAMs were first identified by immunohistochemistry of proteins and subsequently by molecular analysis of their genes.

  1. Racemic protein crystallography.

    Science.gov (United States)

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  2. Toxic proteins in plants.

    Science.gov (United States)

    Dang, Liuyi; Van Damme, Els J M

    2015-09-01

    Plants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins. To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant's resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed.

  3. PROTEIN - WHICH IS BEST?

    Directory of Open Access Journals (Sweden)

    Michael J. Falvo

    2004-09-01

    Full Text Available Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids, whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function are also reviewed

  4. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  5. Protein Electrophoresis/Immunofixation Electrophoresis

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Protein Electrophoresis Immunofixation Electrophoresis Share this page: Was this page helpful? Also known as: Serum Protein Electrophoresis; Protein ELP; SPE; SPEP; Urine Protein Electrophoresis; ...

  6. Protein: FEA4 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEA4 Proteins in gibberellin signaling GID2 F-box protein GID2 Gibberellin-insensitive dwarf protein 2, Prot...ein GIBBERELLIN INSENSITIVE DWARF2 39947 Oryza sativa subsp. japonica Q7XAK4 ...

  7. Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique

    Institute of Scientific and Technical Information of China (English)

    Gui-Qin Bai; Yan Liu; Jun Cheng; Shu-Lin Zhang; Ya-Fei Yue; Yan-Ping Huang; Li-Ying Zhang

    2005-01-01

    AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection.METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BarmH-I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ.After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used.The mRNA of HepG2 cells transfected with pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector was isolated,and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR)method, and cDNA was synthesized. After digestion with restriction enzyme RcaI, cDNA fragments were obtained.Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification.RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete S was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86

  8. Regulation of the Src kinase-associated phosphoprotein 55 homologue by the protein tyrosine phosphatase PTP-PEST in the control of cell motility.

    Science.gov (United States)

    Ayoub, Emily; Hall, Anita; Scott, Adam M; Chagnon, Mélanie J; Miquel, Géraldine; Hallé, Maxime; Noda, Masaharu; Bikfalvi, Andreas; Tremblay, Michel L

    2013-09-06

    PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.

  9. Vibrio cholerae proteome-wide screen for immunostimulatory proteins identifies phosphatidylserine decarboxylase as a novel Toll-like receptor 4 agonist.

    Directory of Open Access Journals (Sweden)

    Ann Thanawastien

    2009-08-01

    Full Text Available Recognition of conserved bacterial components provides immediate and efficient immune responses and plays a critical role in triggering antigen-specific adaptive immunity. To date, most microbial components that are detected by host innate immune system are non-proteinaceous structural components. In order to identify novel bacterial immunostimulatory proteins, we developed a new high-throughput approach called "EPSIA", Expressed Protein Screen for Immune Activators. Out of 3,882 Vibrio cholerae proteins, we identified phosphatidylserine decarboxylase (PSD as a conserved bacterial protein capable of activating host innate immunity. PSD in concentrations as low as 100 ng/ml stimulated RAW264.7 murine macrophage cells and primary peritoneal macrophage cells to secrete TNFalpha and IL-6, respectively. PSD-induced proinflammatory response was dependent on the presence of MyD88, a known adaptor molecule for innate immune response. An enzymatically inactive PSD mutant and heat-inactivated PSD induced approximately 40% and approximately 15% of IL-6 production compared to that by native PSD, respectively. This suggests that PSD induces the production of IL-6, in part, via its enzymatic activity. Subsequent receptor screening determined TLR4 as a receptor mediating the PSD-induced proinflammatory response. Moreover, no detectable IL-6 was produced in TLR4-deficient mouse macrophages by PSD. PSD also exhibited a strong adjuvant activity against a co-administered antigen, BSA. Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo. Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant.

  10. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Amarnath Chtterjee; Ashutosh Kumar; Jeetender Chugh; Sudha Srivastava; Neel S Bhavesh; Ramakrishna V Hosur

    2005-01-01

    In the post-genomic era, as more and more genome sequences are becoming known and hectic efforts are underway to decode the information content in them, it is becoming increasingly evident that flexibility in proteins plays a crucial role in many of the biological functions. Many proteins have intrinsic disorder either wholly or in specific regions. It appears that this disorder may be important for regulatory functions of the proteins, on the one hand, and may help in directing the folding process to reach the compact native state, on the other. Nuclear magnetic resonance (NMR) has over the last two decades emerged as the sole, most powerful technique to help characterize these disordered protein systems. In this review, we first discuss the significance of disorder in proteins and then describe the recent developments in NMR methods for their characterization. A brief description of the results obtained on several disordered proteins is presented at the end.

