Science.gov

Sample records for chaperone chz1p regulates

  1. Chaperoning erythropoiesis

    PubMed Central

    dos Santos, Camila O.

    2009-01-01

    Multisubunit complexes containing molecular chaperones regulate protein production, stability, and degradation in virtually every cell type. We are beginning to recognize how generalized and tissue-specific chaperones regulate specialized aspects of erythropoiesis. For example, chaperones intersect with erythropoietin signaling pathways to protect erythroid precursors against apoptosis. Molecular chaperones also participate in hemoglobin synthesis, both directly and indirectly. Current knowledge in these areas only scratches the surface of what is to be learned. Improved understanding of how molecular chaperones regulate erythropoietic development and hemoglobin homeostasis should identify biochemical pathways amenable to pharmacologic manipulation in a variety of red blood cell disorders including thalassemia and other anemias associated with hemoglobin instability. PMID:19109556

  2. Regulation of organismal proteostasis by trans-cellular chaperone signaling

    PubMed Central

    van Oosten-Hawle, Patricija; Porter, Robert S.; Morimoto, Richard I.

    2013-01-01

    Summary A major challenge for metazoans is to ensure that different tissues each expressing distinctive proteomes are, nevertheless, well protected at an organismal level from proteotoxic stress. We have examined this and show that expression of endogenous metastable protein sensors in muscle cells induces a systemic stress response throughout multiple tissues of C. elegans. Suppression of misfolding in muscle cells can be achieved not only by enhanced expression of HSP90 in muscle cells, but as effective by elevated expression of HSP90 in intestine or neuronal cells. This cell-non-autonomous control of HSP90 expression relies upon transcriptional feedback between somatic tissues that is regulated by the FoxA transcription factor PHA-4. This trans-cellular chaperone signaling response maintains organismal proteostasis when challenged by a local tissue imbalance in folding and provides the basis for a novel form of organismal stress sensing surveillance. PMID:23746847

  3. Regulation of Neuronal Survival Factor MEF2D by Chaperone-Mediated Autophagy

    PubMed Central

    Yang, Qian; She, Hua; Gearing, Marla; Colla, Emanuela; Lee, Michael; Shacka, John J.; Mao, Zixu

    2009-01-01

    Chaperone-mediated autophagy controls the degradation of selective cytosolic proteins and may protect neurons against degeneration. In a neuronal cell line, we found that chaperone-mediated autophagy regulated the activity of myocyte enhancer factor 2D (MEF2D), a transcription factor required for neuronal survival. MEF2D was observed to continuously shuttle to the cytoplasm, interact with the chaperone Hsc70, and undergo degradation. Inhibition of chaperone-mediated autophagy caused accumulation of inactive MEF2D in the cytoplasm. MEF2D levels were increased in the brains of α-synuclein transgenic mice and patients with Parkinson’s disease. Wild-type α-synuclein and a Parkinson’s disease–associated mutant disrupted the MEF2D-Hsc70 binding and led to neuronal death. Thus, chaperone-mediated autophagy modulates the neuronal survival machinery, and dysregulation of this pathway is associated with Parkinson’s disease. PMID:19119233

  4. Organismal proteostasis: role of cell-nonautonomous regulation and transcellular chaperone signaling

    PubMed Central

    van Oosten-Hawle, Patricija; Morimoto, Richard I.

    2014-01-01

    Protein quality control is essential in all organisms and regulated by the proteostasis network (PN) and cell stress response pathways that maintain a functional proteome to promote cellular health. In this review, we describe how metazoans employ multiple modes of cell-nonautonomous signaling across tissues to integrate and transmit the heat-shock response (HSR) for balanced expression of molecular chaperones. The HSR and other cell stress responses such as the unfolded protein response (UPR) can function autonomously in single-cell eukaryotes and tissue culture cells; however, within the context of a multicellular animal, the PN is regulated by cell-nonautonomous signaling through specific sensory neurons and by the process of transcellular chaperone signaling. These newly identified forms of stress signaling control the PN between neurons and nonneuronal somatic tissues to achieve balanced tissue expression of chaperones in response to environmental stress and to ensure that metastable aggregation-prone proteins expressed within any single tissue do not generate local proteotoxic risk. Transcellular chaperone signaling leads to the compensatory expression of chaperones in other somatic tissues of the animal, perhaps preventing the spread of proteotoxic damage. Thus, communication between subcellular compartments and across different cells and tissues maintains proteostasis when challenged by acute stress and upon chronic expression of metastable proteins. We propose that transcellular chaperone signaling provides a critical control step for the PN to maintain cellular and organismal health span. PMID:25030693

  5. Co-chaperone HSJ1a dually regulates the proteasomal degradation of ataxin-3.

    PubMed

    Gao, Xue-Chao; Zhou, Chen-Jie; Zhou, Zi-Ren; Zhang, Yu-Hang; Zheng, Xue-Ming; Song, Ai-Xin; Hu, Hong-Yu

    2011-01-01

    Homo sapiens J domain protein (HSJ1) is a J-domain containing co-chaperone that is known to stimulate ATPase activity of HSP70 chaperone, while it also harbors two ubiquitin (Ub)-interacting motifs (UIMs) that may bind with ubiquitinated substrates and potentially function in protein degradation. We studied the effects of HSJ1a on the protein levels of both normal and the disease--related polyQ-expanded forms of ataxin-3 (Atx3) in cells. The results demonstrate that the N-terminal J-domain and the C-terminal UIM domain of HSJ1a exert opposite functions in regulating the protein level of cellular overexpressed Atx3. This dual regulation is dependent on the binding of the J-domain with HSP70, and the UIM domain with polyUb chains. The J-domain down-regulates the protein level of Atx3 through HSP70 mediated proteasomal degradation, while the UIM domain may alleviate this process via maintaining the ubiquitinated Atx3. We propose that co-chaperone HSJ1a orchestrates the balance of substrates in stressed cells in a Yin-Yang manner.

  6. Molecular Chaperone Calnexin Regulates the Function of Drosophila Sodium Channel Paralytic

    PubMed Central

    Xiao, Xi; Chen, Changyan; Yu, Tian-Ming; Ou, Jiayao; Rui, Menglong; Zhai, Yuanfen; He, Yijing; Xue, Lei; Ho, Margaret S.

    2017-01-01

    Neuronal activity mediated by voltage-gated channels provides the basis for higher-order behavioral tasks that orchestrate life. Chaperone-mediated regulation, one of the major means to control protein quality and function, is an essential route for controlling channel activity. Here we present evidence that Drosophila ER chaperone Calnexin colocalizes and interacts with the α subunit of sodium channel Paralytic. Co-immunoprecipitation analysis indicates that Calnexin interacts with Paralytic protein variants that contain glycosylation sites Asn313, 325, 343, 1463, and 1482. Downregulation of Calnexin expression results in a decrease in Paralytic protein levels, whereas overexpression of the Calnexin C-terminal calcium-binding domain triggers an increase reversely. Genetic analysis using adult climbing, seizure-induced paralysis, and neuromuscular junction indicates that lack of Calnexin expression enhances Paralytic-mediated locomotor deficits, suppresses Paralytic-mediated ghost bouton formation, and regulates minature excitatory junction potentials (mEJP) frequency and latency time. Taken together, our findings demonstrate a need for chaperone-mediated regulation on channel activity during locomotor control, providing the molecular basis for channlopathies such as epilepsy. PMID:28326013

  7. Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle.

    PubMed

    Siligardi, Giuliano; Hu, Bin; Panaretou, Barry; Piper, Peter W; Pearl, Laurence H; Prodromou, Chrisostomos

    2004-12-10

    ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. Central to this is a segment of the structure, which closes like a "lid" over bound ATP, promoting N-terminal dimerization and assembly of a competent active site. Hsp90 mutants that influence these conformational switches have strong effects on ATPase activity. ATPase activity is specifically regulated by Hsp90 co-chaperones, which directly influence the conformational switches. Here we have analyzed the effect of Hsp90 mutations on binding (using isothermal titration calorimetry and difference circular dichroism) and ATPase regulation by the co-chaperones Aha1, Sti1 (Hop), and Sba1 (p23). The ability of Sti1 to bind Hsp90 and arrest its ATPase activity was not affected by any of the mutants screened. Sba1 bound in the presence of AMPPNP to wild-type and ATPase hyperactive mutants with similar affinity but only very weakly to hypoactive mutants despite their wild-type ATP affinity. Unexpectedly, in all cases Sba1 bound to Hsp90 with a 1:2 molar stoichiometry. Aha1 binding to mutants was similar to wild-type, but the -fold activation of their ATPase varied substantially between mutants. Analysis of complex formation with co-chaperone mixtures showed Aha1 and p50cdc37 able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50cdc37 bound independently to Hsp90-AMPPNP but not together. These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not.

  8. Histone H1 chaperone activity of TAF-I is regulated by its subtype-dependent intramolecular interaction.

    PubMed

    Kajitani, Kaori; Kato, Kohsuke; Nagata, Kyosuke

    2017-03-02

    Linker histone H1 is involved in the regulation of gene activity through the maintenance of higher-order chromatin structure. Previously, we have shown that template activating factor-I (TAF-I or protein SET) is involved in linker histone H1 dynamics as a histone H1 chaperone. In human and murine cells, two TAF-I subtypes exist, namely TAF-Iα and TAF-Iβ. TAF-I has a highly acidic amino acid cluster in its C-terminal region and forms homo- or heterodimers through its dimerization domain. Both dimer formation and the C-terminal region of TAF-I are essential for the histone chaperone activity. TAF-Iα exhibits less histone chaperone activity compared with TAF-Iβ even though TAF-Iα and β differ only in their N-terminal regions. However, it is unclear how subtype-specific TAF-I activities are regulated. Here, we have shown that the N-terminal region of TAF-Iα autoinhibits its histone chaperone activity via intramolecular interaction with its C-terminal region. When the interaction between the N- and C-terminal regions of TAF-Iα is disrupted, TAF-Iα shows a histone chaperone activity similar to that of TAF-Iβ. Taken together, these results provide mechanistic insights into the concept that fine tuning of TAF-I histone H1 chaperone activity relies on the subtype compositions of the TAF-I dimer.

  9. Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

    PubMed Central

    Liang, Jingjing; Sagum, Cari A.; Bedford, Mark T.; Sudol, Marius; Han, Ziying

    2017-01-01

    Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles. PMID:28076420

  10. Human protein-disulfide isomerase is a redox-regulated chaperone activated by oxidation of domain a'.

    PubMed

    Wang, Chao; Yu, Jiang; Huo, Lin; Wang, Lei; Feng, Wei; Wang, Chih-chen

    2012-01-06

    Protein-disulfide isomerase (PDI), with domains arranged as abb'xa'c, is a key enzyme and chaperone localized in the endoplasmic reticulum (ER) catalyzing oxidative folding and preventing misfolding/aggregation of proteins. It has been controversial whether the chaperone activity of PDI is redox-regulated, and the molecular basis is unclear. Here, we show that both the chaperone activity and the overall conformation of human PDI are redox-regulated. We further demonstrate that the conformational changes are triggered by the active site of domain a', and the minimum redox-regulated cassette is located in b'xa'. The structure of the reduced bb'xa' reveals for the first time that domain a' packs tightly with both domain b' and linker x to form one compact structural module. Oxidation of domain a' releases the compact conformation and exposes the shielded hydrophobic areas to facilitate its high chaperone activity. Thus, the study unequivocally provides mechanistic insights into the redox-regulated chaperone activity of human PDI.

  11. Human Protein-disulfide Isomerase Is a Redox-regulated Chaperone Activated by Oxidation of Domain a′*

    PubMed Central

    Wang, Chao; Yu, Jiang; Huo, Lin; Wang, Lei; Feng, Wei; Wang, Chih-chen

    2012-01-01

    Protein-disulfide isomerase (PDI), with domains arranged as abb′xa′c, is a key enzyme and chaperone localized in the endoplasmic reticulum (ER) catalyzing oxidative folding and preventing misfolding/aggregation of proteins. It has been controversial whether the chaperone activity of PDI is redox-regulated, and the molecular basis is unclear. Here, we show that both the chaperone activity and the overall conformation of human PDI are redox-regulated. We further demonstrate that the conformational changes are triggered by the active site of domain a′, and the minimum redox-regulated cassette is located in b′xa′. The structure of the reduced bb′xa′ reveals for the first time that domain a′ packs tightly with both domain b′ and linker x to form one compact structural module. Oxidation of domain a′ releases the compact conformation and exposes the shielded hydrophobic areas to facilitate its high chaperone activity. Thus, the study unequivocally provides mechanistic insights into the redox-regulated chaperone activity of human PDI. PMID:22090031

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

    PubMed

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

    2013-04-01

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

  13. The Quaternary Organization and Dynamics of the Molecular Chaperone HSP26 Are Thermally Regulated

    PubMed Central

    Benesch, Justin L.P.; Aquilina, J. Andrew; Baldwin, Andrew J.; Rekas, Agata; Stengel, Florian; Lindner, Robyn A.; Basha, Eman; Devlin, Glyn L.; Horwitz, Joseph; Vierling, Elizabeth; Carver, John A.; Robinson, Carol V.

    2011-01-01

    SUMMARY The function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization of target proteins leads to its activation as a molecular chaperone. We investigate the structural and dynamical properties of ScHSP26 oligomers through a combination of multiangle light scattering, fluorescence spectroscopy, NMR spectroscopy, and mass spec-trometry. We show that ScHSP26 exists as a heterogeneous oligomeric ensemble at room temperature. At heat-shock temperatures, two shifts in equilibria are observed: toward dissociation and to larger oligomers. We examine the quaternary dynamics of these oligomers by investigating the rate of exchange of subunits between them and find that this not only increases with temperature but proceeds via two separate processes. This is consistent with a conformational change of the oligomers at elevated temperatures which regulates the disassembly rates of this thermally activated protein. PMID:20851350

  14. Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation.

    PubMed

    Valdor, Rut; Mocholi, Enric; Botbol, Yair; Guerrero-Ros, Ignacio; Chandra, Dinesh; Koga, Hiroshi; Gravekamp, Claudia; Cuervo, Ana Maria; Macian, Fernando

    2014-11-01

    Chaperone-mediated autophagy (CMA) targets soluble proteins for lysosomal degradation. Here we found that CMA was activated in T cells in response to engagement of the T cell antigen receptor (TCR), which induced expression of the CMA-related lysosomal receptor LAMP-2A. In activated T cells, CMA targeted the ubiquitin ligase Itch and the calcineurin inhibitor RCAN1 for degradation to maintain activation-induced responses. Consequently, deletion of the gene encoding LAMP-2A in T cells caused deficient in vivo responses to immunization or infection with Listeria monocytogenes. Impaired CMA activity also occurred in T cells with age, which negatively affected their function. Restoration of LAMP-2A in T cells from old mice resulted in enhancement of activation-induced responses. Our findings define a role for CMA in regulating T cell activation through the targeted degradation of negative regulators of T cell activation.

  15. Molecular chaperone Hsp27 regulates the Hippo tumor suppressor pathway in cancer

    PubMed Central

    Vahid, Sepideh; Thaper, Daksh; Gibson, Kate F.; Bishop, Jennifer L.; Zoubeidi, Amina

    2016-01-01

    Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers. PMID:27555231

  16. Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin

    PubMed Central

    Sandoz, Patrick A.; Savoglidis, Georgios; Hatzimanikatis, Vassily; van der Goot, F. Gisou

    2016-01-01

    Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non- palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6. PMID:26900856

  17. Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin.

    PubMed

    Dallavilla, Tiziano; Abrami, Laurence; Sandoz, Patrick A; Savoglidis, Georgios; Hatzimanikatis, Vassily; van der Goot, F Gisou

    2016-02-01

    Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non-palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6.

  18. Conformational dynamics of a membrane protein chaperone enables spatially regulated substrate capture and release

    PubMed Central

    Liang, Fu-Cheng; Kroon, Gerard; McAvoy, Camille Z.; Chi, Chris; Wright, Peter E.; Shan, Shu-ou

    2016-01-01

    Membrane protein biogenesis poses enormous challenges to cellular protein homeostasis and requires effective molecular chaperones. Compared with chaperones that promote soluble protein folding, membrane protein chaperones require tight spatiotemporal coordination of their substrate binding and release cycles. Here we define the chaperone cycle for cpSRP43, which protects the largest family of membrane proteins, the light harvesting chlorophyll a/b-binding proteins (LHCPs), during their delivery. Biochemical and NMR analyses demonstrate that cpSRP43 samples three distinct conformations. The stromal factor cpSRP54 drives cpSRP43 to the active state, allowing it to tightly bind substrate in the aqueous compartment. Bidentate interactions with the Alb3 translocase drive cpSRP43 to a partially inactive state, triggering selective release of LHCP’s transmembrane domains in a productive unloading complex at the membrane. Our work demonstrates how the intrinsic conformational dynamics of a chaperone enables spatially coordinated substrate capture and release, which may be general to other ATP-independent chaperone systems. PMID:26951662

  19. Enhancement of stress resilience through Hdac6-mediated regulation of glucocorticoid receptor chaperone dynamics

    PubMed Central

    Jochems, Jeanine; Teegarden, Sarah L; Chen, Yong; Boulden, Janette; Challis, Collin; Ben-Dor, Gabriel A; Kim, Sangwon F; Berton, Olivier

    2014-01-01

    Background Acetylation of Hsp90 regulates downstream hormone signaling via the glucocorticoid receptor (GR), but the role of this molecular mechanism in stress homeostasis remains poorly understood. We tested whether acetylation of Hsp90 in the brain predicts and modulates the behavioral sequelae of a mouse model of social stress. Methods Mice subjected to chronic social defeat stress (CSDS) were stratified into resilient and vulnerable subpopulations. HPA axis function was probed using a DEX/CRF test. Hsp90 acetylation, Hsp90-GR interactions and GR translocation were measured in the dorsal raphe nucleus (DRN). To manipulate Hsp90 acetylation, we pharmacologically inhibited Hdac6, a known deacetylase of Hsp90 or overexpressed a point-mutant that mimics the hyperacetylated state of Hsp90 at lysine K294 Results Lower acetylated Hsp90, higher GR-Hsp90 association and enhanced GR translocation were observed in DRN of vulnerable mice after CSDS. Administration of ACY-738, an Hdac6-selective inhibitor, led to Hsp90 hyperacetylation in brain and in neuronal culture. In cell-based assays, ACY-738 increased the relative association of Hsp90 with FKBP51 versus FKBP52 and inhibited hormone-induced GR translocation. This effect was replicated by overexpressing the acetylation-mimic point-mutant of Hsp90. In vivo, ACY-738 promoted resilience to CSDS and serotonin-selective viral overexpression of the acetylation-mimic mutant of Hsp90 in raphe neurons reproduced the behaviroral effect of ACY-738. Conclusions Hyperacetylation of Hsp90 is a predictor and causal molecular determinant of stress resilience in mice. Brain-penetrant Hdac6 inhibitors increase Hsp90 acetylation and modulate GR chaperone dynamics offering a promising strategy to curtail deleterious socioaffective effects of stress and glucocorticoids. PMID:25442004

  20. Hsp70A and GlsA interact as partner chaperones to regulate asymmetric division in Volvox.

    PubMed

    Cheng, Qian; Pappas, Valeria; Hallmann, Armin; Miller, Stephen M

    2005-10-15

    GlsA, a J-protein chaperone, is required for the asymmetric divisions that set aside germ and somatic cell precursors during embryogenesis in Volvox carteri, and previous evidence indicated that this function requires an intact Hsp70-binding site. To determine if Hsp70A, the only known cytoplasmic Hsp70 in V. carteri, is the chaperone partner of GlsA, we investigated the localization of the two proteins during critical stages of embryogenesis and tested their capacity to interact. We found that a substantial fraction of Hsp70A co-localizes with GlsA, both in interphase and mitotic blastomeres. In addition, Hsp70A coimmunoprecipitated with GlsA, and co-expression of GlsA and Hsp70A variants partially rescued the Gls phenotype of a glsA mutant, whereas neither variant by itself rescued the mutant phenotype. Immunofluorescence analysis demonstrated that GlsA is about equally abundant in all blastomeres at all cleavage stages examined but that Hsp70A is more abundant in anterior (asymmetrically dividing) blastomeres than in posterior (symmetrically dividing) blastomeres during the period of asymmetric division. We conclude that Hsp70A and GlsA function as chaperone partners that regulate asymmetric division and that the relative abundance of Hsp70A in asymmetrically dividing embryos may determine which blastomeres divide asymmetrically and which do not.