  11. Computational Protein Design

    DEFF Research Database (Denmark)

    Johansson, Kristoffer Enøe

    Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

  12. Protein Models Comparator

    CERN Document Server

    Widera, Paweł

    2011-01-01

    The process of comparison of computer generated protein structural models is an important element of protein structure prediction. It has many uses including model quality evaluation, selection of the final models from a large set of candidates or optimisation of parameters of energy functions used in template free modelling and refinement. Although many protein comparison methods are available online on numerous web servers, their ability to handle a large scale model comparison is often very limited. Most of the servers offer only a single pairwise structural comparison, and they usually do not provide a model-specific comparison with a fixed alignment between the models. To bridge the gap between the protein and model structure comparison we have developed the Protein Models Comparator (pm-cmp). To be able to deliver the scalability on demand and handle large comparison experiments the pm-cmp was implemented "in the cloud". Protein Models Comparator is a scalable web application for a fast distributed comp...

  13. Mayaro virus proteins.

    Science.gov (United States)

    Mezencio, J M; Rebello, M A

    1993-01-01

    Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 +/- 2.3 nm in diameter. Three structural virus proteins were identified and designated p1, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in which three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein synthesized at 5 hours post-infection in both cell lines studied.

  14. Mayaro virus proteins

    Directory of Open Access Journals (Sweden)

    J. M. S. Mezencio

    1993-06-01

    Full Text Available Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%. The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 ñ 2.3 nm in diameter. Three structural virus proteins were identified and designated pl, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected. Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in wich three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein sinthesized at 5 hours post-infection in both cell lines studied.

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

    Directory of Open Access Journals (Sweden)

    Bishun D Prasad

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

  16. Proteins: Form and function

    OpenAIRE

    Roy D Sleator

    2012-01-01

    An overwhelming array of structural variants has evolved from a comparatively small number of protein structural domains; which has in turn facilitated an expanse of functional derivatives. Herein, I review the primary mechanisms which have contributed to the vastness of our existing, and expanding, protein repertoires. Protein function prediction strategies, both sequence and structure based, are also discussed and their associated strengths and weaknesses assessed.

  17. Pressure cryocooling protein crystals

    Science.gov (United States)

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  18. Acanthamoeba castellanii STAT Protein

    OpenAIRE

    Anna Kicinska; Jacek Leluk; Wieslawa Jarmuszkiewicz

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyos...

  19. Moonlighting proteins in cancer.

    Science.gov (United States)

    Min, Kyung-Won; Lee, Seong-Ho; Baek, Seung Joon

    2016-01-01

    Since the 1980s, growing evidence suggested that the cellular localization of proteins determined their activity and biological functions. In a classical view, a protein is characterized by the single cellular compartment where it primarily resides and functions. It is now believed that when proteins appear in different subcellular locations, the cells surpass the expected activity of proteins given the same genomic information to fulfill complex biological behavior. Many proteins are recognized for having the potential to exist in multiple locations in cells. Dysregulation of translocation may cause cancer or contribute to poorer cancer prognosis. Thus, quantitative and comprehensive assessment of dynamic proteins and associated protein movements could be a promising indicator in determining cancer prognosis and efficiency of cancer treatment and therapy. This review will summarize these so-called moonlighting proteins, in terms of a coupled intracellular cancer signaling pathway. Determination of the detailed biological intracellular and extracellular transit and regulatory activity of moonlighting proteins permits a better understanding of cancer and identification of potential means of molecular intervention.

  20. Self assembling proteins

    Science.gov (United States)

    Yeates, Todd O.; Padilla, Jennifer; Colovos, Chris

    2004-06-29

    Novel fusion proteins capable of self-assembling into regular structures, as well as nucleic acids encoding the same, are provided. The subject fusion proteins comprise at least two oligomerization domains rigidly linked together, e.g. through an alpha helical linking group. Also provided are regular structures comprising a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of a variety of nanostructures, where such structures include: cages, shells, double-layer rings, two-dimensional layers, three-dimensional crystals, filaments, and tubes.

  1. Acanthamoeba castellanii STAT protein.

    Directory of Open Access Journals (Sweden)

    Anna Kicinska

    Full Text Available STAT (signal transducers and activators of transcription proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil, a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

  2. Acanthamoeba castellanii STAT protein.

    Science.gov (United States)

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

  3. PIC: Protein Interactions Calculator.

    Science.gov (United States)

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside.

  4. Characterization of Metal Proteins

    Science.gov (United States)

    Unno, Masaki; Ikeda-Saito, Masao

    Some metals are essential for life. Most of these metals are associated with biological macromolecule like DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and more often with proteins: metals bind or interact with them. A number of protein molecules intrinsically contain metals in their structure. Some of these proteins catalyze unique chemical reactions and perform specific physiological functions. In this chapter, we will shed light on the several metalcontaining proteins, termed metalloproteins, and other proteins interacting metals. We will also introduce several key techniques which have been used to characterize these proteins. Characterizing these proteins and to understand the relationships between their structures and functions shall continue to be one of the major avenues to solve the mysteries of life. At first, we introduce what are the protein structures and how these proteins interact with metals. In the next section, we discuss the physiological roles of some representative metals. Next, we show two examples of special metal cofactors those help the biological macromolecules to carry out their functions. Then we describe some functions of metalloproteins. Finally, we introduce some physical methods to characterize metalloproteins.