  1. The Hsp90 Co-chaperones Sti1, Aha1, and P23 Regulate Adaptive Responses to Antifungal Azoles

    PubMed Central

    Gu, Xiaokui; Xue, Wei; Yin, Yajing; Liu, Hongwei; Li, Shaojie; Sun, Xianyun

    2016-01-01

    Heat Shock Protein 90 (Hsp90) is essential for tumor progression in humans and drug resistance in fungi. However, the roles of its many co-chaperones in antifungal resistance are unknown. In this study, by susceptibility test of Neurospora crassa mutants lacking each of 18 Hsp90/Calcineurin system member genes (including 8 Hsp90 co-chaperone genes) to antifungal drugs and other stresses, we demonstrate that the Hsp90 co-chaperones Sti1 (Hop1 in yeast), Aha1, and P23 (Sba1 in yeast) were required for the basal resistance to antifungal azoles and heat stress. Deletion of any of them resulted in hypersensitivity to azoles and heat. Liquid chromatography–mass spectrometry (LC-MS) analysis showed that the toxic sterols eburicol and 14α-methyl-3,6-diol were significantly accumulated in the sti1 and p23 deletion mutants after ketoconazole treatment, which has been shown before to led to cell membrane stress. At the transcriptional level, Aha1, Sti1, and P23 positively regulate responses to ketoconazole stress by erg11 and erg6, key genes in the ergosterol biosynthetic pathway. Aha1, Sti1, and P23 are highly conserved in fungi, and sti1 and p23 deletion also increased the susceptibility to azoles in Fusarium verticillioides. These results indicate that Hsp90-cochaperones Aha1, Sti1, and P23 are critical for the basal azole resistance and could be potential targets for developing new antifungal agents. PMID:27761133

  2. Regulation of σ-1 Receptors and Endoplasmic Reticulum Chaperones in the Brain of Methamphetamine Self-Administering Rats

    PubMed Central

    Hayashi, Teruo; Justinova, Zuzana; Hayashi, Eri; Cormaci, Gianfrancesco; Mori, Tomohisa; Tsai, Shang-Yi; Barnes, Chanel; Goldberg, Steven R.

    2010-01-01

    σ-1 Receptors are endoplasmic reticulum (ER) chaperones that are implicated in the neuroplasticity associated with psychostimulant abuse. We immunocytochemically examined the distribution of σ-1 receptors in the brain of drug-naive rats and then examined the dynamics of σ-1 receptors and other ER chaperones in specific brain subregions of rats that self-administered methamphetamine, received methamphetamine passively, or received only saline injections. σ-1 Receptors were found to be expressed in moderate to high levels in the olfactory bulb, striatum, nucleus accumbens shell, olfactory tubercle, amygdala, hippocampus, red nucleus, ventral tegmental area, substantia nigra, and locus ceruleus. Methamphetamine, whether self-administered or passively received, significantly elevated ER chaperones including the σ-1 receptor, BiP, and calreticulin in the ventral tegmental area and substantia nigra. In the olfactory bulb, however, only the σ-1 receptor chaperone was increased, and this increase occurred only in rats that actively self-administered methamphetamine. Consistent with an increase in σ-1 receptors, extracellular signal-regulated kinase was found to be activated and protein kinase A attenuated in the olfactory bulb of methamphetamine self-administering rats. σ-1 Receptors in the olfactory bulb were found to be colocalized with dopamine D1 receptors. These results indicate that methamphetamine induces ER stress in the ventral tegmental area and substantia nigra in rats whether the drug is received actively or passively. However, the changes seen only in rats that actively self-administered methamphetamine suggest that D1 and σ-1 receptors in the olfactory bulb might play an important role in the motivational conditioning/learning aspects of methamphetamine self-administration in the rat. PMID:19940104

  3. An RpoS-dependent sRNA regulates the expression of a chaperone involved in protein folding

    PubMed Central

    Silva, Inês Jesus; Ortega, Álvaro Darío; Viegas, Sandra Cristina; García-del Portillo, Francisco; Arraiano, Cecília Maria

    2013-01-01

    Small noncoding RNAs (sRNAs) are usually expressed in the cell to face a variety of stresses. In this report we disclose the first target for SraL (also known as RyjA), a sRNA present in many bacteria, which is highly induced in stationary phase. We also demonstrate that this sRNA is directly transcribed by the major stress σ factor σS (RpoS) in Salmonella enterica serovar Typhimurium. We show that SraL sRNA down-regulates the expression of the chaperone Trigger Factor (TF), encoded by the tig gene. TF is one of the three major chaperones that cooperate in the folding of the newly synthesized cytosolic proteins and is the only ribosome-associated chaperone known in bacteria. By use of bioinformatic tools and mutagenesis experiments, SraL was shown to directly interact with the 5′ UTR of the tig mRNA a few nucleotides upstream of the Shine-Dalgarno region. Namely, point mutations in the sRNA (SraL*) abolished the repression of tig mRNA and could only down-regulate a tig transcript target with the respective compensatory mutations. We have also validated in vitro that SraL forms a stable duplex with the tig mRNA. This work constitutes the first report of a small RNA affecting protein folding. Taking into account that both SraL and TF are very well conserved in enterobacteria, this work will have important repercussions in the field. PMID:23893734

  4. Molecular chaperones and photoreceptor function

    PubMed Central

    Kosmaoglou, Maria; Schwarz, Nele; Bett, John S.; Cheetham, Michael E.

    2008-01-01

    Molecular chaperones facilitate and regulate protein conformational change within cells. This encompasses many fundamental cellular processes: including the correct folding of nascent chains; protein transport and translocation; signal transduction and protein quality control. Chaperones are, therefore, important in several forms of human disease, including neurodegeneration. Within the retina, the highly specialized photoreceptor cell presents a fascinating paradigm to investigate the specialization of molecular chaperone function and reveals unique chaperone requirements essential to photoreceptor function. Mutations in several photoreceptor proteins lead to protein misfolding mediated neurodegeneration. The best characterized of these are mutations in the molecular light sensor, rhodopsin, which cause autosomal dominant retinitis pigmentosa. Rhodopsin biogenesis is likely to require chaperones, while rhodopsin misfolding involves molecular chaperones in quality control and the cellular response to protein aggregation. Furthermore, the specialization of components of the chaperone machinery to photoreceptor specific roles has been revealed by the identification of mutations in molecular chaperones that cause inherited retinal dysfunction and degeneration. These chaperones are involved in several important cellular pathways and further illuminate the essential and diverse roles of molecular chaperones. PMID:18490186

  5. Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications

    PubMed Central

    Blacklock, Kristin; Verkhivker, Gennady M.

    2014-01-01

    A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This

  6. Juvenile Hormone Differentially Regulates Two Grp78 Genes Encoding Protein Chaperones Required for Insect Fat Body Cell Homeostasis and Vitellogenesis.

    PubMed

    Luo, Maowu; Li, Dong; Wang, Zhiming; Guo, Wei; Kang, Le; Zhou, Shutang

    2017-03-29

    Juvenile hormone (JH) has a well-known role in stimulating insect vitellogenesis (i.e. yolk deposition) and oocyte maturation, but the molecular mechanisms of JH action in insect reproduction are unclear. Glucose-regulated protein of 78 kDa (Grp78) is a heat shock protein 70 kDa family member and one of the most abundant chaperones in the endoplasmic reticulum (ER) where it helps fold newly synthesized peptides. Because of its prominent role in protein folding and also ER stress, we hypothesized that Grp78 might be involved in fat body cell homeostasis and vitellogenesis and a regulatory target of JH. We report here that the migratory locust Locusta migratoria possesses two Grp78 genes that are differentially regulated by JH. We found that Grp78-1 is regulated by JH through Mcm4/7-dependent DNA replication and polyploidization, whereas Grp78-2 expression is directly activated by the JH-receptor complex comprising Methoprene-tolerant and Taiman proteins. Interestingly, Grp78-2 expression in the fat body is about 10-fold higher than that of Grp78-1 Knockdown of either Grp78-1 or Grp78-2 significantly reduced levels of vitellogenin (Vg) protein, accompanied by retarded maturation of oocytes. Depletion of both Grp78-1 and Grp78-2 resulted in ER stress and apoptosis in the fat body and in severely defective Vg synthesis and oocyte maturation. These results indicate a crucial role of Grp78 in JH-dependent vitellogenesis and egg production. The presence and differential regulation of two Grp78 genes in L. migratoria likely help accelerate the production of this chaperone in the fat body to facilitate folding of massively synthesized Vg and other proteins.

  7. Binding of a Small Molecule at a Protein–Protein Interface Regulates the Chaperone Activity of Hsp70–Hsp40

    PubMed Central

    Wisén, Susanne; Bertelsen, Eric B.; Thompson, Andrea D.; Patury, Srikanth; Ung, Peter; Chang, Lyra; Evans, Christopher G.; Walter, Gladis M.; Wipf, Peter; Carlson, Heather A.; Brodsky, Jeffrey L.; Zuiderweg, Erik R. P.; Gestwicki, Jason E.

    2010-01-01

    Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70–Hsp40 complex assembly at a native protein–protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis. PMID:20481474

  8. Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics

    PubMed Central

    Nithianantham, Stanley; Le, Sinh; Seto, Elbert; Jia, Weitao; Leary, Julie; Corbett, Kevin D; Moore, Jeffrey K; Al-Bassam, Jawdat

    2015-01-01

    Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics. DOI: http://dx.doi.org/10.7554/eLife.08811.001 PMID:26208336

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

    PubMed Central

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

    2013-01-01

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

  10. Regulation and Quality Control of Adiponectin Assembly by Endoplasmic Reticulum Chaperone ERp44*

    PubMed Central

    Hampe, Lutz; Radjainia, Mazdak; Xu, Cheng; Harris, Paul W. R.; Bashiri, Ghader; Goldstone, David C.; Brimble, Margaret A.; Wang, Yu; Mitra, Alok K.

    2015-01-01

    Adiponectin, a collagenous hormone secreted abundantly from adipocytes, possesses potent antidiabetic and anti-inflammatory properties. Mediated by the conserved Cys39 located in the variable region of the N terminus, the trimeric (low molecular weight (LMW)) adiponectin subunit assembles into different higher order complexes, e.g. hexamers (middle molecular weight (MMW)) and 12–18-mers (high molecular weight (HMW)), the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. The endoplasmic reticulum (ER) chaperone ERp44 retains adiponectin in the early secretory compartment and tightly controls the oxidative state of Cys39 and the oligomerization of adiponectin. Using cellular and in vitro assays, we show that ERp44 specifically recognizes the LMW and MMW forms but not the HMW form. Our binding assays with short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW form, into reduced trimeric precursors. These ERp44-bound precursors in the cis-Golgi may be transported back to the ER and released to enhance the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control. PMID:26060250

  11. Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

    PubMed Central

    Vaughan, Cara K.; Mollapour, Mehdi; Smith, Jennifer R.; Truman, Andrew; Hu, Bin; Good, Valerie M.; Panaretou, Barry; Neckers, Len; Clarke, Paul A.; Workman, Paul; Piper, Peter W.; Prodromou, Chrisostomos; Pearl, Laurence H.

    2008-01-01

    Summary Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes. PMID:18922470

  12. C-terminal truncation of a bovine B(12) trafficking chaperone enhances the sensitivity of the glutathione-regulated thermostability.

    PubMed

    Jeong, Jinju; Park, Jihyun; Lee, Dong-Yeon; Kim, Jihoe

    2013-03-01

    The human B(12) trafficking chaperone hCblC is well conserved in mammals and non-mammalian eukaryotes. However, the C-terminal ~40 amino acids of hCblC vary significantly and are predicted to be deleted by alternative splicing of the encoding gene. In this study, we examined the thermostability of the bovine CblC truncated at the C-terminal variable region (t-bCblC) and its regulation by glutathione. t-bCblC is highly thermolabile (T(m) = ~42(o)C) similar to the full-length protein (f-bCblC). However, t-bCblC is stabilized to a greater extent than f-bCblC by binding of reduced glutathione (GSH) with increased sensitivity to GSH. In addition, binding of oxidized glutathione (GSSG) destabilizes t-bCblC to a greater extent and with increased sensitivity as compared to f-bCblC. These results indicate that t-bCblC is a more sensitive form to be regulated by glutathione than the full-length form of the protein.

  13. Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes.

    PubMed

    Fisher, Susan H; Wray, Lewis V

    2008-01-22

    The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo. Although stable GS-GlnR-DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein-protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR-DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein-protein interaction that stabilizes GlnR-DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-11-01

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

  16. Cell surface trafficking of TLR1 is differentially regulated by the chaperones PRAT4A and PRAT4B.

    PubMed

    Hart, Bryan E; Tapping, Richard I

    2012-05-11

    The subcellular localization of Toll-like receptors (TLRs) is critical to their ability to function as innate immune sensors of microbial infection. We previously reported that an I602S polymorphism of human TLR1 is associated with aberrant trafficking of the receptor to the cell surface, loss of responses to TLR1 agonists, and differential susceptibility to diseases caused by pathogenic mycobacteria. Through an extensive analysis of receptor deletion and point mutants we have discovered that position 602 resides within a short 6 amino acid cytoplasmic region that is required for TLR1 surface expression. This short trafficking motif, in conjunction with the adjacent transmembrane domain, is sufficient to direct TLR1 to the cell surface. A serine at position 602 interrupts this trafficking motif and prevents cell surface expression of TLR1. Additionally, we have found that ER-resident TLR chaperones, PRAT4A and PRAT4B, act as positive and negative regulators of TLR1 surface trafficking, respectively. Importantly, either over-expression of PRAT4A or knock-down of PRAT4B rescues cell surface expression of the TLR1 602S variant. We also report that IFN-γ treatment of primary human monocytes derived from homozygous 602S individuals rescues TLR1 cell surface trafficking and cellular responses to soluble agonists. This event appears to be mediated by PRAT4A whose expression is strongly induced in human monocytes by IFN-γ. Collectively, these results provide a mechanism for the differential trafficking of TLR1 I602S variants, and highlight the distinct roles for PRAT4A and PRAT4B in the regulation of TLR1 surface expression.

  17. Plasma Membrane Targeting of Protocadherin 15 Is Regulated by the Golgi-Associated Chaperone Protein PIST.

    PubMed

    Nie, Hongyun; Liu, Yueyue; Yin, Xiaolei; Cao, Huiren; Wang, Yanfei; Xiong, Wei; Lin, Yushuang; Xu, Zhigang

    2016-01-01

    Protocadherin 15 (PCDH15) is a core component of hair cell tip-links and crucial for proper function of inner ear hair cells. Mutations of PCDH15 gene cause syndromic and nonsyndromic hearing loss. At present, the regulatory mechanisms responsible for the intracellular transportation of PCDH15 largely remain unknown. Here we show that PIST, a Golgi-associated, PDZ domain-containing protein, interacts with PCDH15. The interaction is mediated by the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI) of PCDH15. Through this interaction, PIST retains PCDH15 in the trans-Golgi network (TGN) and reduces the membrane expression of PCDH15. We have previously showed that PIST regulates the membrane expression of another tip-link component, cadherin 23 (CDH23). Taken together, our finding suggests that PIST regulates the intracellular trafficking and membrane targeting of the tip-link proteins CDH23 and PCDH15.

  18. Chaperones get in touch: the Hip-Hop connection.

    PubMed

    Frydman, J; Höhfeld, J

    1997-03-01

    Recent findings emphasize that different molecular chaperones cooperate during intracellular protein biogenesis. Mechanistic aspects of chaperone cooperation are now emerging from studies on the regulation of certain signal transduction pathways mediated by Hsc70 and Hsp90 in the eukaryotic cytosol. Efficient cooperation appears to be achieved through a defined regulation of Hsc70 activity by the chaperone cofactors Hip and Hop.

  19. Differences in structure and changes in gene regulation of murrel molecular chaperone HSP family during epizootic ulcerative syndrome (EUS) infection.

    PubMed

    Sathyamoorthy, Akila; Chaurasia, Mukesh Kumar; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Harikrishnan, Ramasamy; Arockiaraj, Jesu

    2017-01-01

    Heat shock proteins (HSPs) are immunogenic, ubiquitous class of molecular chaperones, which are induced in response to various environmental and microbial stressful conditions. It plays a vital role in maintaining cellular protein homeostasis in eukaryotic cells. In this study, we described a comprehensive comparative data by bioinformatics approach on three different full length cDNA sequences of HSP family at molecular level. The cDNA sequences of three HSPs were identified from constructed cDNA library of Channa striatus and named as CsCPN60, CsHSP60 and CsHSP70. We have conducted various physicochemical study, which showed that CsHSP70 (666 amino acid) possessed a larger polypeptides followed by CsCPN60 (575) and CsCPN60 (542). Three dimensional structural analysis of these HSPs showed maximum residues in α-helices and least in β-sheets; also CsHSP60 lacks β-sheet and formed helix-turn-helix structure. Further analysis indicated that each HSP carried distinct domains and gene specific signature motif, which showed that each HSP are structurally diverse. Homology and phylogenetic study showed that the sequences taken for analysis shared maximum identity with fish HSP family. Tissue specific mRNA expression analysis revealed that all the HSPs showed maximum expression in one of the major immune organ such as CsCPN60 in kidney, CsHSP60 in spleen and CsHSP70 in head kidney. To understand the function of HSPs in murrel immune system, the elevation in mRNA expression level was analyzed against microbial oxidative stressors such as fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila). It is interesting to note that all the HSP showed a different expression pattern and reached maximum up-regulation at 48 h post-infection (p.i) during fungal stress, whereas in bacterial stress only CsCPN60 showed maximum up-regulation at 48 h p.i, but CsHSP60 and CsHSP70 showed maximum up-regulation at 24 h p.i. The differential expression pattern showed that each

  20. Systems biology of molecular chaperone networks.

    PubMed

    Csermely, Péter; Korcsmáros, Tamás; Kovács, István A; Szalay, Máté S; Soti, Csaba

    2008-01-01

    Molecular chaperones are not only fascinating molecular machines that help the folding, refolding, activation or assembly of other proteins, but also have a number of functions. These functions can be understood only by considering the emergent properties of cellular networks--and that of chaperones as special network constituents. As a notable example for the network-related roles of chaperones they may act as genetic buffers stabilizing the phenotype of various cells and organisms, and may serve as potential regulators of evolvability. Why are chaperones special in the context of cellular networks? Chaperones: (1) have weak links, i.e. low affinity, transient interactions with most of their partners; (2) connect hubs, i.e. act as 'masterminds' of the cell being close to several centre proteins with a lot of neighbours; and (3) are in the overlaps of network modules, which confers upon them a special regulatory role. Importantly, chaperones may uncouple or even quarantine modules of protein-protein interaction networks, signalling networks, genetic regulatory networks and membrane organelle networks during stress, which gives an additional chaperone-mediated protection for the cell at the network-level. Moreover, chaperones are essential to rebuild inter-modular contacts after stress by their low affinity, 'quasi-random' sampling of the potential interaction partners in different cellular modules. This opens the way to the chaperone-regulated modular evolution of cellular networks, and helps us to design novel therapeutic and anti-ageing strategies.