  5. Dietary proteins and angiogenesis.

    Science.gov (United States)

    Medina, Miguel Ángel; Quesada, Ana R

    2014-01-17

    Both defective and persistent angiogenesis are linked to pathological situations in the adult. Compounds able to modulate angiogenesis have a potential value for the treatment of such pathologies. Several small molecules present in the diet have been shown to have modulatory effects on angiogenesis. This review presents the current state of knowledge on the potential modulatory roles of dietary proteins on angiogenesis. There is currently limited available information on the topic. Milk contains at least three proteins for which modulatory effects on angiogenesis have been previously demonstrated. On the other hand, there is some scarce information on the potential of dietary lectins, edible plant proteins and high protein diets to modulate angiogenesis.

  6. [Alternative scaffold proteins].

    Science.gov (United States)

    Petrovskaia, L E; Shingarova, L N; Dolgikh, D A; Kirpichnikov, M P

    2011-01-01

    Review is devoted to the challenging direction in modem molecular biology and bioengineering - the properties of alternative scaffold proteins (ASP) and methods for obtaining ASP binding molecules. ASP molecules incorporate conservative protein core and hypervariable regions, providing for the binding function. Structural classification of ASP includes several types which differ also in their molecular targets and potential applications. Construction of artificial binding proteins on the ASP basis implies a combinatorial library design with subsequent selection of specific binders with the use of phage display or the modem cell-free systems. Alternative binding proteins on non-immunoglobulin scaffolds find broad applications in different fields ofbiotechnology and molecular medicine.

  7. Ultrafiltration of pegylated proteins

    Science.gov (United States)

    Molek, Jessica R.

    There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

  8. Distinct and overlapping functions of the cullin E3 ligase scaffolding proteins CUL4A and CUL4B

    Science.gov (United States)

    Hannah, Jeffrey

    2016-01-01

    The cullin 4 subfamily of genes includes CUL4A and CUL4B, which share a mostly identical amino acid sequence aside from the elongated N-terminal region in CUL4B. Both act as scaffolding proteins for modular cullin RING ligase 4 (CRL4) complexes which promote the ubiquitination of a variety of substrates. CRL4 function is vital to cells as loss of both genes or their shared substrate adaptor protein DDB1 halts proliferation and eventually leads to cell death. Due to their high structural similarity, CUL4A and CUL4B share a substantial overlap in function. However, in some cases, differences in subcellular localization, spatiotemporal expression patterns and stress-inducibility preclude functional compensation. In this review, we highlight the most essential functions of the CUL4 genes in: DNA repair and replication, chromatin-remodeling, cell cycle regulation, embryogenesis, hematopoiesis and spermatogenesis. CUL4 genes are also clinically relevant as dysregulation can contribute to the onset of cancer and CRL4 complexes are often hijacked by certain viruses to promote viral replication and survival. Also, mutations in CUL4B have been implicated in a subset of patients suffering from syndromic X-linked intellectual disability (AKA mental retardation). Interestingly, the antitumor effects of immunomodulatory drugs are caused by their binding to the CRL4CRBN complex and re-directing the E3 ligase towards the Ikaros transcription factors IKZF1 and IKZF3. Because of their influence over key cellular functions and relevance to human disease, CRL4s are considered promising targets for therapeutic intervention. PMID:26344709

  9. Protein oxidation in aquatic foods

    DEFF Research Database (Denmark)

    Baron, Caroline P.

    2014-01-01

    The chapter discusses general considerations about protein oxidation and reviews the mechanisms involved in protein oxidation and consequences of protein oxidation on fish proteins. It presents two case studies, the first deals with protein and lipid oxidation in frozen rainbow trout, and the sec......The chapter discusses general considerations about protein oxidation and reviews the mechanisms involved in protein oxidation and consequences of protein oxidation on fish proteins. It presents two case studies, the first deals with protein and lipid oxidation in frozen rainbow trout...... oxidation. The protein carbonyl group measurement is the widely used method for estimating protein oxidation in foods and has been used in fish muscle. The chapter also talks about the impact of protein oxidation on protein functionality, fish muscle texture, and food nutritional value. Protein oxidation...... may not only induce quality losses but may be desirable in some type of foods, such as salted herring....

  10. SwissProt search result: AK110215 [KOME

    Lifescience Database Archive (English)

    Full Text Available pha-adaptin C) (Adaptor protein complex AP-2 alpha-2 subunit) (Clathrin assembly protein complex 2 alpha-C large chain) (100 kDa coat...ed vesicle protein C) (Plasma membrane adaptor HA AP2A2_RAT 6e-80 ...

  11. SwissProt search result: AK110215 [KOME

    Lifescience Database Archive (English)

    Full Text Available pha-adaptin C) (Adaptor protein complex AP-2 alpha-2 subunit) (Clathrin assembly protein complex 2 alpha-C large chain) (100 kDa coat...ed vesicle protein C) (Plasma membrane adaptor AP2A2_HUMAN 1e-78 ...

  12. SwissProt search result: AK110215 [KOME

    Lifescience Database Archive (English)

    Full Text Available pha-adaptin A) (Adaptor protein complex AP-2 alpha-1 subunit) (Clathrin assembly protein complex 2 alpha-A large chain) (100 kDa coat...ed vesicle protein A) (Plasma membrane adaptor AP2A1_MOUSE 1e-77 ...