  1. RNA Chaperones Step Out of Hfq's Shadow.

    PubMed

    Attaiech, Laetitia; Glover, J N Mark; Charpentier, Xavier

    2017-04-01

    The stability and function of regulatory small RNAs (sRNAs) often require a specialized RNA-binding protein called an RNA chaperone. Recent findings show that proteins containing a ProQ/FinO domain constitute a new class of RNA chaperones that could play key roles in post-transcriptional gene regulation throughout bacterial species.

  2. Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.

    PubMed

    Ballinger, C A; Connell, P; Wu, Y; Hu, Z; Thompson, L J; Yin, L Y; Patterson, C

    1999-06-01

    The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.

  3. Artemin as an efficient molecular chaperone.

    PubMed

    Shahangian, S Shirin; Rasti, Behnam; Sajedi, Reza H; Khodarahmi, Reza; Taghdir, Majid; Ranjbar, Bijan

    2011-12-01

    Artemin is an abundant thermostable protein in Artemia encysted embryos under stress. It is considered as a stress protein, as its highly regulated expression is associated with stress resistance in this crustacea. In the present study, artemin has been shown to be a potent molecular chaperone with high efficacy. Artemin is capable of inhibiting the chemical aggregation of proteins such as carbonic anhydrase (CA) and horseradish peroxidase (HRP) at unique molar ratios of chaperone to substrates (1:40 and 1:26 for CA and HRP, respectively). Furthermore, it can also enhance refolding yield of these substrates by nearly 50%. The refolding promotion of CA is checked and verified through a sensitive fluorimetric technique. Based on these experiments, artemin showed higher chaperone activity than other chaperones. The evaluation of artemin surface using ANS showed it to be highly hydrophobic, probably resulting in its high efficacy. These results suggest that artemin can be considered a novel low molecular weight chaperone.

  4. Arabidopsis COLD SHOCK DOMAIN PROTEIN2 is a RNA chaperone that is regulated by cold and developmental signals

    SciTech Connect

    Sasaki, Kentaro; Kim, Myung-Hee; Imai, Ryozo

    2007-12-21

    Bacterial cold shock proteins (CSPs) are RNA chaperones that unwind RNA secondary structures. Arabidopsis COLD SHOCK DOMAIN PROTEIN2 (AtCSP2) contains a domain that is shared with bacterial CSPs. Here we showed that AtCSP2 binds to RNA and unwinds nucleic acid duplex. Heterologous expression of AtCSP2 complemented cold sensitivity of an Escherichia coli csp quadruple mutant, indicating that AtCSP2 function as a RNA chaperone in E. coli. AtCSP2 mRNA and protein levels increased during cold acclimation, but the protein accumulation was most prominent after 10 days of cold treatment. AtCSP2 promoter::GUS transgenic plants revealed that AtCSP2 is expressed only in root and shoot apical regions during vegetative growth but is expressed in reproductive organs such as pollens, ovules and embryos. These data indicated that AtCSP2 is involved in developmental processes as well as cold adaptation. Localization of AtCSP2::GFP in nucleolus and cytoplasm suggested different nuclear and cytosolic RNA targets.

  5. The U4/U6 Recycling Factor SART3 Has Histone Chaperone Activity and Associates with USP15 to Regulate H2B Deubiquitination*

    PubMed Central

    Long, Lindsey; Thelen, Joseph P.; Furgason, Melonnie; Haj-Yahya, Mahmood; Brik, Ashraf; Cheng, Dongmei; Peng, Junmin; Yao, Tingting

    2014-01-01

    Post-translational modifications of histone proteins produce dynamic signals that regulate the structure and function of chromatin. Mono-ubiquitination of H2B in the histone tail (at Lys-123 in yeast or Lys-120 in humans) is a conserved modification that has been implicated in the regulation of transcription, replication, and DNA repair processes. In a search for direct effectors of ubH2B, we identified a deubiquitinating enzyme, Usp15, through affinity purification with a nonhydrolyzable ubH2B mimic. In the nucleus, Usp15 indirectly associates with the ubH2B E3 ligase, RNF20/RNF40, and directly associates with a component of the splicing machinery, SART3 (also known as TIP110 or p110). These physical interactions place Usp15 in the vicinity of actively transcribed DNA. Importantly we found that SART3 has previously unrecognized histone chaperone activities. SART3, but not the well-characterized histone chaperone Nap1, enhances Usp15 binding to ubH2B and facilitates deubiquitination of ubH2B in free histones but not in nucleosomes. These results suggest that SART3 recruits ubH2B, which may be evicted from DNA during transcription, for deubiquitination by Usp15. In light of the function played by SART3 in U4/U6 di-snRNP formation, our discovery points to a direct link between eviction-coupled erasure of the ubiquitin mark from ubH2B and co-transcriptional pre-mRNA splicing. PMID:24526689

  6. Study on the chaperone properties of conserved GTPases.

    PubMed

    Wang, Xiang; Xue, Jiaying; Sun, Zhe; Qin, Yan; Gong, Weimin

    2012-01-01

    As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.

  7. O-linked N-acetylglucosamine transferase (OGT) interacts with the histone chaperone HIRA complex and regulates nucleosome assembly and cellular senescence

    PubMed Central

    Lee, Jong-Sun; Zhang, Zhiguo

    2016-01-01

    The histone chaperone HIRA complex, consisting of histone cell cycle regulator (HIRA), Ubinuclein1 (UBN1), and calcineurin binding protein 1 (CABIN1), deposits histone variant H3.3 to genic regions and regulates gene expression in various cellular processes, including cellular senescence. How HIRA-mediated nucleosome assembly of H3.3–H4 is regulated remains not well understood. Here, we show that O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), an enzyme that catalyzes O-GlcNAcylation of serine or threonine residues, interacts with UBN1, modifies HIRA, and promotes nucleosome assembly of H3.3. Depletion of OGT or expression of the HIRA S231A O-GlcNAcylation–deficient mutant compromises formation of the HIRA–H3.3 complex and H3.3 nucleosome assembly. Importantly, OGT depletion or expression of the HIRA S231A mutant delays premature cellular senescence in primary human fibroblasts, whereas overexpression of OGT accelerates senescence. Taken together, these results support a model in which OGT modifies HIRA to regulate HIRA–H3.3 complex formation and H3.3 nucleosome assembly and reveal the mechanism by which OGT functions in cellular senescence. PMID:27217568

  8. The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

    PubMed

    Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

    2017-02-24

    The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3'UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2'-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome.

  9. The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization

    PubMed Central

    Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

    2017-01-01

    The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3′UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2′-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome. PMID:28233845

  10. Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication

    PubMed Central

    Stetz, Gabrielle; Verkhivker, Gennady M.

    2017-01-01

    Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we

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

    PubMed Central

    Zorzi, Elisa; Bonvini, Paolo

    2011-01-01

    Understanding the mechanisms that control stress is central to realize how cells respond to environmental and physiological insults. All the more important is to reveal how tumour cells withstand their harsher growth conditions and cope with drug-induced apoptosis, since resistance to chemotherapy is the foremost complication when curing cancer. Intensive research on tumour biology over the past number of years has provided significant insights into the molecular events that occur during oncogenesis, and resistance to anti-cancer drugs has been shown to often rely on stress response and expression of inducible heat shock proteins (HSPs). However, with respect to the mechanisms guarding cancer cells against proteotoxic stresses and the modulatory effects that allow their survival, much remains to be defined. Heat shock proteins are molecules responsible for folding newly synthesized polypeptides under physiological conditions and misfolded proteins under stress, but their role in maintaining the transformed phenotype often goes beyond their conventional chaperone activity. Expression of inducible HSPs is known to correlate with limited sensitivity to apoptosis induced by diverse cytotoxic agents and dismal prognosis of several tumour types, however whether cancer cells survive because of the constitutive expression of heat shock proteins or the ability to induce them when adapting to the hostile microenvironment remains to be elucidated. Clear is that tumours appear nowadays more “addicted” to heat shock proteins than previously envisaged, and targeting HSPs represents a powerful approach and a future challenge for sensitizing tumours to therapy. This review will focus on the anti-apoptotic role of heat shock 70kDa protein (Hsp70), and how regulatory factors that control inducible Hsp70 synthesis, expression and activity may be relevant for response to stress and survival of cancer cells. PMID:24213118

  12. The RNA chaperone Hfq is involved in stress response and virulence in Neisseria meningitidis and is a pleiotropic regulator of protein expression.

    PubMed

    Fantappiè, Laura; Metruccio, Matteo M E; Seib, Kate L; Oriente, Francesca; Cartocci, Elena; Ferlicca, Francesca; Giuliani, Marzia M; Scarlato, Vincenzo; Delany, Isabel

    2009-05-01

    The well-conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. This protein is thought to function as an RNA chaperone and to facilitate base pairing between small regulatory RNA (sRNA) and mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis, we generated an Hfq mutant of the MC58 strain, and the knockout mutant has pleiotropic phenotypes; it has a general growth phenotype in vitro in culture media, and it is sensitive to a wide range of stresses, including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant, and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence, and we propose a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which so far is largely unexplored.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

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

  15. Deletion of the Small RNA Chaperone Protein Hfq down Regulates Genes Related to Virulence and Confers Protection against Wild-Type Brucella Challenge in Mice

    PubMed Central

    Lei, Shuangshuang; Zhong, Zhijun; Ke, Yuehua; Yang, Mingjuan; Xu, Xiaoyang; Ren, Hang; An, Chang; Yuan, Jiuyun; Yu, Jiuxuan; Xu, Jie; Qiu, Yefeng; Shi, Yanchun; Wang, Yufei; Peng, Guangneng; Chen, Zeliang

    2016-01-01

    Brucellosis is one of the most common zoonotic epidemics worldwide. Brucella, the etiological pathogen of brucellosis, has unique virulence characteristics, including the ability to survive within the host cell. Hfq is a bacterial chaperone protein that is involved in the survival of the pathogen under stress conditions. Moreover, hfq affects the expression of a large number of target genes. In the present study, we characterized the expression and regulatory patterns of the target genes of Hfq during brucellosis. The results revealed that hfq expression is highly induced in macrophages at the early infection stage and at the late stage of mouse infection. Several genes related to virulence, including omp25, omp31, vjbR, htrA, gntR, and dnaK, were found to be regulated by hfq during infection in BALB/c mice. Gene expression and cytokine secretion analysis revealed that an hfq-deletion mutant induced different cytokine profiles compared with that induced by 16M. Infection with the hfq-deletion mutant induced protective immune responses against 16M challenge. Together, these results suggest that hfq is induced during infection and its deletion results in significant attenuation which affects the host immune response caused by Brucella infection. By regulating genes related to virulence, hfq promotes the virulence of Brucella. The unique characteristics of the hfq-deletion mutant, including its decreased virulence and the ability to induce protective immune response upon infection, suggest that it represents an attractive candidate for the design of a live attenuated vaccine against Brucella. PMID:26834720

  16. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways

    PubMed Central

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-01-01

    Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of co-factors (co-chaperones) that regulate their specificity and function. However, how these co-chaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We have combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone/co-chaperone/client interaction network in human cells. We uncover hundreds of novel chaperone clients, delineate their participation in specific co-chaperone complexes, and establish a surprisingly distinct network of protein/protein interactions for co-chaperones. As a salient example of the power of such analysis, we establish that NUDC family co-chaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network, its regulation in development and disease, and expand the use of chaperones as sensors for drug/target engagement. PMID:25036637

  17. Molecular chaperones as rational drug targets for Parkinson's disease therapeutics.

    PubMed

    Kalia, S K; Kalia, L V; McLean, P J

    2010-12-01

    Parkinson's disease is a neurodegenerative movement disorder that is caused, in part, by the loss of dopaminergic neurons within the substantia nigra pars compacta of the basal ganglia. The presence of intracellular protein aggregates, known as Lewy bodies and Lewy neurites, within the surviving nigral neurons is the defining neuropathological feature of the disease. Accordingly, the identification of specific genes mutated in families with Parkinson's disease and of genetic susceptibility variants for idiopathic Parkinson's disease has implicated abnormalities in proteostasis, or the handling and elimination of misfolded proteins, in the pathogenesis of this neurodegenerative disorder. Protein folding and the refolding of misfolded proteins are regulated by a network of interactive molecules, known as the chaperone system, which is composed of molecular chaperones and co-chaperones. The chaperone system is intimately associated with the ubiquitin-proteasome system and the autophagy-lysosomal pathway which are responsible for elimination of misfolded proteins and protein quality control. In addition to their role in proteostasis, some chaperone molecules are involved in the regulation of cell death pathways. Here we review the role of the molecular chaperones Hsp70 and Hsp90, and the cochaperones Hsp40, BAG family members such as BAG5, CHIP and Hip in modulating neuronal death with a focus on dopaminergic neurodegeneration in Parkinson's disease. We also review current progress in preclinical studies aimed at targetting the chaperone system to prevent neurodegeneration. Finally, we discuss potential future chaperone-based therapeutics for the symptomatic treatment and possible disease modification of Parkinson's disease.

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

    PubMed Central

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

    2016-01-01

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

  19. Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation

    PubMed Central

    Zhao, Rongmin; Kakihara, Yoshito; Gribun, Anna; Huen, Jennifer; Yang, Guocheng; Khanna, May; Costanzo, Michael; Brost, Renée L.; Boone, Charles; Hughes, Timothy R.; Yip, Christopher M.; Houry, Walid A.

    2008-01-01

    Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs. PMID:18268103

  20. Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone sis1

    SciTech Connect

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

    2015-02-13

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD-binding adaptor proteins. Finally, these interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.

  1. Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone sis1

    DOE PAGES

    Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; ...

    2015-02-13

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activitymore » with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD-binding adaptor proteins. Finally, these interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.« less

  2. Histone chaperone-mediated nucleosome assembly process.

    PubMed

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

    2015-01-01

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

  3. Molecular chaperones and neuronal proteostasis

    PubMed Central

    Smith, Heather L.; Li, Wenwen; Cheetham, Michael E.

    2015-01-01

    Protein homeostasis (proteostasis) is essential for maintaining the functionality of the proteome. The disruption of proteostasis, due to genetic mutations or an age-related decline, leads to aberrantly folded proteins that typically lose their function. The accumulation of misfolded and aggregated protein is also cytotoxic and has been implicated in the pathogenesis of neurodegenerative diseases. Neurons have developed an intrinsic protein quality control network, of which molecular chaperones are an essential component. Molecular chaperones function to promote efficient folding and target misfolded proteins for refolding or degradation. Increasing molecular chaperone expression can suppress protein aggregation and toxicity in numerous models of neurodegenerative disease; therefore, molecular chaperones are considered exciting therapeutic targets. Furthermore, mutations in several chaperones cause inherited neurodegenerative diseases. In this review, we focus on the importance of molecular chaperones in neurodegenerative diseases, and discuss the advances in understanding their protective mechanisms. PMID:25770416

  4. The C. elegans UNC-23 protein, a member of the BCL-2-associated athanogene (BAG) family of chaperone regulators, interacts with HSP-1 to regulate cell attachment and maintain hypodermal integrity

    PubMed Central

    Rahmani, Poupak; Rogalski, Teresa; Moerman, Donald G

    2015-01-01

    Mutations in the unc-23 gene in the free-living nematode, Caenorhabditis elegans result in detachment and dystrophy of the anterior body wall musculature and a bent-head phenotype when grown on solid substrate. We have determined that the unc-23 gene product is the nematode ortholog of the human BAG-2 protein, a member of the Bcl-2 associated athanogene (BAG) family of molecular chaperone regulators. We show that a functional GFP-tagged UNC-23 protein is expressed throughout development in several tissues of the animal, including body wall muscle and hypodermis, and associates with adhesion complexes and attachment structures within these 2 tissues. In humans, the BAG protein family consists of 6 members that all contain a conserved 45 amino acid BAG domain near their C-termini. These proteins bind to and modulate the activity of the ATPase domain of the heat shock cognate protein 70, Hsc70. We have isolated missense mutations in the ATPase domain of the C. elegans heat shock 70 protein, HSP-1 that suppress the phenotype exhibited by unc-23(e25) mutant hermaphrodites and we show that UNC-23 and HSP-1 interact in a yeast-2-hybrid system. The interaction of UNC-23 with HSP-1 defines a role for HSP-1 function in the maintenance of muscle attachment during development. PMID:26435886

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

    PubMed

    Alsford, Sam; Horn, David

    2012-11-01

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

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

    PubMed

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

    2014-01-01

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

  7. Chaperone activation by unfolding.

    PubMed

    Foit, Linda; George, Jenny S; Zhang, Bin W; Brooks, Charles L; Bardwell, James C A

    2013-04-02

    Conditionally disordered proteins can alternate between highly ordered and less ordered configurations under physiological conditions. Whereas protein function is often associated with the ordered conformation, for some of these conditionally unstructured proteins, the opposite applies: Their activation is associated with their unfolding. An example is the small periplasmic chaperone HdeA, which is critical for the ability of enteric bacterial pathogens like Escherichia coli to survive passage through extremely acidic environments, such as the human stomach. At neutral pH, HdeA is a chaperone-inactive dimer. On a shift to low pH, however, HdeA monomerizes, partially unfolds, and becomes rapidly active in preventing the aggregation of substrate proteins. By mutating two aspartic acid residues predicted to be responsible for the pH-dependent monomerization of HdeA, we have succeeded in isolating an HdeA mutant that is active at neutral pH. We find this HdeA mutant to be substantially destabilized, partially unfolded, and mainly monomeric at near-neutral pH at a concentration at which it prevents aggregation of a substrate protein. These results provide convincing evidence for direct activation of a protein by partial unfolding.

  8. The right place at the right time: chaperoning core histone variants.

    PubMed

    Mattiroli, Francesca; D'Arcy, Sheena; Luger, Karolin

    2015-11-01

    Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics.

  9. The right place at the right time: chaperoning core histone variants

    PubMed Central

    Mattiroli, Francesca; D’Arcy, Sheena; Luger, Karolin

    2015-01-01

    Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics. PMID:26459557

  10. Molecular chaperones and hypoxic-ischemic encephalopathy

    PubMed Central

    Hua, Cong; Ju, Wei-na; Jin, Hang; Sun, Xin; Zhao, Gang

    2017-01-01

    Hypoxic-ischemic encephalopathy (HIE) is a disease that occurs when the brain is subjected to hypoxia, resulting in neuronal death and neurological deficits, with a poor prognosis. The mechanisms underlying hypoxic-ischemic brain injury include excitatory amino acid release, cellular proteolysis, reactive oxygen species generation, nitric oxide synthesis, and inflammation. The molecular and cellular changes in HIE include protein misfolding, aggregation, and destruction of organelles. The apoptotic pathways activated by ischemia and hypoxia include the mitochondrial pathway, the extrinsic Fas receptor pathway, and the endoplasmic reticulum stress-induced pathway. Numerous treatments for hypoxic-ischemic brain injury caused by HIE have been developed over the last half century. Hypothermia, xenon gas treatment, the use of melatonin and erythropoietin, and hypoxic-ischemic preconditioning have proven effective in HIE patients. Molecular chaperones are proteins ubiquitously present in both prokaryotes and eukaryotes. A large number of molecular chaperones are induced after brain ischemia and hypoxia, among which the heat shock proteins are the most important. Heat shock proteins not only maintain protein homeostasis; they also exert anti-apoptotic effects. Heat shock proteins maintain protein homeostasis by helping to transport proteins to their target destinations, assisting in the proper folding of newly synthesized polypeptides, regulating the degradation of misfolded proteins, inhibiting the aggregation of proteins, and by controlling the refolding of misfolded proteins. In addition, heat shock proteins exert anti-apoptotic effects by interacting with various signaling pathways to block the activation of downstream effectors in numerous apoptotic pathways, including the intrinsic pathway, the endoplasmic reticulum-stress mediated pathway and the extrinsic Fas receptor pathway. Molecular chaperones play a key role in neuroprotection in HIE. In this review, we

  11. The Molecular Chaperone HSPA2 Plays a Key Role in Regulating the Expression of Sperm Surface Receptors That Mediate Sperm-Egg Recognition

    PubMed Central

    Redgrove, Kate A.; Nixon, Brett; Baker, Mark A.; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R. John

    2012-01-01

    A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility. PMID:23209833

  12. Histone Chaperone Nap1 Is a Major Regulator of Histone H2A-H2B Dynamics at the Inducible GAL Locus

    PubMed Central

    Chen, Xu; D'Arcy, Sheena; Radebaugh, Catherine A.; Krzizike, Daniel D.; Giebler, Holli A.; Huang, Liangquan; Nyborg, Jennifer K.; Luger, Karolin

    2016-01-01

    Histone chaperones, like nucleosome assembly protein 1 (Nap1), play a critical role in the maintenance of chromatin architecture. Here, we use the GAL locus in Saccharomyces cerevisiae to investigate the influence of Nap1 on chromatin structure and histone dynamics during distinct transcriptional states. When the GAL locus is not expressed, cells lacking Nap1 show an accumulation of histone H2A-H2B but not histone H3-H4 at this locus. Excess H2A-H2B interacts with the linker DNA between nucleosomes, and the interaction is independent of the inherent DNA-binding affinity of H2A-H2B for these particular sequences as measured in vitro. When the GAL locus is transcribed, excess H2A-H2B is reversed, and levels of all chromatin-bound histones are depleted in cells lacking Nap1. We developed an in vivo system to measure histone exchange at the GAL locus and observed considerable variability in the rate of exchange across the locus in wild-type cells. We recapitulate this variability with in vitro nucleosome reconstitutions, which suggests a contribution of DNA sequence to histone dynamics. We also find that Nap1 is required for transcription-dependent H2A-H2B exchange. Altogether, these results indicate that Nap1 is essential for maintaining proper chromatin composition and modulating the exchange of H2A-H2B in vivo. PMID:26884462

  13. Histone Chaperone Nap1 Is a Major Regulator of Histone H2A-H2B Dynamics at the Inducible GAL Locus.

    PubMed

    Chen, Xu; D'Arcy, Sheena; Radebaugh, Catherine A; Krzizike, Daniel D; Giebler, Holli A; Huang, Liangquan; Nyborg, Jennifer K; Luger, Karolin; Stargell, Laurie A

    2016-04-01

    Histone chaperones, like nucleosome assembly protein 1 (Nap1), play a critical role in the maintenance of chromatin architecture. Here, we use the GAL locus in Saccharomyces cerevisiae to investigate the influence of Nap1 on chromatin structure and histone dynamics during distinct transcriptional states. When the GAL locus is not expressed, cells lacking Nap1 show an accumulation of histone H2A-H2B but not histone H3-H4 at this locus. Excess H2A-H2B interacts with the linker DNA between nucleosomes, and the interaction is independent of the inherent DNA-binding affinity of H2A-H2B for these particular sequences as measured in vitro When the GAL locus is transcribed, excess H2A-H2B is reversed, and levels of all chromatin-bound histones are depleted in cells lacking Nap1. We developed an in vivo system to measure histone exchange at the GAL locus and observed considerable variability in the rate of exchange across the locus in wild-type cells. We recapitulate this variability with in vitro nucleosome reconstitutions, which suggests a contribution of DNA sequence to histone dynamics. We also find that Nap1 is required for transcription-dependent H2A-H2B exchange. Altogether, these results indicate that Nap1 is essential for maintaining proper chromatin composition and modulating the exchange of H2A-H2B in vivo.

  14. Regulation of the myosin-directed chaperone UNC-45 by a novel E3/E4-multiubiquitylation complex in C. elegans.

    PubMed

    Hoppe, Thorsten; Cassata, Giuseppe; Barral, José M; Springer, Wolfdieter; Hutagalung, Alex H; Epstein, Henry F; Baumeister, Ralf

    2004-08-06

    The organization of the motor protein myosin into motile cellular structures requires precise temporal and spatial control. Caenorhabditis elegans UNC-45 facilitates this by functioning both as a chaperone and as a Hsp90 cochaperone for myosin during thick filament assembly. Consequently, mutations in C. elegans unc-45 result in paralyzed animals with severe myofibril disorganization in striated body wall muscles. Here, we report a new E3/E4 complex, formed by CHN-1, the C. elegans ortholog of CHIP (carboxyl terminus of Hsc70-interacting protein), and UFD-2, an enzyme known to have ubiquitin conjugating E4 activity in yeast, as necessary and sufficient to multiubiquitylate UNC-45 in vitro. The phenotype of unc-45 temperature-sensitive animals is partially suppressed by chn-1 loss of function, while UNC-45 overexpression in worms deficient for chn-1 results in severely disorganized muscle cells. These results identify CHN-1 and UFD-2 as a functional E3/E4 complex and UNC-45 as its physiologically relevant substrate.

  15. The molecular chaperone HSPA2 plays a key role in regulating the expression of sperm surface receptors that mediate sperm-egg recognition.

    PubMed

    Redgrove, Kate A; Nixon, Brett; Baker, Mark A; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R John

    2012-01-01

    A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm-egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm-zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.

  16. Histone chaperone networks shaping chromatin function.

    PubMed

    Hammond, Colin M; Strømme, Caroline B; Huang, Hongda; Patel, Dinshaw J; Groth, Anja

    2017-03-01

    The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin.

  17. Conformational dynamics of the molecular chaperone Hsp90

    PubMed Central

    Krukenberg, Kristin A.; Street, Timothy O.; Lavery, Laura A.; Agard, David A.

    2016-01-01

    The molecular chaperone Hsp90 is an essential eukaryotic protein that makes up 1–2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have also been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 is a highly dynamic and flexible molecule that can adapt its conformation to the wide variety of substrate proteins with which it acts. Large conformational rearrangements are also required for the activation of these client proteins. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shifts the equilibrium between a pre-existing set of conformational states in an organism-dependent manner. In vivo Hsp90 functions as part of larger heterocomplexes. The binding partners of Hsp90, co-chaperones, assist in the recruitment and activation of substrates, and many co-chaperones further regulate the conformational dynamics of Hsp90 by shifting the conformational equilibrium towards a particular state. Studies have also suggested alternative mechanisms for the regulation of Hsp90’s conformation. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90 and the role that nucleotide, co-chaperones, post-translational modification and clients play in regulating Hsp90’s conformation. We also discuss the effects of current Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics. PMID:21414251

  18. Specific Chaperones and Regulatory Domains in Control of Amyloid Formation*

    PubMed Central

    Landreh, Michael; Rising, Anna; Presto, Jenny; Jörnvall, Hans; Johansson, Jan

    2015-01-01

    Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology. PMID:26354437

  19. Control of cell cycle and cell growth by molecular chaperones.

    PubMed

    Aldea, Martí; Garí, Eloi; Colomina, Neus

    2007-11-01

    Cells adapt their size to both intrinsic and extrinsic demands and, among them, those that stem from growth and proliferation rates are crucial for cell size homeostasis. Here we revisit mechanisms that regulate cell cycle and cell growth in budding yeast. Cyclin Cln3, the most upstream activator of Start, is retained at the endoplasmic reticulum in early G(1) and released by specific chaperones in late G(1) to initiate the cell cycle. On one hand, these chaperones are rate-limiting for release of Cln3 and cell cycle entry and, on the other hand, they are required for key biosynthetic processes. We propose a model whereby the competition for specialized chaperones between growth and cycle machineries could gauge biosynthetic rates and set a critical size threshold at Start.

  20. ER chaperones in neurodegenerative disease: Folding and beyond.

    PubMed

    Garcia-Huerta, Paula; Bargsted, Leslie; Rivas, Alexis; Matus, Soledad; Vidal, Rene L

    2016-10-01

    Proteins along the secretory pathway are co-translationally translocated into the lumen of the endoplasmic reticulum (ER) as unfolded polypeptide chains. Afterwards, they are usually modified with N-linked glycans, correctly folded and stabilized by disulfide bonds. ER chaperones and folding enzymes control these processes. The accumulation of unfolded proteins in the ER activates a signaling response, termed the unfolded protein response (UPR). The hallmark of this response is the coordinated transcriptional up-regulation of ER chaperones and folding enzymes. In order to discuss the importance of the proper folding of certain substrates we will address the role of ER chaperones in normal physiological conditions and examine different aspects of its contribution in neurodegenerative disease. This article is part of a Special Issue entitled SI:ER stress.

  1. The conformational dynamics of the mitochondrial Hsp70 chaperone.

    PubMed

    Mapa, Koyeli; Sikor, Martin; Kudryavtsev, Volodymyr; Waegemann, Karin; Kalinin, Stanislav; Seidel, Claus A M; Neupert, Walter; Lamb, Don C; Mokranjac, Dejana

    2010-04-09

    Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments.

  2. Direct Measurement of Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator to the Cell Surface and Binding to a Chemical Chaperone.

    PubMed

    Zhang, Zhihui; Baksh, Michael M; Finn, M G; Heidary, David K; Richards, Christopher I

    2017-01-10

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in the disease cystic fibrosis. Deletion of Phe508, the most prevalent mutation associated with this disease, disrupts trafficking of the protein. Small molecule correctors yield moderate improvements in the trafficking of ΔF508-CFTR to the plasma membrane. It is currently not known if correctors increase the level of trafficking through improved cargo loading of transport vesicles or through direct binding to CFTR. Real-time measurements of trafficking were utilized to identify the mechanistic details of chemical, biochemical, and thermal factors that impact CFTR correction, using the corrector molecule VX-809, a secondary mutation (I539T), and low-temperature conditions. Each individually improved trafficking of ΔF508-CFTR to approximately 10% of wild-type levels. The combination of VX-809 with either low temperature or the I539T mutation increased the amount of CFTR on the plasma membrane to nearly 40%, indicating synergistic activity. The number of vesicles reaching the surface was significantly altered; however, the amount of channel in each vesicle remained the same. Direct binding measurements of VX-809 in native membranes using backscattering interferometry indicate tight binding to CFTR, which occurred in a manner independent of mutation. The similar values obtained for all forms of the channel indicate that the binding site is not compromised or enhanced by these mutations.

  3. Pharmacological Targeting of the Hsp70 Chaperone

    PubMed Central

    Patury, Srikanth; Miyata, Yoshinari; Gestwicki, Jason E.

    2009-01-01

    The molecular chaperone, heat shock protein 70 (Hsp70), acts at multiple steps in a protein’s life cycle, including during the processes of folding, trafficking, remodeling and degradation. To accomplish these various tasks, the activity of Hsp70 is shaped by a host of co-chaperones, which bind to the core chaperone and influence its functions. Genetic studies have strongly linked Hsp70 and its co-chaperones to numerous diseases, including cancer, neurodegeneration and microbial pathogenesis, yet the potential of this chaperone as a therapeutic target remains largely underexplored. Here, we review the current state of Hsp70 as a drug target, with a special emphasis on the important challenges and opportunities imposed by its co-chaperones, protein-protein interactions and allostery. PMID:19860737

  4. Catalysis of protein folding by chaperones accelerates evolutionary dynamics in adapting cell populations.

    PubMed

    Cetinbaş, Murat; Shakhnovich, Eugene I

    2013-01-01

    Although molecular chaperones are essential components of protein homeostatic machinery, their mechanism of action and impact on adaptation and evolutionary dynamics remain controversial. Here we developed a physics-based ab initio multi-scale model of a living cell for population dynamics simulations to elucidate the effect of chaperones on adaptive evolution. The 6-loci genomes of model cells encode model proteins, whose folding and interactions in cellular milieu can be evaluated exactly from their genome sequences. A genotype-phenotype relationship that is based on a simple yet non-trivially postulated protein-protein interaction (PPI) network determines the cell division rate. Model proteins can exist in native and molten globule states and participate in functional and all possible promiscuous non-functional PPIs. We find that an active chaperone mechanism, whereby chaperones directly catalyze protein folding, has a significant impact on the cellular fitness and the rate of evolutionary dynamics, while passive chaperones, which just maintain misfolded proteins in soluble complexes have a negligible effect on the fitness. We find that by partially releasing the constraint on protein stability, active chaperones promote a deeper exploration of sequence space to strengthen functional PPIs, and diminish the non-functional PPIs. A key experimentally testable prediction emerging from our analysis is that down-regulation of chaperones that catalyze protein folding significantly slows down the adaptation dynamics.

  5. Agrobacterium tumefaciens Zur Regulates the High-Affinity Zinc Uptake System TroCBA and the Putative Metal Chaperone YciC, along with ZinT and ZnuABC, for Survival under Zinc-Limiting Conditions

    PubMed Central

    Chaoprasid, Paweena; Dokpikul, Thanittra; Johnrod, Jaruwan; Sirirakphaisarn, Sirin; Nookabkaew, Sumontha; Mongkolsuk, Skorn

    2016-01-01

    ABSTRACT Agrobacterium tumefaciens has a cluster of genes (Atu3178, Atu3179, and Atu3180) encoding an ABC-type transporter, here named troA, troB, and troC, respectively, which is shown here to be a zinc-specific uptake system. Reverse transcription (RT)-PCR analysis confirmed that troA, troB, and troC are cotranscribed, with troC as the first gene of the operon. The yciC (Atu3181) gene is transcribed in the opposite orientation to that of the troCBA operon and belongs to a metal-binding GTPase family. Expression of troCBA and yciC was inducible under zinc-limiting conditions and was controlled by the zinc uptake regulator, Zur. Compared to the wild type, the mutant strain lacking troC was hypersensitive to a metal chelator, EDTA, and the phenotype could be rescued by the addition of zinc, while the strain with a single yciC mutation showed no phenotype. However, yciC was important for survival under zinc limitation when either troC or zinT was inactivated. The periplasmic zinc-binding protein, ZinT, could not function when TroC was inactivated, suggesting that ZinT may interact with TroCBA in zinc uptake. Unlike many other bacteria, the ABC-type transporter ZnuABC was not the major zinc uptake system in A. tumefaciens. However, the important role of A. tumefaciens ZnuABC was revealed when TroCBA was impaired. The strain containing double mutations in the znuA and troC genes exhibited a growth defect in minimal medium. A. tumefaciens requires cooperation of zinc uptake systems and zinc chaperones, including TroCBA, ZnuABC, ZinT, and YciC, for survival under a wide range of zinc-limiting conditions. IMPORTANCE Both host and pathogen battle over access to essential metals, including zinc. In low-zinc environments, physiological responses that make it possible to acquire enough zinc are important for bacterial survival and could determine the outcome of host-pathogen interactions. A. tumefaciens was found to operate a novel pathway for zinc uptake in which Zin

  6. Review: Beta-thalassemia and molecular chaperones.

    PubMed

    Sumera, Afshan; Radhakrishnan, Ammu; Baba, Abdul Aziz; George, Elizabeth

    2015-04-01

    Thalassemia is known as a diverse single gene disorder, which is prevalent worldwide. The molecular chaperones are set of proteins that help in two important processes while protein synthesis and degradation include folding or unfolding and assembly or disassembly, thereby helping in cell homeostasis. This review recaps current knowledge regarding the role of molecular chaperones in thalassemia, with a focus on beta thalassemia.

  7. Chaperone addiction of toxin–antitoxin systems

    PubMed Central

    Bordes, Patricia; Sala, Ambre Julie; Ayala, Sara; Texier, Pauline; Slama, Nawel; Cirinesi, Anne-Marie; Guillet, Valérie; Mourey, Lionel; Genevaux, Pierre

    2016-01-01

    Bacterial toxin–antitoxin (TA) systems, in which a labile antitoxin binds and inhibits the toxin, can promote adaptation and persistence by modulating bacterial growth in response to stress. Some atypical TA systems, known as tripartite toxin–antitoxin–chaperone (TAC) modules, include a molecular chaperone that facilitates folding and protects the antitoxin from degradation. Here we use a TAC module from Mycobacterium tuberculosis as a model to investigate the molecular mechanisms by which classical TAs can become ‘chaperone-addicted'. The chaperone specifically binds the antitoxin at a short carboxy-terminal sequence (chaperone addiction sequence, ChAD) that is not present in chaperone-independent antitoxins. In the absence of chaperone, the ChAD sequence destabilizes the antitoxin, thus preventing toxin inhibition. Chaperone–ChAD pairs can be transferred to classical TA systems or to unrelated proteins and render them chaperone-dependent. This mechanism might be used to optimize the expression and folding of heterologous proteins in bacterial hosts for biotechnological or medical purposes. PMID:27827369

  8. Chaperone-mediated specificity in Ras and Rap signaling.

    PubMed

    Azoulay-Alfaguter, Inbar; Strazza, Marianne; Mor, Adam

    2015-01-01

    Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.

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

    PubMed

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

    2011-02-18

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

  10. Lipid Chaperones and Metabolic Inflammation

    PubMed Central

    Furuhashi, Masato; Ishimura, Shutaro; Ota, Hideki; Miura, Tetsuji

    2011-01-01

    Over the past decade, a large body of evidence has emerged demonstrating an integration of metabolic and immune response pathways. It is now clear that obesity and associated disorders such as insulin resistance and type 2 diabetes are associated with a metabolically driven, low-grade, chronic inflammatory state, referred to as “metaflammation.” Several inflammatory cytokines as well as lipids and metabolic stress pathways can activate metaflammation, which targets metabolically critical organs and tissues including adipocytes and macrophages to adversely affect systemic homeostasis. On the other hand, inside the cell, fatty acid-binding proteins (FABPs), a family of lipid chaperones, as well as endoplasmic reticulum (ER) stress, and reactive oxygen species derived from mitochondria play significant roles in promotion of metabolically triggered inflammation. Here, we discuss the molecular and cellular basis of the roles of FABPs, especially FABP4 and FABP5, in metaflammation and related diseases including obesity, diabetes, and atherosclerosis. PMID:22121495

  11. Chaperone receptors: guiding proteins to intracellular compartments.

    PubMed

    Kriechbaumer, Verena; von Löffelholz, Ottilie; Abell, Ben M

    2012-01-01

    Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519-530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529-535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface of both these organelles (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein targeting (Young et al., Cell 112:41-50, 2003; Qbadou et al., EMBO J 25:1836-1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly, at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors.

  12. Molecular chaperones: functional mechanisms and nanotechnological applications

    NASA Astrophysics Data System (ADS)

    Rosario Fernández-Fernández, M.; Sot, Begoña; María Valpuesta, José

    2016-08-01

    Molecular chaperones are a group of proteins that assist in protein homeostasis. They not only prevent protein misfolding and aggregation, but also target misfolded proteins for degradation. Despite differences in structure, all types of chaperones share a common general feature, a surface that recognizes and interacts with the misfolded protein. This and other, more specialized properties can be adapted for various nanotechnological purposes, by modification of the original biomolecules or by de novo design based on artificial structures.

  13. mTORC1 links protein quality and quantity control by sensing chaperone availability.

    PubMed

    Qian, Shu-Bing; Zhang, Xingqian; Sun, Jun; Bennink, Jack R; Yewdell, Jonathan W; Patterson, Cam

    2010-08-27

    Balanced protein synthesis and degradation are crucial for proper cellular function. Protein synthesis is tightly coupled to energy status and nutrient levels by the mammalian target of rapamycin complex 1 (mTORC1). Quality of newly synthesized polypeptides is maintained by the molecular chaperone and ubiquitin-proteasome systems. Little is known about how cells integrate information about the quantity and quality of translational products simultaneously. We demonstrate that cells distinguish moderate reductions in protein quality from severe protein misfolding using molecular chaperones to differentially regulate mTORC1 signaling. Moderate reduction of chaperone availability enhances mTORC1 signaling, whereas stress-induced complete depletion of chaperoning capacity suppresses mTORC1 signaling. Molecular chaperones regulate mTORC1 assembly in coordination with nutrient availability. This mechanism enables mTORC1 to rapidly detect and respond to environmental cues while also sensing intracellular protein misfolding. The tight linkage between protein quality and quantity control provides a plausible mechanism coupling protein misfolding with metabolic dyshomeostasis.