  13. SwissProt search result: AK110375 [KOME

    Lifescience Database Archive (English)

    Full Text Available pha-adaptin C) (Adaptor protein complex AP-2 alpha-2 subunit) (Clathrin assembly protein complex 2 alpha-C large chain) (100 kDa coat...ed vesicle protein C) (Plasma membrane adaptor HA AP2A2_RAT 2e-15 ...

  14. SwissProt search result: AK110375 [KOME

    Lifescience Database Archive (English)

    Full Text Available pha-adaptin A) (Adaptor protein complex AP-2 alpha-1 subunit) (Clathrin assembly protein complex 2 alpha-A large chain) (100 kDa coat...ed vesicle protein A) (Plasma membrane adaptor AP2A1_MOUSE 1e-16 ...

  15. SwissProt search result: AK110375 [KOME

    Lifescience Database Archive (English)

    Full Text Available pha-adaptin C) (Adaptor protein complex AP-2 alpha-2 subunit) (Clathrin assembly protein complex 2 alpha-C large chain) (100 kDa coat...ed vesicle protein C) (Plasma membrane adaptor AP2A2_HUMAN 3e-15 ...

  16. A SNARE-like superfamily protein SbSLSP from the halophyte Salicornia brachiata confers salt and drought tolerance by maintaining membrane stability, K+/Na+ ratio, and antioxidant machinery

    Directory of Open Access Journals (Sweden)

    Dinkar eSingh

    2016-06-01

    Full Text Available About 1000 salt-responsive ESTs were identified from an extreme halophyte Salicornia brachiata. Among these, a novel salt-inducible gene SbSLSP, (Salicornia brachiata SNARE-like superfamily protein showed up-regulation upon salinity and dehydration stress. The presence of cis-regulatory motifs related to abiotic stress in the putative promoter region supports our finding that SbSLSP gene is inducible by abiotic stress. The SbSLSP protein showed a high sequence identity to hypothetical/uncharacterised proteins from Beta vulgaris, Spinacia oleracea, Eucalyptus grandis and Prunus persica and with SNARE-like superfamily proteins from Zostera marina and Arabidopsis thaliana. Bioinformatics analysis predicted a clathrin adaptor complex small-chain domain and N-myristoylation site in the SbSLSP protein. Subcellular localisation studies indicated that the SbSLSP protein is mainly localised in the plasma membrane. Using transgenic tobacco lines, we establish that overexpression of SbSLSP resulted in elevated tolerance to salt and drought stress. The improved tolerance was confirmed by alterations in a range of physiological parameters, including high germination and survival rate, higher leaf chlorophyll contents, and reduced accumulation of Na+ ion and reactive oxygen species (ROS. Furthermore, overexpressing lines also showed lower water loss, higher cell membrane stability and increased accumulation of proline and ROS-scavenging enzymes. Overexpression of SbSLSP also enhanced the transcript levels of ROS-scavenging and signalling enzyme genes. This study is the first investigation of the function of the SbSLSP gene as a novel determinant of salinity/drought tolerance. The results suggest that SbSLSP could be a potential candidate to increase salinity and drought tolerance in crop plants for sustainable agriculture in semi-arid saline soil.

  17. A SNARE-Like Superfamily Protein SbSLSP from the Halophyte Salicornia brachiata Confers Salt and Drought Tolerance by Maintaining Membrane Stability, K(+)/Na(+) Ratio, and Antioxidant Machinery.

    Science.gov (United States)

    Singh, Dinkar; Yadav, Narendra Singh; Tiwari, Vivekanand; Agarwal, Pradeep K; Jha, Bhavanath

    2016-01-01

    About 1000 salt-responsive ESTs were identified from an extreme halophyte Salicornia brachiata. Among these, a novel salt-inducible gene SbSLSP (Salicornia brachiata SNARE-like superfamily protein), showed up-regulation upon salinity and dehydration stress. The presence of cis-regulatory motifs related to abiotic stress in the putative promoter region supports our finding that SbSLSP gene is inducible by abiotic stress. The SbSLSP protein showed a high sequence identity to hypothetical/uncharacterized proteins from Beta vulgaris, Spinacia oleracea, Eucalyptus grandis, and Prunus persica and with SNARE-like superfamily proteins from Zostera marina and Arabidopsis thaliana. Bioinformatics analysis predicted a clathrin adaptor complex small-chain domain and N-myristoylation site in the SbSLSP protein. Subcellular localization studies indicated that the SbSLSP protein is mainly localized in the plasma membrane. Using transgenic tobacco lines, we establish that overexpression of SbSLSP resulted in elevated tolerance to salt and drought stress. The improved tolerance was confirmed by alterations in a range of physiological parameters, including high germination and survival rate, higher leaf chlorophyll contents, and reduced accumulation of Na(+) ion and reactive oxygen species (ROS). Furthermore, overexpressing lines also showed lower water loss, higher cell membrane stability, and increased accumulation of proline and ROS-scavenging enzymes. Overexpression of SbSLSP also enhanced the transcript levels of ROS-scavenging and signaling enzyme genes. This study is the first investigation of the function of the SbSLSP gene as a novel determinant of salinity/drought tolerance. The results suggest that SbSLSP could be a potential candidate to increase salinity and drought tolerance in crop plants for sustainable agriculture in semi-arid saline soil.