  14. Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

    PubMed Central

    Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna

    2016-01-01

    The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023

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

    ERIC Educational Resources Information Center

    Brandt, Brian

    2016-01-01

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

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

    PubMed

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

    2014-09-01

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

  17. Visualizing chaperone-assisted protein folding

    PubMed Central

    Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; Ahlstrom, Logan S.; Martin, Raoul; Quan, Shu; Afonine, Pavel V.; van den Bedem, Henry; Wang, Lili; Xu, Qingping; Trievel, Raymond C.; Brooks, Charles L.; Bardwell, James CA

    2016-01-01

    Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. PMID:27239796

  18. Chaperones in hepatitis C virus infection

    PubMed Central

    Khachatoorian, Ronik; French, Samuel W

    2016-01-01

    The hepatitis C virus (HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases: (1) binding and internalization; (2) cytoplasmic release and uncoating; (3) viral polyprotein translation and processing; (4) RNA genome replication; (5) encapsidation (packaging) and assembly; and (6) virus morphogenesis (maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses. PMID:26783419

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

    PubMed Central

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

    2016-01-01

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

  20. Molecular chaperones in lactic acid bacteria: physiological consequences and biochemical properties.

    PubMed

    Sugimoto, Shinya; Abdullah-Al-Mahin; Sonomoto, Kenji

    2008-10-01

    Recently, lactic acid bacteria (LAB) have attracted much attention because of their potential application to probiotics and industrial applications as starters for dairy products or lactic acid fermentation. Additional emphasis is also being paid to them as commensal bacteria in gastrointestinal tract. Since LAB exhibit a stress response, insight into the relationship between stress proteins such as molecular chaperones and stress tolerance or adaptation is increasing gradually along with current research examining these important bacteria. Similar to other bacteria, one of the major stress-response systems in LAB is the expression of molecular chaperones. The recently completed genome sequencing of various LAB strains, combined with the development of advanced molecular techniques, have enabled us to identify molecular chaperones and to understand their regulation systems in response to various stresses. Furthermore, recent biochemical studies provided novel insight into the molecular mechanisms of LAB chaperone systems. This review highlights the physiological consequences and biochemical properties of molecular chaperones (especially sHsps, Hsp70, and Hsp100) in LAB and their use in biotechnological applications.

  1. Inhibitors of the AAA+ Chaperone p97

    PubMed Central

    Chapman, Eli; Maksim, Nick; de la Cruz, Fabian; La Clair, James J.

    2015-01-01

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

  2. Structure of Vps75 and Implications for Histone Chaperone Function

    SciTech Connect

    Tang,Y.; Meeth, K.; Jiang, E.; Luo, c.; Marmostein, R.

    2008-01-01

    The vacuolar protein sorting 75 (Vps75) histone chaperone participates in chromatin assembly and disassembly at both active and inactive genes through the preferential binding to histone H3-H4. Vps75 is also one of two histone chaperones, along with antisilencing factor 1, that promotes histone H3-Lys-56 acetylation by the regulation of Ty1 transposition protein 109 (Rtt109) histone acetyltransferase. Here, we report the x-ray crystal structure of Vps75 and carry out biochemical studies to characterize its interaction with Rtt109. We find that the Vps75 structure forms a homodimeric 'headphone' architecture that includes an extended helical dimerization domain and earmuff domains at opposite ends and sides of the dimerization domain. Despite the similar overall architecture with the yeast nucleosome assembly protein 1 and human SET/TAF-1{beta}/INHAT histone chaperones, Vps75 shows several unique features including the relative disposition of the earmuff domains to the dimerization domain, characteristics of the earmuff domains, and a pronounced cleft at the center of the Vps75 dimer. These differences appear to correlate with the unique function of Vps75 to interact with Rtt109 for histone acetylation. Our biochemical studies reveal that two surfaces on the earmuff domain of Vps75 participate in Rtt109 interaction with a stoichiometry of 2:1, thus leaving the pronounced central cleft of the Vps75 dimer largely accessible for histone binding. Taken together, our data provide a structural framework for understanding how Vps75 mediates both nucleosome assembly and histone acetylation by Rtt109.

  3. Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation

    PubMed Central

    Zheng, Xu; Krakowiak, Joanna; Patel, Nikit; Beyzavi, Ali; Ezike, Jideofor; Khalil, Ahmad S; Pincus, David

    2016-01-01

    Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.18638.001 PMID:27831465

  4. Phenylalanine hydroxylase misfolding and pharmacological chaperones.

    PubMed

    Underhaug, Jarl; Aubi, Oscar; Martinez, Aurora

    2012-01-01

    Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism. As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant phenylalanine hydroxylase (PAH) to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilization of PKU-mutants in vitro, in cells, and in mice by pharmacological chaperones, which have been found either by using the tetrahydrobiopterin (BH(4)) cofactor as query structure for shape-focused virtual screening or by high-throughput screening of small compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH(4). Furthermore, PAH shares a number of ligands, such as BH(4), amino acid substrates and inhibitors, with the other aromatic amino acid hydroxylases: the neuronal/neuroendocrine enzymes tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatized to avoid unwanted interactions with these other enzymes. After derivatization and validation in animal models, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU.

  5. Consequences of the selective blockage of chaperone-mediated autophagy

    PubMed Central

    Massey, Ashish C.; Kaushik, Susmita; Sovak, Guy; Kiffin, Roberta; Cuervo, Ana Maria

    2006-01-01

    Chaperone-mediated autophagy (CMA) is a selective pathway for the degradation of cytosolic proteins in lysosomes. CMA declines with age because of a decrease in the levels of lysosome-associated membrane protein (LAMP) type 2A, a lysosomal receptor for this pathway. We have selectively blocked the expression of LAMP-2A in mouse fibroblasts in culture and analyzed the cellular consequences of reduced CMA activity. CMA-defective cells maintain normal rates of long-lived protein degradation by up-regulating macroautophagy, the major form of autophagy. Constitutive up-regulation of macroautophagy is unable, however, to compensate for all CMA functions. Thus, CMA-defective cells are more sensitive to stressors, suggesting that, although protein turnover is maintained, the selectivity of CMA is necessary as part of the cellular response to stress. Our results also denote the existence of cross-talk among different forms of autophagy. PMID:16585521

  6. Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity.

    PubMed

    Tomoyasu, Toshifumi; Tabata, Atsushi; Imaki, Hidenori; Tsuruno, Keigo; Miyazaki, Aya; Sonomoto, Kenji; Whiley, Robert Alan; Nagamune, Hideaki

    2012-01-01

    Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that causes purulent infections and abscess formation. The DnaK chaperone system has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity. However, the role of DnaK in S. intermedius remains unclear. Therefore, we constructed a dnaK knockout mutant that exhibited slow growth, thermosensitivity, accumulation of GroEL in the cell, and reduced cytotoxicity to HepG2 cells. The level of secretion of a major pathogenic factor, intermedilysin, was not affected by dnaK mutation. We further examined the function and property of the S. intermedius DnaK chaperone system by using Escherichia coli ΔdnaK and ΔrpoH mutant strains. S. intermedius DnaK could not complement the thermosensitivity of E. coli ΔdnaK mutant. However, the intact S. intermedius DnaK chaperone system could complement the thermosensitivity and acid sensitivity of E. coli ΔdnaK mutant. The S. intermedius DnaK chaperone system could regulate the activity and stability of the heat shock transcription factor σ(32) in E. coli, although S. intermedius does not utilize σ(32) for heat shock transcription. The S. intermedius DnaK chaperone system was also able to efficiently eliminate the aggregated proteins from ΔrpoH mutant cells. Overall, our data showed that the S. intermedius DnaK chaperone system has important functions in quality control of cellular proteins but has less participation in the modulation of expression of pathogenic factors.

  7. Emerging novel concept of chaperone therapies for protein misfolding diseases

    PubMed Central

    SUZUKI, Yoshiyuki

    2014-01-01

    Chaperone therapy is a newly developed molecular therapeutic approach to protein misfolding diseases. Among them we found unstable mutant enzyme proteins in a few lysosomal diseases, resulting in rapid intracellular degradation and loss of function. Active-site binding low molecular competitive inhibitors (chemical chaperones) paradoxically stabilized and enhanced the enzyme activity in somatic cells by correction of the misfolding of enzyme protein. They reached the brain through the blood-brain barrier after oral administration, and corrected pathophysiology of the disease. In addition to these inhibitory chaperones, non-competitive chaperones without inhibitory bioactivity are being developed. Furthermore molecular chaperone therapy utilizing the heat shock protein and other chaperone proteins induced by small molecules has been experimentally tried to handle abnormally accumulated proteins as a new approach particularly to neurodegenerative diseases. These three types of chaperones are promising candidates for various types of diseases, genetic or non-genetic, and neurological or non-neurological, in addition to lysosomal diseases. PMID:24814990

  8. Degradation of AF1Q by chaperone-mediated autophagy

    SciTech Connect

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru; Li, Huanjie; Cui, Taixing; Li Wang, Xing; Tang, Dongqi; Ji, Chunyan

    2014-09-10

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

  9. Plant Leucine Aminopeptidases Moonlight as Molecular Chaperones to Alleviate Stress-induced Damage*

    PubMed Central

    Scranton, Melissa A.; Yee, Ashley; Park, Sang-Youl; Walling, Linda L.

    2012-01-01

    Leucine aminopeptidases (LAPs) are present in animals, plants, and microbes. In plants, there are two classes of LAPs. The neutral LAPs (LAP-N and its orthologs) are constitutively expressed and detected in all plants, whereas the stress-induced acidic LAPs (LAP-A) are expressed only in a subset of the Solanaceae. LAPs have a role in insect defense and act as a regulator of the late branch of wound signaling in Solanum lycopersicum (tomato). Although the mechanism of LAP-A action is unknown, it has been presumed that LAP peptidase activity is essential for regulating wound signaling. Here we show that plant LAPs are bifunctional. Using three assays to monitor protein protection from heat-induced damage, it was shown that the tomato LAP-A and LAP-N and the Arabidopsis thaliana LAP1 and LAP2 are molecular chaperones. Assays using LAP-A catalytic site mutants demonstrated that LAP-A chaperone activity was independent of its peptidase activity. Furthermore, disruption of the LAP-A hexameric structure increased chaperone activity. Together, these data identify a new class of molecular chaperones and a new function for the plant LAPs as well as suggesting new mechanisms for LAP action in the defense of solanaceous plants against stress. PMID:22493451

  10. Plant leucine aminopeptidases moonlight as molecular chaperones to alleviate stress-induced damage.

    PubMed

    Scranton, Melissa A; Yee, Ashley; Park, Sang-Youl; Walling, Linda L

    2012-05-25

    Leucine aminopeptidases (LAPs) are present in animals, plants, and microbes. In plants, there are two classes of LAPs. The neutral LAPs (LAP-N and its orthologs) are constitutively expressed and detected in all plants, whereas the stress-induced acidic LAPs (LAP-A) are expressed only in a subset of the Solanaceae. LAPs have a role in insect defense and act as a regulator of the late branch of wound signaling in Solanum lycopersicum (tomato). Although the mechanism of LAP-A action is unknown, it has been presumed that LAP peptidase activity is essential for regulating wound signaling. Here we show that plant LAPs are bifunctional. Using three assays to monitor protein protection from heat-induced damage, it was shown that the tomato LAP-A and LAP-N and the Arabidopsis thaliana LAP1 and LAP2 are molecular chaperones. Assays using LAP-A catalytic site mutants demonstrated that LAP-A chaperone activity was independent of its peptidase activity. Furthermore, disruption of the LAP-A hexameric structure increased chaperone activity. Together, these data identify a new class of molecular chaperones and a new function for the plant LAPs as well as suggesting new mechanisms for LAP action in the defense of solanaceous plants against stress.

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

    PubMed Central

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

    2016-01-01

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

  12. Histone chaperone specificity in Rtt109 activation

    PubMed Central

    Park, Young-Jun; Sudhoff, Keely B; Andrews, Andrew J; Stargell, Laurie A; Luger, Karolin

    2008-01-01

    Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone 3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. Here we show that both Rtt109 and Vps75 bind histones with high affinity, but only the complex is efficient for catalysis. The C-terminal acidic domain of Vps75 contributes to activation of Rtt109 and is necessary for in vivo functionality of Vps75, but it is not required for interaction with either Rtt109 or histones. We demonstrate that Vps75 is a structural homolog of yeast Nap1 by solving its crystal structure. Nap1 and Vps75 interact with histones and Rtt109 with comparable affinities. However, only Vps75 stimulates Rtt109 enzymatic activity. Our data highlight the functional specificity of Vps75 in Rtt109 activation. PMID:19172749

  13. Recombination of ozone via the chaperon mechanism

    NASA Astrophysics Data System (ADS)

    Ivanov, Mikhail V.; Schinke, Reinhard

    2006-03-01

    The recombination of ozone via the chaperon mechanism, i.e., ArO +O2→Ar+O3 and ArO2+O→Ar+O3, is studied by means of classical trajectories and a pairwise additive Ar -O3 potential energy surface. The recombination rate coefficient has a strong temperature dependence, which approximately can be described by T-n with n ≈3. It is negligible for temperatures above 700 K or so, but it becomes important for low temperatures. The calculations unambiguously affirm the conclusions of Hippler et al. [J. Chem. Phys. 93, 6560 (1990)] and Luther et al. [Phys. Chem. Chem. Phys. 7, 2764 (2005)] that the chaperon mechanism makes a sizable contribution to the recombination of O3 at room temperature and below. The dependence of the chaperon recombination rate coefficient on the isotopomer, studied for two different isotope combinations, is only in rough qualitative agreement with the experimental data. The oxygen atom isotope exchange reaction involving ArO and ArO2 van der Waals complexes is also investigated; the weak binding of O or O2 to Ar has only a small effect.

  14. Characterization of the human sigma-1 receptor chaperone domain structure and binding immunoglobulin protein (BiP) interactions.

    PubMed

    Ortega-Roldan, Jose Luis; Ossa, Felipe; Schnell, Jason R

    2013-07-19

    The sigma-1 receptor (S1R) is a ligand-regulated membrane protein chaperone involved in the ER stress response. S1R activity is implicated in diseases of the central nervous system including amnesia, schizophrenia, depression, Alzheimer disease, and addiction. S1R has been shown previously to regulate the Hsp70 binding immunoglobulin protein (BiP) and the inositol triphosphate receptor calcium channel through a C-terminal domain. We have developed methods for bacterial expression and reconstitution of the chaperone domain of human S1R into detergent micelles that enable its study by solution NMR spectroscopy. The chaperone domain is found to contain a helix at the N terminus followed by a largely dynamic region and a structured, helical C-terminal region that encompasses a membrane associated domain containing four helices. The helical region at residues ∼198-206 is strongly amphipathic and proposed to anchor the chaperone domain to micelles and membranes. Three of the helices in the C-terminal region closely correspond to previously identified cholesterol and drug recognition sites. In addition, it is shown that the chaperone domain interacts with full-length BiP or the isolated nucleotide binding domain of BiP, but not the substrate binding domain, suggesting that the nucleotide binding domain is sufficient for S1R interactions.

  15. Characterization of the Human Sigma-1 Receptor Chaperone Domain Structure and Binding Immunoglobulin Protein (BiP) Interactions*

    PubMed Central

    Ortega-Roldan, Jose Luis; Ossa, Felipe; Schnell, Jason R.

    2013-01-01

    The sigma-1 receptor (S1R) is a ligand-regulated membrane protein chaperone involved in the ER stress response. S1R activity is implicated in diseases of the central nervous system including amnesia, schizophrenia, depression, Alzheimer disease, and addiction. S1R has been shown previously to regulate the Hsp70 binding immunoglobulin protein (BiP) and the inositol triphosphate receptor calcium channel through a C-terminal domain. We have developed methods for bacterial expression and reconstitution of the chaperone domain of human S1R into detergent micelles that enable its study by solution NMR spectroscopy. The chaperone domain is found to contain a helix at the N terminus followed by a largely dynamic region and a structured, helical C-terminal region that encompasses a membrane associated domain containing four helices. The helical region at residues ∼198–206 is strongly amphipathic and proposed to anchor the chaperone domain to micelles and membranes. Three of the helices in the C-terminal region closely correspond to previously identified cholesterol and drug recognition sites. In addition, it is shown that the chaperone domain interacts with full-length BiP or the isolated nucleotide binding domain of BiP, but not the substrate binding domain, suggesting that the nucleotide binding domain is sufficient for S1R interactions. PMID:23760505

  16. A molecular mechanism of chaperone-client recognition

    PubMed Central

    He, Lichun; Sharpe, Timothy; Mazur, Adam; Hiller, Sebastian

    2016-01-01

    Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature. PMID:28138538

  17. Stress and molecular chaperones in disease.

    PubMed

    Macario, A J; Conway de Macario, E

    2000-01-01

    Stress, a common phenomenon in today's society, is suspected of playing a role in the development of disease. Stressors of various types, psychological, physical, and biological, abound. They occur in the working and social environments, in air, soil, water, food, and medicines. Stressors impact on cells directly or indirectly, cause protein denaturation, and elicit a stress response. This is mediated by stress (heat-shock) genes and proteins, among which are those named molecular chaperones because they assist other proteins to achieve and maintain a functional shape (the native configuration), and to recover it when partially lost due to stress. Denatured proteins tend to aggregate and precipitate. The same occurs with abnormal proteins due to mutations, or to failure of post-transcriptional or post-translational mechanisms. These abnormal proteins need the help of molecular chaperones as much as denatured molecules do, especially during stress. A cell with normal antistress mechanisms, including a complete and functional set of chaperones, may be able to withstand stress if its intensity is not beyond that which will cause irreversible protein damage. There is a certain threshold that normal cells have above which they cannot cope with stress. A cell with an abnormal protein that has an intrinsic tendency to misfold and aggregate is more vulnerable to stress than normal counterparts. Furthermore, these abnormal proteins may precipitate even in the absence of stress and cause diseases named proteinopathies. It is possible that stress contributes to the pathogenesis of proteinopathies by promoting protein aggregation, even in cells that possess a normal chaperoning system. Examples of proteinopathies are age-related degenerative disorders with protein deposits in various tissues, most importantly in the brain where the deposits are associated with neuronal degeneration. It is conceivable that stress enhances the progression of these diseases by facilitating

  18. Type III secretion chaperones of Pseudomonas syringae protect effectors from Lon-associated degradation.

    PubMed

    Losada, Liliana C; Hutcheson, Steven W

    2005-02-01

    The hrp type III secretion system (TTSS) of Pseudomonas syringae translocates effector proteins into the cytoplasm of host cells. Proteolysis of HrpR by Lon has been shown to negatively regulate the hrp TTSS. The inability to bypass Lon-associated effects on the regulatory system by ectopic expression of the known regulators suggested a second site of action for Lon in TTSS-dependent effector secretion. In this study we report that TTSS-dependent effectors are subject to the proteolytic degradation that appears to be rate-limiting to secretion. The half-lives of the effectors AvrPto, AvrRpt2, HopPsyA, HopPsyB1, HopPtoB2, HopPsyV1, HopPtoG and HopPtoM were substantially higher in bacteria lacking Lon. TTSS-dependent secretion of several effectors was enhanced from Lon mutants. A primary role for chaperones appears to be protection of effectors from Lon-associated degradation prior to secretion. When coexpressed with their cognate chaperone, HopPsyB1, HopPsyV1 and HopPtoM were at least 10 times more stable in strains expressing Lon. Distinct Lon-targeting and chaperone-binding domains were identified in HopPtoM. The results imply that Lon is involved at two distinct levels in the regulation of the P. syringae TTSS: regulation of assembly of the secreton and modulation of effector secretion.