  18. A SNARE-Like Superfamily Protein SbSLSP from the Halophyte Salicornia brachiata Confers Salt and Drought Tolerance by Maintaining Membrane Stability, K+/Na+ Ratio, and Antioxidant Machinery

    Science.gov (United States)

    Singh, Dinkar; Yadav, Narendra Singh; Tiwari, Vivekanand; Agarwal, Pradeep K.; Jha, Bhavanath

    2016-01-01

    About 1000 salt-responsive ESTs were identified from an extreme halophyte Salicornia brachiata. Among these, a novel salt-inducible gene SbSLSP (Salicornia brachiata SNARE-like superfamily protein), showed up-regulation upon salinity and dehydration stress. The presence of cis-regulatory motifs related to abiotic stress in the putative promoter region supports our finding that SbSLSP gene is inducible by abiotic stress. The SbSLSP protein showed a high sequence identity to hypothetical/uncharacterized proteins from Beta vulgaris, Spinacia oleracea, Eucalyptus grandis, and Prunus persica and with SNARE-like superfamily proteins from Zostera marina and Arabidopsis thaliana. Bioinformatics analysis predicted a clathrin adaptor complex small-chain domain and N-myristoylation site in the SbSLSP protein. Subcellular localization studies indicated that the SbSLSP protein is mainly localized in the plasma membrane. Using transgenic tobacco lines, we establish that overexpression of SbSLSP resulted in elevated tolerance to salt and drought stress. The improved tolerance was confirmed by alterations in a range of physiological parameters, including high germination and survival rate, higher leaf chlorophyll contents, and reduced accumulation of Na+ ion and reactive oxygen species (ROS). Furthermore, overexpressing lines also showed lower water loss, higher cell membrane stability, and increased accumulation of proline and ROS-scavenging enzymes. Overexpression of SbSLSP also enhanced the transcript levels of ROS-scavenging and signaling enzyme genes. This study is the first investigation of the function of the SbSLSP gene as a novel determinant of salinity/drought tolerance. The results suggest that SbSLSP could be a potential candidate to increase salinity and drought tolerance in crop plants for sustainable agriculture in semi-arid saline soil. PMID:27313584

  19. An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.

    Directory of Open Access Journals (Sweden)

    Mariana Serpeloni

    Full Text Available In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II, but not RNA polymerase I (RNA pol I or Spliced Leader (SL transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and

  20. Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates.

    Science.gov (United States)

    McGregor, Lynn M; Jain, Tara; Liu, David R

    2014-02-26

    We describe the development and validation of interaction determination using unpurified proteins (IDUP), a method that selectively amplifies DNA sequences identifying ligand+target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. By operating in cell lysates, IDUP preserves native post-translational modifications and interactions with endogenous binding partners, thereby enabling the study of difficult-to-purify targets and increasing the potential biological relevance of detected interactions compared with methods that require purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-linked antibody or covalently using a SNAP-tag. Ligand-target binding promotes hybridization of a self-priming hairpin that is extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing. IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+FKBP, Kd ≈ 100 fM; FKBP·rapamycin+FRB, Kd = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, Kd ≈ 0.2 nM; rapamcyin+FRB, Kd = 26 μM). Finally, IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences corresponding to five known ligand+target pairs ranging in binding

  1. The protein ATG16L1 suppresses inflammatory cytokines induced by the intracellular sensors Nod1 and Nod2 in an autophagy-independent manner.

    Science.gov (United States)

    Sorbara, Matthew T; Ellison, Lisa K; Ramjeet, Mahendrasingh; Travassos, Leonardo H; Jones, Nicola L; Girardin, Stephen E; Philpott, Dana J

    2013-11-14

    The peptidoglycan sensor Nod2 and the autophagy protein ATG16L1 have been linked to Crohn's disease (CD). Although Nod2 and the related sensor, Nod1, direct ATG16L1 to initiate anti-bacterial autophagy, whether ATG16L1 affects Nod-driven inflammation has not been examined. Here, we uncover an unanticipated autophagy-independent role for ATG16L1 in negatively regulating Nod-driven inflammatory responses. Knockdown of ATG16L1 expression, but not that of ATG5 or ATG9a, specifically enhanced Nod-driven cytokine production. In addition, autophagy-incompetent truncated forms of ATG16L1 regulated Nod-driven cytokine responses. Mechanistically, we demonstrated that ATG16L1 interfered with poly-ubiquitination of the Rip2 adaptor and recruitment of Rip2 into large signaling complexes. The CD-associated allele of ATG16L1 was impaired in its ability to regulate Nod-driven inflammatory responses. Overall, these results suggest that ATG16L1 is critical for Nod-dependent regulation of cytokine responses and that disruption of this Nod1- or Nod2-ATG16L1 signaling axis could contribute to the chronic inflammation associated with CD.

  2. TRIM14 inhibits hepatitis C virus infection by SPRY domain-dependent targeted degradation of the viral NS5A protein

    Science.gov (United States)

    Wang, Shanshan; Chen, Yongzhi; Li, Chunfeng; Wu, Yaoxing; Guo, Lei; Peng, Changwei; Huang, Yueping; Cheng, Genhong; Qin, F. Xiao-Feng

    2016-01-01

    Tripartite motif 14 (TRIM14) was reported to function as a mitochondrial signaling adaptor in mediating innate immune responses. However, the involvement of TRIM14 in host defense against viral infection and molecular mechanisms remain unclear. Here, we demonstrated that enforced expression of TRIM14 could potently inhibit the infection and replication of HCV in hepatocytes, whereas TRIM14 knockout cells became more susceptible to HCV infection. Interestingly, further experiments revealed that such anti-HCV activity was independent of activating the NF-κB or interferon pathways but required the C-terminal SPRY domain of no signaling capacity. In searching for mechanisms how TRIM14 exerts its antiviral function we found that TRIM14 interacted with HCV encoded non-structural protein NS5A and could strongly induce its degradation dependent on the NS5A1 subdomain. Interestingly extensive domain mapping analyses revealed that NS5A degradation was mediated by the highly conserved SPRY domain of TRIM14, which might involve the K48 ubiquitination pathway. Collectively, our work uncovered a new mechanism responsible for host defense against HCV infection, and could potentially aid the development of novel anti-HCV therapeutics. PMID:27578425

  3. Effect of proline rich domain of an RNA-binding protein Sam68 in cell growth process, death and B cell signal transduction

    Institute of Scientific and Technical Information of China (English)

    LI Qing-hua; FAN Tian-xue; PANG Tian-xiang; YUAN Wen-su; HAN Zhong-chao

    2006-01-01

    Background Sam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line.Methods By using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometfic analyses were performed to investigate the Sam68 functions.Results A slower growth rate (2.1 hours growth elongation) and longer S phase (1.7 hours elongation) was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells.Conclusions The proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.

  4. The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

    Science.gov (United States)

    Warner, T S; Sinclair, D A; Fitzpatrick, K A; Singh, M; Devlin, R H; Honda, B M

    1998-04-01

    Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

  5. Protein and Heart Health

    Science.gov (United States)

    ... of food? Drink an 8-ounce glass of milk, and you’ll log 8 grams of protein. Add a cup of yogurt for another 11 ... low-fat options, such as lean meats, skim milk or other foods with high levels of protein. Legumes, for example, can pack about 16 grams ...

  6. Brushes and proteins

    NARCIS (Netherlands)

    Bosker, W.T.E.

    2011-01-01

      Brushes and Proteins   Wouter T. E. Bosker         Protein adsorption at solid surfaces can be prevented by applying a polymer brush at the surface. A polymer brush consists of polymer chains end-grafted to the surface at such a grafting density that th

  7. Poxviral Ankyrin Proteins

    Directory of Open Access Journals (Sweden)

    Michael H. Herbert

    2015-02-01

    Full Text Available Multiple repeats of the ankyrin motif (ANK are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.

  8. Learning about Proteins

    Science.gov (United States)

    ... Emergency Room? What Happens in the Operating Room? Learning About Proteins KidsHealth > For Kids > Learning About Proteins A A A What's in this ... Nutrition & Fitness Center Minerals Vitamins Eating for Sports Learning About Fats MyPlate Food Guide Word! Nutrition Contact ...

  9. Protein Attachment on Nanodiamonds.

    Science.gov (United States)

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  10. Engineered Protein Polymers

    Science.gov (United States)

    2010-05-31

    of each pure polymer, we plan to combine the various polymer solutions in different ratios to tune the composition and physico-chemical properties...protein materials as vehicles for storage and delivery of small molecules. Each protein polymer under concentrations for particle formation ( vida

  11. Sensitizing properties of proteins

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott;

    2014-01-01

    The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding...... the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein...... Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary...

  12. Unconventional protein secretion.

    Science.gov (United States)

    Ding, Yu; Wang, Juan; Wang, Junqi; Stierhof, York-Dieter; Robinson, David G; Jiang, Liwen

    2012-10-01

    It is generally believed that protein secretion or exocytosis is achieved via a conventional ER (endoplasmic reticulum)-Golgi-TGN (trans-Golgi network)-PM (plasma membrane) pathway in the plant endomembrane system. However, such signal peptide (SP)-dependent protein secretion cannot explain the increasing number of SP-lacking proteins which are found outside of the PM in plant cells. The process by which such leaderless secretory proteins (LSPs) gain access to the cell exterior is termed unconventional protein secretion (UPS) and has been well-studied in animal and yeast cells, but largely ignored by the plant community. Here, we review the evidence for UPS in plants especially in regard to the recently discovered EXPO (exocyst-positive-organelle).

  13. Protein Unfolding and Alzheimer's

    Science.gov (United States)

    Cheng, Kelvin

    2012-10-01

    Early interaction events of beta-amyloid (Aβ) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single Aβ interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of Aβ in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the Aβ interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the Aβ proteins.