  19. Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding

    PubMed Central

    Aremu, David A.; Ezomo, Ojeiru F.

    2010-01-01

    Objectives Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. Methods Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. Results Gene expression analysis revealed that Ire1β was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca2+-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. Conclusions The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery. PMID:21432213

  20. Copper accumulation and compartmentalization in mouse fibroblast lacking metallothionein and copper chaperone, Atox1

    SciTech Connect

    Miyayama, Takamitsu; Suzuki, Kazuo T.; Ogra, Yasumitsu

    2009-06-01

    Copper (Cu) is the active center of some enzymes because of its redox-active property, although that property could have harmful effects. Because of this, cells have strict regulation/detoxification systems for this metal. In this study, multi-disciplinary approaches, such as speciation and elemental imaging of Cu, were applied to reveal the detoxification mechanisms for Cu in cells bearing a defect in Cu-regulating genes. Although Cu concentration in metallothionein (MT)-knockout cells was increased by the knockdown of the Cu chaperone, Atox1, the concentrations of the Cu influx pump, Ctr1, and another Cu chaperone, Ccs, were paradoxically increased; namely, the cells responded to the Cu deficiency despite the fact that cellular Cu concentration was actually increased. Cu imaging showed that the elevated Cu was compartmentalized in cytoplasmic vesicles. Together, the results point to the novel roles of MT and cytoplasmic vesicles in the detoxification of Cu in mammalian cells.

  1. N. meningitidis 1681 is a member of the FinO family of RNA chaperones

    PubMed Central

    Chaulk, Steven; Lu, Jun; Tan, Kemin; Arthur, David C; Edwards, Ross A; Frost, Laura S; Joachimiak, Andrzej

    2010-01-01

    The conjugative transfer of F-like plasmids between bacteria is regulated by the plasmid-encoded RNA chaperone, FinO, which facilitates sense—antisense RNA interactions to regulate plasmid gene expression. FinO was thought to adopt a unique structure, however many putative homologs have been identified in microbial genomes and are considered members of the FinO_conjugation_repressor superfamily. We were interested in determining whether other members were also able to bind RNA and promote duplex formation, suggesting that this motif does indeed identify a putative RNA chaperone. We determined the crystal structure of the N. meningitidis MC58 protein NMB1681. It revealed striking similarity to FinO, with a conserved fold and a large, positively charged surface that could function in RNA interactions. Using assays developed to study FinO-FinP sRNA interactions, NMB1681, like FinO, bound tightly to FinP RNA stem-loops with short 5′ and 3′ single-stranded tails but not to ssRNA. It also was able to catalyze strand exchange between an RNA duplex and a complementary single-strand, and facilitated duplexing between complementary RNA hairpins. Finally, NMB1681 was able to rescue a finO deficiency and repress F plasmid conjugation. This study strongly suggests that NMB1681 is a FinO-like RNA chaperone that likely regulates gene expression through RNA-based mechanisms in N. meningitidis. PMID:21045552

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

    PubMed Central

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

    2016-01-01

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

  3. THE PROTEIN TARGETING FACTOR GET3 FUNCTIONS AS AN ATP-INDEPENDENT CHAPERONE UNDER OXIDATIVE STRESS CONDITIONS

    PubMed Central

    Voth, Wilhelm; Schick, Markus; Gates, Stephanie; Li, Sheng; Vilardi, Fabio; Gostimskaya, Irina; Southworth, Daniel R.; Schwappach, Blanche; Jakob, Ursula

    2014-01-01

    Summary Exposure of cells to reactive oxygen species (ROS) causes a rapid and significant drop in intracellular ATP-levels. This energy depletion negatively affects ATP-dependent chaperone systems, making ROS-mediated protein unfolding and aggregation a potentially very challenging problem. Here we show that Get3, a protein involved in ATP-dependent targeting of tail-anchored (TA) proteins under non-stress conditions, turns into an effective ATP-in dependent chaperone when oxidized. Activation of Get3’s chaperone function, which is a fully reversible process, involves disulfide bond formation, metal release and its conversion into distinct, higher oligomeric structures. Mutational studies demonstrate that the chaperone activity of Get3 is functionally distinct from and likely mutually exclusive with its targeting function, and responsible for the oxidative stress sensitive phenotype that has long been noted for yeast cells lacking functional Get3. These results provide convincing evidence that Get3 functions as a redox regulated chaperone, effectively protecting eukaryotic cells against oxidative protein damage. PMID:25242142

  4. Bacterial proteostasis balances energy and chaperone utilization efficiently.

    PubMed

    Santra, Mantu; Farrell, Daniel W; Dill, Ken A

    2017-03-28

    Chaperones are protein complexes that help to fold and disaggregate a cell's proteins. It is not understood how four major chaperone systems of Escherichia coli work together in proteostasis: the recognition, sorting, folding, and disaggregating of the cell's many different proteins. Here, we model this machine. We combine extensive data on chaperoning, folding, and aggregation rates with expression levels of proteins and chaperones measured at different growth rates. We find that the proteostasis machine recognizes and sorts a client protein based on two biophysical properties of the client's misfolded state (M state): its stability and its kinetic accessibility from its unfolded state (U state). The machine is energy-efficient (the sickest proteins use the most ATP-expensive chaperones), comprehensive (it can handle any type of protein), and economical (the chaperone concentrations are just high enough to keep the whole proteome folded and disaggregated but no higher). The cell needs higher chaperone levels in two situations: fast growth (when protein production rates are high) and very slow growth (to mitigate the effects of protein degradation). This type of model complements experimental knowledge by showing how the various chaperones work together to achieve the broad folding and disaggregation needs of the cell.

  5. Chaperones rescue luciferase folding by separating its domains.

    PubMed

    Scholl, Zackary N; Yang, Weitao; Marszalek, Piotr E

    2014-10-10

    Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process.

  6. Chaperones Rescue Luciferase Folding by Separating Its Domains*

    PubMed Central

    Scholl, Zackary N.; Yang, Weitao; Marszalek, Piotr E.

    2014-01-01

    Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process. PMID:25160632

  7. Bacterial proteostasis balances energy and chaperone utilization efficiently

    PubMed Central

    Santra, Mantu; Farrell, Daniel W.; Dill, Ken A.

    2017-01-01

    Chaperones are protein complexes that help to fold and disaggregate a cell’s proteins. It is not understood how four major chaperone systems of Escherichia coli work together in proteostasis: the recognition, sorting, folding, and disaggregating of the cell’s many different proteins. Here, we model this machine. We combine extensive data on chaperoning, folding, and aggregation rates with expression levels of proteins and chaperones measured at different growth rates. We find that the proteostasis machine recognizes and sorts a client protein based on two biophysical properties of the client’s misfolded state (M state): its stability and its kinetic accessibility from its unfolded state (U state). The machine is energy-efficient (the sickest proteins use the most ATP-expensive chaperones), comprehensive (it can handle any type of protein), and economical (the chaperone concentrations are just high enough to keep the whole proteome folded and disaggregated but no higher). The cell needs higher chaperone levels in two situations: fast growth (when protein production rates are high) and very slow growth (to mitigate the effects of protein degradation). This type of model complements experimental knowledge by showing how the various chaperones work together to achieve the broad folding and disaggregation needs of the cell. PMID:28292901

  8. Toward Instituting a Chaperone Policy in Outpatient Pediatric Clinics

    ERIC Educational Resources Information Center

    Feldman, Kenneth W.; Jenkins, Carol; Laney, Tyler; Seidel, Kristy

    2009-01-01

    Objectives: We sought to evaluate child, parent and medical provider preferences for chaperones for outpatient encounters and to evaluate the acceptability and frequency of utilization following institution of a chaperone policy. Secondarily, we sought to understand what medical history and examinations teens consider "sensitive." Design: We…

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

    Cancer.gov

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

  10. Chaperones as potential therapeutics for Krabbe disease.

    PubMed

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

    2016-11-01

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

  11. The RNA-Binding Chaperone Hfq Is an Important Global Regulator of Gene Expression in Pasteurella multocida and Plays a Crucial Role in Production of a Number of Virulence Factors, Including Hyaluronic Acid Capsule

    PubMed Central

    Mégroz, Marianne; Kleifeld, Oded; Wright, Amy; Powell, David; Harrison, Paul; Adler, Ben; Harper, Marina

    2016-01-01

    The Gram-negative bacterium Pasteurella multocida is the causative agent of a number of economically important animal diseases, including avian fowl cholera. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS), and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA (sRNA) molecules and their mRNA targets. Here, we show that a P. multocida hfq mutant produces significantly less hyaluronic acid capsule during all growth phases and displays reduced in vivo fitness. Transcriptional and proteomic analyses of the hfq mutant during mid-exponential-phase growth revealed altered transcript levels for 128 genes and altered protein levels for 78 proteins. Further proteomic analyses of the hfq mutant during the early exponential growth phase identified 106 proteins that were produced at altered levels. Both the transcript and protein levels for genes/proteins involved in capsule biosynthesis were reduced in the hfq mutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, there were increased expression levels of three LPS biosynthesis genes, encoding proteins involved in phosphocholine and phosphoethanolamine addition to LPS, suggesting that these genes are negatively regulated by Hfq-dependent mechanisms. Taken together, these data provide the first evidence that Hfq plays a crucial role in regulating the global expression of P. multocida genes, including the regulation of key P. multocida virulence factors, capsule, LPS, and filamentous hemagglutinin. PMID:26883595

  12. Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes

    PubMed Central

    Angelov, Dimitar; Bondarenko, Vladimir A; Almagro, Sébastien; Menoni, Hervé; Mongélard, Fabien; Hans, Fabienne; Mietton, Flore; Studitsky, Vasily M; Hamiche, Ali; Dimitrov, Stefan; Bouvet, Philippe

    2006-01-01

    Remodeling machines play an essential role in the control of gene expression, but how their activity is regulated is not known. Here we report that the nuclear protein nucleolin possesses a histone chaperone activity and that this factor greatly enhances the activity of the chromatin remodeling machineries SWI/SNF and ACF. Interestingly, nucleolin is able to induce the remodeling by SWI/SNF of macroH2A, but not of H2ABbd nucleosomes, which are otherwise resistant to remodeling. This new histone chaperone promotes the destabilization of the histone octamer, helping the dissociation of a H2A–H2B dimer, and stimulates the SWI/SNF-mediated transfer of H2A–H2B dimers. Furthermore, nucleolin facilitates transcription through the nucleosome, which is reminiscent of the activity of the FACT complex. This work defines new functions for histone chaperones in chromatin remodeling and regulation of transcription and explains how nucleolin could act on transcription. PMID:16601700

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

    PubMed Central

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

    2016-01-01

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

  14. Goniothalamin enhances the ATPase activity of the molecular chaperone Hsp90 but inhibits its chaperone activity.

    PubMed

    Yokoyama, Yuhei; Ohtaki, Aguru; Jantan, Ibrahim; Yohda, Masafumi; Nakamoto, Hitoshi

    2015-03-01

    Hsp90 is an ATP-dependent molecular chaperone that is involved in important cellular pathways such as signal transduction pathways. It is a potential cancer drug target because it plays a critical role for stabilization and activation of oncoproteins. Thus, small molecule compounds that control the Hsp90 function are useful to elucidate potential lead compounds against cancer. We studied effect of a naturally occurring styryl-lactone goniothalamin on the activity of Hsp90. Although many drugs targeting Hsp90 inhibit the ATPase activity of Hsp90, goniothalamin enhanced rather than inhibited the ATPase activity of a cyanobacterial Hsp90 (HtpG) and a yeast Hsp90. It increased both K(m) and k(cat) of the Hsp90s. Domain competition assays and tryptophan fluorescence measurements with various truncated derivatives of HtpG indicated that goniothalamin binds to the N-terminal domain of HtpG. Goniothalamin did not influence on the interaction of HtpG with a non-native protein or the anti-aggregation activity of HtpG significantly. However, it inhibited the activity of HtpG that assists refolding of a non-native protein in cooperation with the Hsp70 chaperone system. This is the first report to show that a small molecule that binds to the N-terminal domain of Hsp90 activates its ATPase activity, while inhibiting the chaperone function of Hsp90.

  15. NMR characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa

    PubMed Central

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

    2017-01-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS) to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologs are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by CD and NMR spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residues 16–41 and 55–76. NMR backbone dynamics data show that the helices in PcrG have semi-rigid flexibility and they tumble as a single entity with similar backbone dynamics. NMR titrations show that the entire length of PcrG residues from 9–76 is involved in binding to PcrV. Thus the PcrG family of T3SS chaperone proteins is essentially partially folded. PMID:26451841

  16. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.

    PubMed

    González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A

    2015-08-11

    Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ's histone chaperone activity.

  17. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c

    PubMed Central

    González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A.

    2015-01-01

    Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ’s histone chaperone activity. PMID:26216969

  18. Inhibition of endoplasmic reticulum chaperone protein glucose-regulated protein 78 potentiates anti-angiogenic therapy in renal cell carcinoma through inactivation of the PERK/eIF2α pathway

    PubMed Central

    Han, Kyung Seok; Li, Na; Raven, Pater A.; Fazli, Ladan; Frees, Sebastian; Ettinger, Susan; Park, Ki Chung; Hong, Sung Joon; Gleave, Martin E.; So, Alan I.

    2015-01-01

    Tumor microenvironments are characterized by decreased oxygen and nutrition due to the rapid and progressive nature of tumors and also stresses induced by several anti-tumor therapies. These intense cell stressors trigger a protective cell survival mechanism heralded by the unfolded protein response (UPR). The UPR is induced by an accumulation of unfolded proteins in the endoplasmic reticulum (ER) following cell starvation. Although the ER stress response is implicated in cytoprotection, its precise role during anti-angiogenic therapy remains unclear. One of the major proteins involved in ER stress is glucose-regulated protein 78 (GRP78), which binds to unfolded proteins and dissociates from membrane-bound ER stress sensors. To determine the role of ER stress responses during anti-angiogenic therapy and the potential role of GRP78 in combined therapy in renal cell carcinoma (RCC), we used GRP78 overexpressing or knockdown RCC cells under hypoxic or hypoglycemic conditions in vitro and in animal models treated with sunitinib. Here, we report that GRP78 plays a crucial role in protecting RCC cells from hypoxic and hypoglycemic stress induced by anti-angiogenic therapy. Knockdown of GRP78 using siRNA inhibited cancer cell survival and induced apoptosis in RCC cells in vitro and also resulted in ER stress-induced apoptosis and hypoxic/hypoglycemic stress-induced apoptosis by inactivating the PERK/eIF-2α pathway. Finally, GRP78 knockdown showed potent suppression of tumor growth and enhanced the antitumor effect of sunitinib in RCC xenografts. Our findings suggest that GRP78 may serve as a novel therapeutic target in combination with anti-angiogenic therapy for the management of RCC. PMID:26472187

  19. New insights into the roles of molecular chaperones in Chlamydomonas and Volvox.

    PubMed

    Nordhues, André; Miller, Stephen M; Mühlhaus, Timo; Schroda, Michael

    2010-01-01

    The unicellular green alga Chlamydomonas reinhardtii has been used as a model organism for many decades, mainly to study photosynthesis and flagella/cilia. Only recently, Chlamydomonas has received much attention because of its ability to produce hydrogen and nonpolar lipids that have promise as biofuels. The best-studied multicellular cousin of Chlamydomonas reinhardtii is Volvox carteri, whose life cycle comprises events that have clear parallels in higher plants and/or animals, making it an excellent system in which to study fundamental developmental processes. Molecular chaperones are proteins that guide other cellular proteins through their life cycle. They assist in de novo folding of nascent chains, mediate assembly and disassembly of protein complexes, facilitate protein transport across membranes, disassemble protein aggregates, fold denatured proteins back to the native state, and transfer unfoldable proteins to proteolytic degradation. Hence, molecular chaperones regulate protein function under all growth conditions and play important roles in many basic cellular and developmental processes. The aim of this chapter is to describe recent advances toward understanding molecular chaperone biology in Chlamydomonas and Volvox.

  20. Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75

    SciTech Connect

    Berndsen, Christopher E; Tsubota, Toshiaki; Lindner, Scott E; Lee, Susan; Holton, James M; Kaufman, Paul D; Keck, James L; Denu, John M

    2010-01-12

    Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by {approx}100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

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

    SciTech Connect

    Marcianò, G.; Huang, D. T.

    2016-01-22

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

  2. Copper Chaperone Antioxidant Protein1 Is Essential for Copper Homeostasis1[W][OA

    PubMed Central

    Shin, Lung-Jiun; Lo, Jing-Chi; Yeh, Kuo-Chen

    2012-01-01

    Copper (Cu) is essential for plant growth but toxic in excess. Specific molecular mechanisms maintain Cu homeostasis to facilitate its use and avoid the toxicity. Cu chaperones, proteins containing a Cu-binding domain(s), are thought to assist Cu intracellular homeostasis by their Cu-chelating ability. In Arabidopsis (Arabidopsis thaliana), two Cu chaperones, Antioxidant Protein1 (ATX1) and ATX1-Like Copper Chaperone (CCH), share high sequence homology. Previously, their Cu-binding capabilities were demonstrated and interacting molecules were identified. To understand the physiological functions of these two chaperones, we characterized the phenotype of atx1 and cch mutants and the cchatx1 double mutant in Arabidopsis. The shoot and root growth of atx1 and cchatx1 but not cch was specifically hypersensitive to excess Cu but not excess iron, zinc, or cadmium. The activities of antioxidant enzymes in atx1 and cchatx1 were markedly regulated in response to excess Cu, which confirms the phenotype of Cu hypersensitivity. Interestingly, atx1 and cchatx1 were sensitive to Cu deficiency. Overexpression of ATX1 not only enhanced Cu tolerance and accumulation in excess Cu conditions but also tolerance to Cu deficiency. In addition, the Cu-binding motif MXCXXC of ATX1 was required for these physiological functions. ATX1 was previously proposed to be involved in Cu homeostasis by its Cu-binding activity and interaction with the Cu transporter Heavy metal-transporting P-type ATPase5. In this study, we demonstrate that ATX1 plays an essential role in Cu homeostasis in conferring tolerance to excess Cu and Cu deficiency. The possible mechanism is discussed. PMID:22555879

  3. Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase.

    PubMed

    Paul, Subhankar; Punam, Shashikala; Chaudhuri, Tapan K

    2007-11-01

    In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escherichia coli protein, was studied in the presence of glycerol, dimethylsulfoxide, trimethylamine-N-oxide, ethylene glycol, trehalose, proline and chaperonins GroEL and GroES. Different osmolytes, namely proline, glycerol, trimethylamine-N-oxide and dimethylsulfoxide, also known as chemical chaperones, assist in protein folding through effective inhibition of the aggregation process. In the present study, it was observed that a few chemical chaperones effectively reduced the aggregation process of maltodextrin glucosidase and hence the in vitro refolding was substantially enhanced, with ethylene glycol being the exception. Although, the highest recovery of active maltodextrin glucosidase was achieved through the ATP-mediated GroEL/GroES-assisted refolding of denatured protein, the yield of correctly folded protein from glycerol- or proline-assisted spontaneous refolding process was closer to the chaperonin-assisted refolding. It was also observed that the combined application of chemical chaperones and molecular chaperone was more productive than their individual contribution towards the in vitro refolding of maltodextrin glucosidase. The chemical chaperones, except ethylene glycol, were found to provide different degrees of protection to maltodextrin glucosidase from thermal denaturation, whereas proline caused the highest protection. The observations from the present studies conclusively demonstrate that chemical or molecular chaperones, or the combination of both chaperones, could be used in the efficient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying or designing suitable small molecules that can act as chemical chaperones in the efficient refolding of various aggregate-prone proteins of commercial and medical importance.