  14. Manipulating and Visualizing Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Simon, Horst D.

    2003-12-05

    ProteinShop Gives Researchers a Hands-On Tool for Manipulating, Visualizing Protein Structures. The Human Genome Project and other biological research efforts are creating an avalanche of new data about the chemical makeup and genetic codes of living organisms. But in order to make sense of this raw data, researchers need software tools which let them explore and model data in a more intuitive fashion. With this in mind, researchers at Lawrence Berkeley National Laboratory and the University of California, Davis, have developed ProteinShop, a visualization and modeling program which allows researchers to manipulate protein structures with pinpoint control, guided in large part by their own biological and experimental instincts. Biologists have spent the last half century trying to unravel the ''protein folding problem,'' which refers to the way chains of amino acids physically fold themselves into three-dimensional proteins. This final shape, which resembles a crumpled ribbon or piece of origami, is what determines how the protein functions and translates genetic information. Understanding and modeling this geometrically complex formation is no easy matter. ProteinShop takes a given sequence of amino acids and uses visualization guides to help generate predictions about the secondary structures, identifying alpha helices and flat beta strands, and the coil regions that bind them. Once secondary structures are in place, researchers can twist and turn these pre-configurations until they come up with a number of possible tertiary structure conformations. In turn, these are fed into a computationally intensive optimization procedure that tries to find the final, three-dimensional protein structure. Most importantly, ProteinShop allows users to add human knowledge and intuition to the protein structure prediction process, thus bypassing bad configurations that would otherwise be fruitless for optimization. This saves compute cycles and accelerates

  15. Transdermal delivery of proteins.

    Science.gov (United States)

    Kalluri, Haripriya; Banga, Ajay K

    2011-03-01

    Transdermal delivery of peptides and proteins avoids the disadvantages associated with the invasive parenteral route of administration and other alternative routes such as the pulmonary and nasal routes. Since proteins have a large size and are hydrophilic in nature, they cannot permeate passively across the skin due to the stratum corneum which allows the transport of only small lipophilic drug molecules. Enhancement techniques such as chemical enhancers, iontophoresis, microneedles, electroporation, sonophoresis, thermal ablation, laser ablation, radiofrequency ablation and noninvasive jet injectors aid in the delivery of proteins by overcoming the skin barrier in different ways. In this review, these enhancement techniques that can enable the transdermal delivery of proteins are discussed, including a discussion of mechanisms, sterility requirements, and commercial development of products. Combination of enhancement techniques may result in a synergistic effect allowing increased protein delivery and these are also discussed.

  16. Extracellular BCL2 proteins are danger-associated molecular patterns that reduce tissue damage in murine models of ischemia-reperfusion injury.

    Directory of Open Access Journals (Sweden)

    Akiko Iwata

    Full Text Available BACKGROUND: Ischemia-reperfusion (I/R injury contributes to organ dysfunction in a variety of clinical disorders, including myocardial infarction, stroke, organ transplantation, and hemorrhagic shock. Recent investigations have demonstrated that apoptosis as an important mechanism of cell death leading to organ dysfunction following I/R. Intracellular danger-associated molecular patterns (DAMPs released during cell death can activate cytoprotective responses by engaging receptors of the innate immune system. METHODOLOGY/PRINCIPAL FINDINGS: Ischemia was induced in the mouse hind limb by tourniquet or in the heart by coronary artery ligation. Reperfusion injury of skeletal or cardiac muscle was markedly reduced by intraperitoneal or subcutaneous injection of recombinant human (rhBCL2 protein or rhBCL2-related protein A1 (BCL2A1 (50 ng/g given prior to ischemia or at the time of reperfusion. The cytoprotective activity of extracellular rhBCL2 or rhBCL2A1 protein was mapped to the BH4 domain, as treatment with a mutant BCL2 protein lacking the BH4 domain was not protective, whereas peptides derived from the BH4 domain of BCL2 or the BH4-like domain of BCL2A1 were. Protection by extracellular rhBCL2 or rhBCL2A1 was associated with a reduction in apoptosis in skeletal and cardiac muscle following I/R, concomitant with increased expression of endogenous mouse BCL2 (mBCL2 protein. Notably, treatment with rhBCL2A1 protein did not protect mice deficient in toll-like receptor-2 (TLR2 or the adaptor protein, myeloid differentiation factor-88 (MyD88. CONCLUSIONS/SIGNIFICANCE: Treatment with cytokine-like doses of rhBCL2 or rhBCL2A1 protein or BH4-domain peptides reduces apoptosis and tissue injury following I/R by a TLR2-MyD88-dependent mechanism. These findings establish a novel extracellular cytoprotective activity of BCL2 BH4-domain proteins as potent cytoprotective DAMPs.

  17. Endocytosis of the Aspartic Acid/Glutamic Acid Transporter Dip5 Is Triggered by Substrate-Dependent Recruitment of the Rsp5 Ubiquitin Ligase via the Arrestin-Like Protein Aly2 ▿

    Science.gov (United States)

    Hatakeyama, Riko; Kamiya, Masao; Takahara, Terunao; Maeda, Tatsuya

    2010-01-01

    Endocytosis of nutrient transporters is stimulated under various conditions, such as elevated nutrient availability. In Saccharomyces cerevisiae, endocytosis is triggered by ubiquitination of transporters catalyzed by the E3 ubiquitin ligase Rsp5. However, how the ubiquitination is accelerated under certain conditions remains obscure. Here we demonstrate that closely related proteins Aly2/Art3 and Aly1/Art6, which are poorly characterized members of the arrestin-like protein family, mediate endocytosis of the aspartic acid/glutamic acid transporter Dip5. In aly2Δ cells, Dip5 is stabilized at the plasma membrane and is not endocytosed efficiently. Efficient ubiquitination of Dip5 is dependent on Aly2. aly1Δ cells also show deficiency in Dip5 endocytosis, although less remarkably than aly2Δ cells. Aly2 physically interacts in vivo with Rsp5 at its PY motif and also with Dip5, thus serving as an adaptor linking Rsp5 with Dip5 to achieve Dip5 ubiquitination. Importantly, the interaction between Aly2 and Dip5 is accelerated in response to elevated aspartic acid availability. This result indicates that the regulation of Dip5 endocytosis is accomplished by dynamic recruitment of Rsp5 via Aly2. PMID:20956561

  18. Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) negatively regulates the TRAF2 ubiquitin-dependent pathway by suppressing the TRAF2-sphingosine 1-phosphate (S1P) interaction.

    Science.gov (United States)

    Park, Eui-Soon; Choi, Seunga; Shin, Bongjin; Yu, Jungeun; Yu, Jiyeon; Hwang, Jung-Me; Yun, Hyeongseok; Chung, Young-Ho; Choi, Jong-Soon; Choi, Yongwon; Rho, Jaerang

    2015-04-10

    The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys(63)-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.

  19. Coarse-grain modelling of protein-protein interactions

    NARCIS (Netherlands)

    Baaden, Marc; Marrink, Siewert J.

    2013-01-01

    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are separate

  20. Protein Binding Pocket Dynamics.

    Science.gov (United States)

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  1. Anchored design of protein-protein interfaces.

    Directory of Open Access Journals (Sweden)

    Steven M Lewis

    Full Text Available BACKGROUND: Few existing protein-protein interface design methods allow for extensive backbone rearrangements during the design process. There is also a dichotomy between redesign methods, which take advantage of the native interface, and de novo methods, which produce novel binders. METHODOLOGY: Here, we propose a new method for designing novel protein reagents that combines advantages of redesign and de novo methods and allows for extensive backbone motion. This method requires a bound structure of a target and one of its natural binding partners. A key interaction in this interface, the anchor, is computationally grafted out of the partner and into a surface loop on the design scaffold. The design scaffold's surface is then redesigned with backbone flexibility to create a new binding partner for the target. Careful choice of a scaffold will bring experimentally desirable characteristics into the new complex. The use of an anchor both expedites the design process and ensures that binding proceeds against a known location on the target. The use of surface loops on the scaffold allows for flexible-backbone redesign to properly search conformational space. CONCLUSIONS AND SIGNIFICANCE: This protocol was implemented within the Rosetta3 software suite. To demonstrate and evaluate this protocol, we have developed a benchmarking set of structures from the PDB with loop-mediated interfaces. This protocol can recover the correct loop-mediated interface in 15 out of 16 tested structures, using only a single residue as an anchor.

  2. PSC: protein surface classification.

    Science.gov (United States)

    Tseng, Yan Yuan; Li, Wen-Hsiung

    2012-07-01

    We recently proposed to classify proteins by their functional surfaces. Using the structural attributes of functional surfaces, we inferred the pairwise relationships of proteins and constructed an expandable database of protein surface classification (PSC). As the functional surface(s) of a protein is the local region where the protein performs its function, our classification may reflect the functional relationships among proteins. Currently, PSC contains a library of 1974 surface types that include 25,857 functional surfaces identified from 24,170 bound structures. The search tool in PSC empowers users to explore related surfaces that share similar local structures and core functions. Each functional surface is characterized by structural attributes, which are geometric, physicochemical or evolutionary features. The attributes have been normalized as descriptors and integrated to produce a profile for each functional surface in PSC. In addition, binding ligands are recorded for comparisons among homologs. PSC allows users to exploit related binding surfaces to reveal the changes in functionally important residues on homologs that have led to functional divergence during evolution. The substitutions at the key residues of a spatial pattern may determine the functional evolution of a protein. In PSC (http://pocket.uchicago.edu/psc/), a pool of changes in residues on similar functional surfaces is provided.

  3. Bacterial Ice Crystal Controlling Proteins

    Directory of Open Access Journals (Sweden)

    Janet S. H. Lorv

    2014-01-01

    Full Text Available Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

  4. The anaphase-promoting complex protein 5 (AnapC5 associates with A20 and inhibits IL-17-mediated signal transduction.

    Directory of Open Access Journals (Sweden)

    Allen W Ho

    Full Text Available IL-17 is the founding member of a family of cytokines and receptors with unique structures a