  4. Diabetic Peripheral Neuropathy: Should a Chaperone Accompany Our Therapeutic Approach?

    PubMed Central

    Farmer, Kevin L.; Li, Chengyuan

    2012-01-01

    Diabetic peripheral neuropathy (DPN) is a common complication of diabetes that is associated with axonal atrophy, demyelination, blunted regenerative potential, and loss of peripheral nerve fibers. The development and progression of DPN is due in large part to hyperglycemia but is also affected by insulin deficiency and dyslipidemia. Although numerous biochemical mechanisms contribute to DPN, increased oxidative/nitrosative stress and mitochondrial dysfunction seem intimately associated with nerve dysfunction and diminished regenerative capacity. Despite advances in understanding the etiology of DPN, few approved therapies exist for the pharmacological management of painful or insensate DPN. Therefore, identifying novel therapeutic strategies remains paramount. Because DPN does not develop with either temporal or biochemical uniformity, its therapeutic management may benefit from a multifaceted approach that inhibits pathogenic mechanisms, manages inflammation, and increases cytoprotective responses. Finally, exercise has long been recognized as a part of the therapeutic management of diabetes, and exercise can delay and/or prevent the development of painful DPN. This review presents an overview of existing therapies that target both causal and symptomatic features of DPN and discusses the role of up-regulating cytoprotective pathways via modulating molecular chaperones. Overall, it may be unrealistic to expect that a single pharmacologic entity will suffice to ameliorate the multiple symptoms of human DPN. Thus, combinatorial therapies that target causal mechanisms and enhance endogenous reparative capacity may enhance nerve function and improve regeneration in DPN if they converge to decrease oxidative stress, improve mitochondrial bioenergetics, and increase response to trophic factors. PMID:22885705

  5. A new perspective in Parkinson's disease, chaperone-mediated autophagy.

    PubMed

    Li, Boyu; Zhang, Yun; Yuan, Yuhe; Chen, Naihong

    2011-05-01

    Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by loss of dopaminergic neurons and aggregation of alpha-synuclein. Although the role of alpha-synuclein in the pathology of PD is still unclear, the fact that its aggregation contributes to the loss of dopaminergic neurons has been confirmed. Therefore, controlling the alpha-synuclein protein level may be critical for PD pathogenesis and may provide potential therapeutics. Wild-type alpha-synuclein is physiologically degraded by chaperone-mediated autophagy (CMA), and dysfunction of CMA results in alpha-synuclein aggregation and compensative macroautophagy activation which finally leads to cell death. Therefore, CMA may participate in PD pathogenesis as a very important factor, and up-regulating CMA activity could degrade overloaded alpha-synuclein. In view of potential compensative effects, maintenance of the balance of CMA activity will be another major challenge in the future development of the therapeutic strategy. Herein we review the current knowledge of the role of CMA in PD.

  6. Structural basis for the antifolding activity of a molecular chaperone

    PubMed Central

    Huang, Chengdong; Rossi, Paolo; Saio, Tomohide; Kalodimos, Charalampos G.

    2016-01-01

    Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase (PhoA) and maltose binding protein (MBP) captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of the non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone. PMID:27501151

  7. Structural basis for the antifolding activity of a molecular chaperone

    NASA Astrophysics Data System (ADS)

    Huang, Chengdong; Rossi, Paolo; Saio, Tomohide; Kalodimos, Charalampos G.

    2016-09-01

    Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase and maltose-binding protein captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone.

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

    PubMed

    Zunder, Rachel M; Rine, Jasper

    2012-11-01

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

  9. Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase

    PubMed Central

    Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph; Marquardt, Iris; Donsante, Anthony; Yi, Ling; Hicks, Julia D.; Steinbach, Peter J.; Wilson, Callum; Elpeleg, Orly; Møller, Lisbeth Birk; Christodoulou, John; Kaler, Stephen G.; Gärtner, Jutta

    2012-01-01

    Copper is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular copper metabolism, an elaborate system of chaperones and transporters has evolved, although no human copper chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited a homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the copper chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163 with tryptophan in domain II of CCS, which interacts directly with SOD1. Biochemical analyses of the patient’s fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal copper homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a copper chaperone. PMID:22508683

  10. OprD Repression upon Metal Treatment Requires the RNA Chaperone Hfq in Pseudomonas aeruginosa

    PubMed Central

    Ducret, Verena; Gonzalez, Manuel R.; Scrignari, Tiziana; Perron, Karl

    2016-01-01

    The metal-specific CzcRS two-component system in Pseudomonas aeruginosa is involved in the repression of the OprD porin, causing in turn carbapenem antibiotic resistance in the presence of high zinc concentration. It has also been shown that CzcR is able to directly regulate the expression of multiple genes including virulence factors. CzcR is therefore an important regulator connecting (i) metal response, (ii) pathogenicity and (iii) antibiotic resistance in P. aeruginosa. Recent data have suggested that other regulators could negatively control oprD expression in the presence of zinc. Here we show that the RNA chaperone Hfq is a key factor acting independently of CzcR for the repression of oprD upon Zn treatment. Additionally, we found that an Hfq-dependent mechanism is necessary for the localization of CzcR to the oprD promoter, mediating oprD transcriptional repression. Furthermore, in the presence of Cu, CopR, the transcriptional regulator of the CopRS two-component system also requires Hfq for oprD repression. Altogether, these results suggest important roles for this RNA chaperone in the context of environment-sensing and antibiotic resistance in P. aeruginosa. PMID:27706108

  11. Molecular chaperone-mediated nuclear protein dynamics.

    PubMed

    Echtenkamp, Frank J; Freeman, Brian C

    2014-05-01

    Homeostasis requires effective action of numerous biological pathways including those working along a genome. The variety of processes functioning in the nucleus is considerable, yet the number of employed factors eclipses this total. Ideally, individual components assemble into distinct complexes and serially operate along a pathway to perform work. Adding to the complexity is a multitude of fluctuating internal and external signals that must be monitored to initiate, continue or halt individual activities. While cooperative interactions between proteins of the same process provide a mechanism for rapid and precise assembly, the inherent stability of such organized structures interferes with the proper timing of biological events. Further prolonging the longevity of biological complexes are crowding effects resulting from the high concentration of intracellular macromolecules. Hence, accessory proteins are required to destabilize the various assemblies to efficiently transition between structures, avoid off-pathway competitive interactions, and to terminate pathway activity. We suggest that molecular chaperones have evolved, in part, to manage these challenges by fostering a general and continuous dynamic protein environment within the nucleus.

  12. Molecular chaperones encoded by a reduced nucleus: the cryptomonad nucleomorph.

    PubMed

    Archibald, J M; Cavalier-Smith, T; Maier, U; Douglas, S

    2001-06-01

    Molecular chaperones mediate the correct folding of nascent or denatured proteins and are found in both the organelles and cytoplasm of eukaryotic cells. Cryptomonad algae are unusual in possessing an extra cytoplasmic compartment (the periplastid space), the result of having engulfed and retained a photosynthetic eukaryote. Within the periplastid space is a diminutive nucleus (the nucleomorph) that encodes mostly genes for its own expression as well as a few needed by the plastid. Two plastid-encoded chaperones (GroEL and DnaK) and a nucleomorph-encoded chaperone (Cpn60) have been reported from the cryptomonad, Guillardia theta. Here we analyse G. theta nucleomorph genes for members of the cytosolic HSP70 and HSP90 families of molecular chaperones, a heat shock transcription factor (HSF), and all eight subunits of the group II chaperonin, CCT. These are presumably all active in the periplastid space, assisting in the maturation of polypeptides required by the cell; we propose a central role for them also in the structure and assembly of a putative relict mitotic apparatus. Curiously, none of the genes for co-chaperones of HSP70, HSP90, or CCT have been detected in the nucleomorph genome; they are either not needed or are encoded in the host nuclear genome and targeted back into the periplastid space. Endoplasmic reticulum (ER) homologs of HSP70 and HSP90 are also not present. Striking differences in the degree of conservation of the various nucleomorph-encoded molecular chaperones were observed. While the G. theta HSP70 and HSP90 homologs are well conserved, each of the eight CCT subunits (alpha, beta, gamma, delta, epsilon, eta, theta, and zeta) is remarkably divergent. Such differences are likely evidence for reduced/different functional constraints on the various molecular chaperones functioning in the periplastid space.

  13. Targeting Hsp90-Cdc37: a promising therapeutic strategy by inhibiting Hsp90 chaperone function.

    PubMed

    Wang, Lei; Li, Li; Gu, Kai; Xu, Xiao-Li; You, Qi-Dong; Sun, Hao-Peng

    2016-05-27

    The Hsp90 chaperone protein regulates the folding, maturation and stability of a wide variety of oncoproteins. In recent years, many Hsp90 inhibitors have entered into the clinical trials while all of them target ATPase showing similar binding capacity and kinds of side-effects so that none have reached to the market. During the regulation progress, numerous protein-protein interactions (PPI) such as Hsp90 and client proteins or cochaperones are involved. With the Hsp90-cochaperones PPI networks being more and more clear, many cancerous proteins have been reported to be tightly correlated to Hsp90-cochaperones PPI. Among them, Hsp90-Cdc37 PPI has been widely reported to associate with numerous protein kinases, making it a novel target for the treatment of cancers. In this paper, we briefly review the strategies and modulators targeting Hsp90-Cdc37 complex including direct and indirect regulation mechanism. Through these discussions we expect to present inspirations for new insights into an alternative way to inhibit Hsp90 chaperone function.

  14. A novel mechanism of post-translational modulation of HMGA functions by the histone chaperone nucleophosmin

    PubMed Central

    Arnoldo, Laura; Sgarra, Riccardo; Chiefari, Eusebio; Iiritano, Stefania; Arcidiacono, Biagio; Pegoraro, Silvia; Pellarin, Ilenia; Brunetti, Antonio; Manfioletti, Guidalberto

    2015-01-01

    High Mobility Group A are non-histone nuclear proteins that regulate chromatin plasticity and accessibility, playing an important role both in physiology and pathology. Their activity is controlled by transcriptional, post-transcriptional, and post-translational mechanisms. In this study we provide evidence for a novel modulatory mechanism for HMGA functions. We show that HMGAs are complexed in vivo with the histone chaperone nucleophosmin (NPM1), that this interaction requires the histone-binding domain of NPM1, and that NPM1 modulates both DNA-binding affinity and specificity of HMGAs. By focusing on two human genes whose expression is directly regulated by HMGA1, the Insulin receptor (INSR) and the Insulin-like growth factor-binding protein 1 (IGFBP1) genes, we demonstrated that occupancy of their promoters by HMGA1 was NPM1-dependent, reflecting a mechanism in which the activity of these cis-regulatory elements is directly modulated by NPM1 leading to changes in gene expression. HMGAs need short stretches of AT-rich nucleosome-free regions to bind to DNA. Therefore, many putative HMGA binding sites are present within the genome. Our findings indicate that NPM1, by exerting a chaperoning activity towards HMGAs, may act as a master regulator in the control of DNA occupancy by these proteins and hence in HMGA-mediated gene expression. PMID:25711412

  15. A Novel Method for Assessing the Chaperone Activity of Proteins

    PubMed Central

    Hristozova, Nevena; Tompa, Peter; Kovacs, Denes

    2016-01-01

    Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families–molecules expressed during adverse conditions, infection, and diseases–chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function. PMID:27564234

  16. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

    PubMed Central

    Vertommen, Didier; Silhavy, Thomas J.; Collet, Jean-Francois

    2013-01-01

    β-barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the non-viable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli. PMID:22589188

  17. The G Protein α Chaperone Ric-8 as a Potential Therapeutic Target

    PubMed Central

    Papasergi, Makaía M.; Patel, Bharti R.

    2015-01-01

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

  18. Histone chaperones ASF1 and NAP1 differentially modulate removal of active histone marks by LID-RPD3 complexes during NOTCH silencing.

    PubMed

    Moshkin, Yuri M; Kan, Tsung Wai; Goodfellow, Henry; Bezstarosti, Karel; Maeda, Robert K; Pilyugin, Maxim; Karch, Francois; Bray, Sarah J; Demmers, Jeroen A A; Verrijzer, C Peter

    2009-09-24

    Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, we identified the interaction networks of histone chaperones ASF1, CAF1, HIRA, and NAP1. Here, we analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) NOTCH-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. We conclude that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing.

  19. Hsp70-Hsp40 Chaperone Complex Functions in Controlling Polarized Growth by Repressing Hsf1-Driven Heat Stress-Associated Transcription

    PubMed Central

    Liu, Jianhua; Oliferenko, Snezhana

    2013-01-01

    How the molecular mechanisms of stress response are integrated at the cellular level remains obscure. Here we show that the cellular polarity machinery in the fission yeast Schizosaccharomyces pombe undergoes dynamic adaptation to thermal stress resulting in a period of decreased Cdc42 activity and altered, monopolar growth. Cells where the heat stress-associated transcription was genetically upregulated exhibit similar growth patterning in the absence of temperature insults. We identify the Ssa2-Mas5/Hsp70-Hsp40 chaperone complex as repressor of the heat shock transcription factor Hsf1. Cells lacking this chaperone activity constitutively activate the heat-stress-associated transcriptional program. Interestingly, they also exhibit intermittent monopolar growth within a physiological temperature range and are unable to adapt to heat stress. We propose that by negatively regulating the heat stress-associated transcription, the Ssa2-Mas5 chaperone system could optimize cellular growth under different temperature regiments. PMID:24146635

  20. Orchestration of secretory protein folding by ER chaperones

    PubMed Central

    Gidalevitz, Tali; Stevens, Fred; Argon, Yair

    2013-01-01

    The endoplasmic reticulum is a major compartment of protein biogenesis in the cell, dedicated to production of secretory, membrane and organelle proteins. The secretome has distinct structural and post-translational characteristics, since folding in the ER occurs in an environment that is distinct in terms of its ionic composition, dynamics and requirements for quality contol. The folding machinery in the ER therefore includes chaperones and folding enzymes that introduce, monitor and react to disulfide bonds, glycans, and fluctuations of luminal calcium. We describe the major chaperone networks in the lumen and discuss how they have distinct modes of operation that enable cells to accomplish highly efficient production of the secretome. PMID:23507200

  1. The ubiquitin-selective chaperone CDC-48/p97 links myosin assembly to human myopathy.

    PubMed

    Janiesch, Philipp Christoph; Kim, Johnny; Mouysset, Julien; Barikbin, Roja; Lochmüller, Hanns; Cassata, Giuseppe; Krause, Sabine; Hoppe, Thorsten

    2007-04-01

    Protein degradation in eukaryotes often requires the ubiquitin-selective chaperone p97 for substrate recruitment and ubiquitin-chain assembly. However, the physiological relevance of p97, and its role in developmental processes, remain unclear. Here, we discover an unanticipated function for CDC-48/p97 in myosin assembly and myofibril organization, both in Caenorhabditis elegans and humans. The developmentally regulated assembly of a CDC-48-UFD-2-CHN-1 complex links turnover of the myosin-directed chaperone UNC-45 to functional muscle formation. Our data suggest a similarly conserved pathway regulating myosin assembly in humans. Remarkably, mutations in human p97, known to cause hereditary inclusion-body myopathy, abrogate UNC-45 degradation and result in severely disorganized myofibrils, detrimental towards sarcomeric function. These results identify a key role for CDC-48/p97 in the process of myofibre differentiation and maintenance, which is abolished during pathological conditions leading to protein aggregation and inclusion-body formation in human skeletal muscle.

  2. The Endoplasmic Reticulum Chaperone GRP170: From Immunobiology to Cancer Therapeutics

    PubMed Central

    Wang, Hongxia; Pezeshki, Abdul Mohammad; Yu, Xiaofei; Guo, Chunqing; Subjeck, John R.; Wang, Xiang-Yang

    2014-01-01

    Glucose-regulated protein 170 (GRP170) is the largest member of glucose-regulated protein family that resides in the endoplasmic reticulum (ER). As a component of the ER chaperone network, GRP170 assists in protein folding, assembly, and transportation of secretory or transmembrane proteins. The well documented cytoprotective activity of intracellular GRP170 due to its intrinsic chaperoning property has been shown to provide a survival benefit in cancer cells during tumor progression or metastasis. Accumulating evidence shows that extracellular GRP170 displays a superior capacity in delivering tumor antigens to specialized antigen-presenting cells for cross-presentation, resulting in generation of an anti-tumor immune response dependent on cytotoxic CD8+ T cells. This unique feature of GRP170 provides a molecular basis for using GRP170 as an immunostimulatory adjuvant to develop a recombinant vaccine for therapeutic immunization against cancers. This review summarizes the latest findings in understanding the biological effects of GRP170 on cell functions and tumor progression. The immunomodulating activities of GRP170 during interactions with the innate and adaptive arms of the immune system as well as its therapeutic applications in cancer immunotherapy will be discussed. PMID:25629003

  3. Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins

    PubMed Central

    Stetz, Gabrielle; Verkhivker, Gennady M.

    2015-01-01

    Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations

  4. Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

    PubMed

    Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

    2014-12-19

    Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

  5. Transcription initiation factor IID-interactive histone chaperone CIA-II implicated in mammalian spermatogenesis.

    PubMed

    Umehara, Takashi; Horikoshi, Masami

    2003-09-12

    Histones are thought to have specific roles in mammalian spermatogenesis, because several subtypes of histones emerge that are post-translationally modified during spermatogenesis. Though regular assembly of nucleosome is guaranteed by histone chaperones, their involvement in spermatogenesis is yet to be characterized. Here we identified a histone chaperone-related factor, which we designated as CCG1-interacting factor A-II (CIA-II), through interaction with bromodomains of TAFII250/CCG1, which is the largest subunit of human transcription initiation factor IID (TFIID). We found that human CIA-II (hCIA-II) localizes in HeLa nuclei and is highly expressed in testis and other proliferating cell-containing tissues. Expression of mouse CIA-II (mCIA-II) does not occur in the germ cell-lacking testes of adult WBB6F1-W/Wv mutant mice, indicating its expression in testis to be specific to germ cells. Fractionation of testicular germ cells revealed that mCIA-II transcripts accumulate in pachytene spermatocytes but not in spermatids. In addition, the mCIA-II transcripts in testis were present as early as 4 days after birth and decreased at 56 days after birth. These findings indicate that mCIA-II expression in testis is restricted to premeiotic to meiotic stages during spermatogenesis. Also, we found that hCIA-II interacts with histone H3 in vivo and with histones H3/H4 in vitro and that it facilitates supercoiling of circular DNA when it is incubated with core histones and topoisomerase I in vitro. These data suggest that CIA-II is a histone chaperone and is implicated in the regulation of mammalian spermatogenesis.

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

    PubMed Central

    Rodriguez, Karl A.; Dodds, Sherry G.; Strong, Randy; Galvan, Veronica; Sharp, Z. D.; Buffenstein, Rochelle

    2014-01-01

    Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24 mg/kg (14 ppm) rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS), heat shock factor 1 (HSF1), and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome. PMID:25414638

  7. Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking

    PubMed Central

    Bird, Jonathan E.; Takagi, Yasuharu; Billington, Neil; Strub, Marie-Paule; Sellers, James R.; Friedman, Thomas B.

    2014-01-01

    Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin–specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end–directed motor that moves actin filaments in a gliding assay (∼430 nm·s−1 at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (kcat ∼6 s−1) was similar to the actin-detachment rate (kdet = 6.2 s−1) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells. PMID:25114250

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

    PubMed

    Rodriguez, Karl A; Dodds, Sherry G; Strong, Randy; Galvan, Veronica; Sharp, Z D; Buffenstein, Rochelle

    2014-01-01

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

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

    PubMed

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

    2014-06-01

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

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

    PubMed Central

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

    1998-01-01

    Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA and protein levels in unstressed embryos during early development. More important, Hsc70 levels were found to depend on endogenous (pro)insulin, as shown by using antisense oligodeoxynucleotides against (pro)insulin mRNA in cultured neurulating embryos. Further, in the cultured embryos, apoptosis affected mainly cells with the lowest level of Hsc70, as shown by simultaneous Hsc70 immunostaining and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling. These results argue in favor of Hsc70 involvement, modulated by embryonic (pro)insulin, in the prevention of apoptosis during early development and suggest a role for a molecular chaperone in normal embryogenesis. PMID:9707581

  11. RNA chaperones buffer deleterious mutations in E. coli

    PubMed Central

    Rudan, Marina; Schneider, Dominique; Warnecke, Tobias; Krisko, Anita

    2015-01-01

    Both proteins and RNAs can misfold into non-functional conformations. Protein chaperones promote native folding of nascent polypeptides and refolding of misfolded species, thereby buffering mutations that compromise protein structure and function. Here, we show that RNA chaperones can also act as mutation buffers that enhance organismal fitness. Using competition assays, we demonstrate that overexpression of select RNA chaperones, including three DEAD box RNA helicases (DBRHs) (CsdA, SrmB, RhlB) and the cold shock protein CspA, improves fitness of two independently evolved Escherichia coli mutator strains that have accumulated deleterious mutations during short- and long-term laboratory evolution. We identify strain-specific mutations that are deleterious and subject to buffering when introduced individually into the ancestral genotype. For DBRHs, we show that buffering requires helicase activity, implicating RNA structural remodelling in the buffering process. Our results suggest that RNA chaperones might play a fundamental role in RNA evolution and evolvability. DOI: http://dx.doi.org/10.7554/eLife.04745.001 PMID:25806682

  12. Reconfiguration of the proteasome during chaperone-mediated assembly.

    PubMed

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

    2013-05-23

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

  13. Pharmacological chaperones for human α-N-acetylgalactosaminidase

    PubMed Central

    Clark, Nathaniel E.; Metcalf, Matthew C.; Best, Daniel; Fleet, George W. J.; Garman, Scott C.

    2012-01-01

    Schindler/Kanzaki disease is an inherited metabolic disease with no current treatment options. This neurologic disease results from a defect in the lysosomal α-N-acetylgalactosaminidase (α-NAGAL) enzyme. In this report, we show evidence that the iminosugar DGJNAc can inhibit, stabilize, and chaperone human α-NAGAL both in vitro and in vivo. We demonstrate that a related iminosugar DGJ (currently in phase III clinical trials for another metabolic disorder, Fabry disease) can also chaperone human α-NAGAL in Schindler/Kanzaki disease. The 1.4- and 1.5-Å crystal structures of human α-NAGAL complexes reveal the different binding modes of iminosugars compared with glycosides. We show how differences in two functional groups result in >9 kcal/mol of additional binding energy and explain the molecular interactions responsible for the unexpectedly high affinity of the pharmacological chaperones. These results open two avenues for treatment of Schindler/Kanzaki disease and elucidate the atomic basis for pharmacological chaperoning in the entire family of lysosomal storage diseases. PMID:23045655

  14. Hsp100/ClpB Chaperone Function and Mechanism

    SciTech Connect

    Vierling, Elizabeth

    2015-01-27

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

  15. Super Spy variants implicate flexibility in chaperone action

    PubMed Central

    Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl AT; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James CA

    2014-01-01

    Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001 PMID:24497545

  16. Insights from Bacterial Subtilases into the Mechanisms of Intramolecular Chaperone-Mediated Activation of Furin

    PubMed Central

    Shinde, Ujwal; Thomas, Gary

    2015-01-01

    Prokaryotic subtilisins and eukaryotic proprotein convertases (PCs) are two homologous protease subfamilies that belong to the larger ubiquitous super-family called subtilases. Members of the subtilase super-family are produced as zymogens wherein their propeptide domains function as dedicated intramolecular chaperones (IMCs) that facilitate correct folding and regulate precise activation of their cognate catalytic domains. The molecular and cellular determinants that modulate IMC-dependent folding and activation of PCs are poorly understood. In this chapter we review what we have learned from the folding and activation of prokaryotic subtilisin, discuss how this has molded our understanding of furin maturation, and foray into the concept of pH sensors, which may represent a paradigm that PCs (and possibly other IMC-dependent eukaryotic proteins) follow for regulating their biological functions using the pH gradient in the secretory pathway. PMID:21805238

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

    PubMed

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

    2010-06-01

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

  18. Oridonin Triggers Chaperon-mediated Proteasomal Degradation of BCR-ABL in Leukemia

    PubMed Central

    Huang, Huilin; Weng, Hengyou; Dong, Bowen; Zhao, Panpan; Zhou, Hui; Qu, Lianghu

    2017-01-01

    Inducing degradation of oncoproteins by small molecule compounds has the potential to avoid drug resistance and therefore deserves to be exploited for new therapies. Oridonin is a natural compound with promising antitumor efficacy that can trigger the degradation of oncoproteins; however, the direct cellular targets and underlying mechanisms remain unclear. Here we report that oridonin depletes BCR-ABL through chaperon-mediated proteasomal degradation in leukemia. Mechanistically, oridonin poses oxidative stress in cancer cells and directly binds to cysteines of HSF1, leading to the activation of this master regulator of the chaperone system. The resulting induction of HSP70 and ubiquitin proteins and the enhanced binding to CHIP E3 ligase hence target BCR-ABL for ubiquitin-proteasome degradation. Both wild-type and mutant forms of BCR-ABL can be efficiently degraded by oridonin, supporting its efficacy observed in cultured cells as well as mouse tumor xenograft assays with either imatinib-sensitive or -resistant cells. Collectively, our results identify a novel mechanism by which oridonin induces rapid degradation of BCR-ABL as well as a novel pharmaceutical activator of HSF1 that represents a promising treatment for leukemia. PMID:28128329

  19. Identification of small molecules that inhibit the histone chaperone Asf1 and its chromatin function

    PubMed Central

    Seol, Ja-Hwan; Song, Tae-Yang; Oh, Se Eun; Jo, Chanhee; Choi, Ahreum; Kim, Byungho; Park, Jinyoung; Hong, Suji; Song, Ilrang; Jung, Kwan Young; Yang, Jae-Hyun; Park, Hwangseo; Ahn, Jin-Hyun; Han, Jeung-Whan; Cho, Eun-Jung

    2015-01-01

    The eukaryotic genome is packed into chromatin, which is important for the genomic integrity and gene regulation. Chromatin structures are maintained through assembly and disassembly of nucleosomes catalyzed by histone chaperones. Asf1 (anti-silencing function 1) is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation at globular core domain of histone H3. To elucidate the role of Asf1 in the modulation of chromatin structure, we screened and identified small molecules that inhibit Asf1 and H3K56 acetylation without affecting other histone modifications. These pyrimidine-2,4,6-trione derivative molecules inhibited the nucleosome assembly mediated by Asf1 in vitro, and reduced the H3K56 acetylation in HeLa cells. Furthermore, production of HSV viral particles was reduced by these compounds. As Asf1 is implicated in genome integrity, cell proliferation, and cancer, current Asf1 inhibitor molecules may offer an opportunity for the therapeutic development for treatment of diseases. [BMB Reports 2015; 48(12): 685-690] PMID:26058396

  20. Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation

    PubMed Central

    Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd

    2016-01-01

    Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380

  1. ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load

    PubMed Central

    Petrova, Kseniya; Tomba, Giulia; Vendruscolo, Michele

    2012-01-01

    Gene expression programs that regulate the abundance of the chaperone BiP adapt the endoplasmic reticulum (ER) to unfolded protein load. However, such programs are slow compared with physiological fluctuations in secreted protein synthesis. While searching for mechanisms that fill this temporal gap in coping with ER stress, we found elevated levels of adenosine diphosphate (ADP)–ribosylated BiP in the inactive pancreas of fasted mice and a rapid decline in this modification in the active fed state. ADP ribosylation mapped to Arg470 and Arg492 in the substrate-binding domain of hamster BiP. Mutations that mimic the negative charge of ADP-ribose destabilized substrate binding and interfered with interdomain allosteric coupling, marking ADP ribosylation as a rapid posttranslational mechanism for reversible inactivation of BiP. A kinetic model showed that buffering fluctuations in unfolded protein load with a recruitable pool of inactive chaperone is an efficient strategy to minimize both aggregation and costly degradation of unfolded proteins. PMID:22869598

  2. Modulating Molecular Chaperones Improves Mitochondrial Bioenergetics and Decreases the Inflammatory Transcriptome in Diabetic Sensory Neurons

    PubMed Central

    Ma, Jiacheng; Pan, Pan; Anyika, Mercy; Blagg, Brian S. J.; Dobrowsky, Rick T.

    2015-01-01

    We have previously demonstrated that modulating molecular chaperones with KU-32, a novobiocin derivative, ameliorates physiologic and bioenergetic deficits of diabetic peripheral neuropathy (DPN). Replacing the coumarin core of KU-32 with a meta-fluorinated biphenyl ring system created KU-596, a novobiocin analogue (novologue) that showed neuroprotective activity in a cell-based assay. The current study sought to determine whether KU-596 offers similar therapeutic potential for treating DPN. Administration of 2–20 mg/kg of KU-596 improved diabetes induced hypoalgesia and sensory neuron bioenergetic deficits in a dose-dependent manner. However, the drug could not improve these neuropathic deficits in diabetic heat shock protein 70 knockout (Hsp70 KO) mice. To gain further insight into the mechanisms by which KU-596 improved DPN, we performed transcriptomic analysis of sensory neuron RNA obtained from diabetic wild-type and Hsp70 KO mice using RNA sequencing. Bioinformatic analysis of the differentially expressed genes indicated that diabetes strongly increased inflammatory pathways and that KU-596 therapy effectively reversed these increases independent of Hsp70. In contrast, the effects of KU-596 on decreasing the expression of genes regulating the production of reactive oxygen species were more Hsp70-dependent. These data indicate that modulation of molecular chaperones by novologue therapy offers an effective approach toward correcting nerve dysfunction in DPN but that normalization of inflammatory pathways alone by novologue therapy seems to be insufficient to reverse sensory deficits associated with insensate DPN. PMID:26161583

  3. In vitro aggregation of the regulated secretory protein chromogranin A.

    PubMed Central

    Jain, Renu K; Chang, Wen Tzu; Geetha, Chitta; Joyce, Paul B M; Gorr, Sven-Ulrik

    2002-01-01

    Aggregation chaperones, consisting of secretory proteins that contain a hexa-histidine epitope tag, enhance the calcium-induced aggregation of regulated secretory proteins and their sorting to secretory granules. The goal of this study was to gain a better understanding of this unusual aggregation mechanism. Hexa-histidine-epitope-tagged secreted alkaline phosphatase, an aggregation chaperone, enhanced the in vitro aggregation of chromogranin A in the presence of calcium, but not in the presence of magnesium or other divalent cations. As an exception, chromogranin was completely aggregated by zinc, even in the absence of the aggregation chaperone. In addition, fluorescence spectroscopy of the aggregation reaction mixture showed an increase in fluorescence intensity consistent with the formation of protein aggregates. The calcium-induced aggregation of chromogranin A was completely inhibited by 0.2% Triton X-100, suggesting that it involves hydrophobic interactions. In contrast, the detergent did not affect chaperone-enhanced aggregation, suggesting that this aggregation does not depend on hydrophobic interactions. EDTA-treated chaperone did not enhance chromogranin A aggregation, indicating that divalent cations are necessary for chaperone action. Although the structure of the aggregation chaperone was not important, the size of the chaperone was. Thus the free His-hexapeptide could not substitute for the aggregation chaperone. Based on these results, we propose that the hexa-histidine tag, in the context of a polypeptide, acts as a divalent cation-dependent nucleation site for chromogranin A aggregation. PMID:12175332

  4. C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

    PubMed Central

    Santiago-Frangos, Andrew; Kavita, Kumari; Schu, Daniel J.; Gottesman, Susan

    2016-01-01

    The bacterial Sm protein and RNA chaperone Hfq stabilizes small noncoding RNAs (sRNAs) and facilitates their annealing to mRNA targets involved in stress tolerance and virulence. Although an arginine patch on the Sm core is needed for Hfq’s RNA chaperone activity, the function of Hfq’s intrinsically disordered C-terminal domain (CTD) has remained unclear. Here, we use stopped flow spectroscopy to show that the CTD of Escherichia coli Hfq is not needed to accelerate RNA base pairing but is required for the release of dsRNA. The Hfq CTD also mediates competition between sRNAs, offering a kinetic advantage to sRNAs that contact both the proximal and distal faces of the Hfq hexamer. The change in sRNA hierarchy caused by deletion of the Hfq CTD in E. coli alters the sRNA accumulation and the kinetics of sRNA regulation in vivo. We propose that the Hfq CTD displaces sRNAs and annealed sRNA⋅mRNA complexes from the Sm core, enabling Hfq to chaperone sRNA–mRNA interactions and rapidly cycle between competing targets in the cell. PMID:27681631

  5. Chemical chaperon 4-phenylbutyrate protects against the endoplasmic reticulum stress-mediated renal fibrosis in vivo and in vitro.

    PubMed

    Liu, Shing-Hwa; Yang, Ching-Chin; Chan, Ding-Cheng; Wu, Cheng-Tien; Chen, Li-Ping; Huang, Jenq-Wen; Hung, Kuan-Yu; Chiang, Chih-Kang

    2016-04-19

    Renal tubulointerstitial fibrosis is the common and final pathologic change of kidney in end-stage renal disease. Interesting, endoplasmic reticulum (ER) stress is known to contribute to the pathophysiological mechanisms during the development of renal fibrosis. Here, we investigated the effects of chemical chaperon sodium 4-phenylbutyrate (4-PBA) on renal fibrosis in vivo and in vitro. In a rat unilateral ureteral obstruction (UUO) model, 4-PBA mimicked endogenous ER chaperon in the kidneys and significantly reduced glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), activating transcription factor 4 (ATF4), and phosphorylated JNK protein expressions as well as restored spliced X-box-binding protein 1 (XBP1) expressions in the kidneys of UUO rats. 4-PBA also attenuated the increases of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) protein expressions, tubulointerstitial fibrosis, and apoptosis in the kidneys of UUO rats. Moreover, transforming growth factor (TGF)-β markedly increased ER stress-associated molecules, profibrotic factors, and apoptotic markers in the renal tubular cells (NRK-52E), all of which could be significantly counteracted by 4-PBA treatment. 4-PBA also diminished TGF-β-increased CTGF promoter activity and CTGF mRNA expression in NRK-52E cells. Taken together, our results indicated that 4-PBA acts as an ER chaperone to ameliorate ER stress-induced renal tubular cell apoptosis and renal fibrosis.

  6. Chemical chaperon 4-phenylbutyrate protects against the endoplasmic reticulum stress-mediated renal fibrosis in vivo and in vitro

    PubMed Central

    Wu, Cheng-Tien; Chen, Li-Ping; Huang, Jenq-Wen; Hung, Kuan-Yu; Chiang, Chih-Kang

    2016-01-01

    Renal tubulointerstitial fibrosis is the common and final pathologic change of kidney in end-stage renal disease. Interesting, endoplasmic reticulum (ER) stress is known to contribute to the pathophysiological mechanisms during the development of renal fibrosis. Here, we investigated the effects of chemical chaperon sodium 4-phenylbutyrate (4-PBA) on renal fibrosis in vivo and in vitro. In a rat unilateral ureteral obstruction (UUO) model, 4-PBA mimicked endogenous ER chaperon in the kidneys and significantly reduced glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), activating transcription factor 4 (ATF4), and phosphorylated JNK protein expressions as well as restored spliced X-box-binding protein 1 (XBP1) expressions in the kidneys of UUO rats. 4-PBA also attenuated the increases of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) protein expressions, tubulointerstitial fibrosis, and apoptosis in the kidneys of UUO rats. Moreover, transforming growth factor (TGF)-β markedly increased ER stress-associated molecules, profibrotic factors, and apoptotic markers in the renal tubular cells (NRK-52E), all of which could be significantly counteracted by 4-PBA treatment. 4-PBA also diminished TGF-β-increased CTGF promoter activity and CTGF mRNA expression in NRK-52E cells. Taken together, our results indicated that 4-PBA acts as an ER chaperone to ameliorate ER stress-induced renal tubular cell apoptosis and renal fibrosis. PMID:26959118

  7. The chaperone like function of the nonhistone protein HMGB1

    SciTech Connect

    Osmanov, Taner; Ugrinova, Iva; Pasheva, Evdokia

    2013-03-08

    Highlights: ► The HMGB1 protein strongly enhanced the formation of nucleosome particles. ► The target of HMGB1 action as a chaperone is the DNA not the histone octamer. ► The acetylation of HMGB1 decreases the stimulating effect of the protein. -- Abstract: Almost all essential nuclear processes as replication, repair, transcription and recombination require the chromatin template to be correctly unwound and than repackaged. The major strategy that the cell uses to overcome the nucleosome barrier is the proper removal of the histone octamer and subsequent deposition onto DNA. Important factors in this multi step phenomenon are the histone chaperones that can assemble nucleosome arrays in vitro in the absence of ATP. The nonhistone protein HMGB1 is a good candidate for a chaperone as its molecule consists of two DNA binding motives, Box’s A and B, and a long nonstructured C tail highly negatively charged. HMGB1 protein is known as a nuclear “architectural” factor for its property to bind preferentially to distorted DNA structures and was reported to kink the double helix. Our experiments show that in the classical stepwise dialysis method for nucleosome assembly the addition of HMGB1 protein stimulates more than two times the formation of middle-positioned nucleosomes. The stimulation effect persists in dialysis free experiment when the reconstitution is possible only in the presence of a chaperone. The addition of HMGB1 protein strongly enhanced the formation of a nucleosome in a dose dependant manner. Our results show that the target of HMGB1 action as a chaperone is the DNA fragment not the histone octamer. One possible explanation for the stimulating effect of HMGB1 is the “architectural” property of the protein to associate with the middle of the DNA fragment and to kink it. The acquired V shaped DNA structure is probably conformationals more favorable to wrap around the prefolded histone octamer. We tested also the role of the post

  8. Chaperoning osteogenesis: new protein-folding disease paradigms.

    PubMed

    Makareeva, Elena; Aviles, Nydea A; Leikin, Sergey

    2011-03-01

    Recent discoveries of severe bone disorders in patients with deficiencies in several endoplasmic reticulum chaperones are reshaping the discussion of type I collagen folding and related diseases. Type I collagen is the most abundant protein in all vertebrates and a crucial structural molecule for bone and other connective tissues. Its misfolding causes bone fragility, skeletal deformity and other tissue failures. Studies of newly discovered bone disorders indicate that collagen folding, chaperones involved in the folding process, cellular responses to misfolding and related bone pathologies might not follow conventional protein folding paradigms. In this review, we examine the features that distinguish collagen folding from that of other proteins and describe the findings that are beginning to reveal how cells manage collagen folding and misfolding. We discuss implications of these studies for general protein folding paradigms, unfolded protein response in cells and protein folding diseases.

  9. Evaluation of Quinazoline analogues as Glucocerebrosidase Inhibitors with Chaperone activity

    PubMed Central

    Marugan, Juan J.; Zheng, Wei; Motabar, Omid; Southall, Noel; Goldin, Ehud; Westbroek, Wendy; K.Stubblefield, Barbara; Sidransky, Ellen; Aungst, Ronald A.; Lea, Wendy A.; Simeonov, Anton; Leister, William; Austin, Christopher P.

    2011-01-01

    Gaucher disease is a Lysosomal Storage Disorder (LSD) caused by deficiency in the enzyme glucocerebrosidase (GC). Small molecule chaperones of protein folding and translocation have been proposed as a promising therapeutic approach to this LSD. Most small molecule chaperones described in the literature contain an iminosugar scaffold. Here we present the discovery and evaluation of a new series of GC inhibitors with a quinazoline core. We demonstrate that this series can improve the translocation of GC to the lysosome in patient-derived cells. To optimize this chemical series, systematic synthetic modifications were performed and the SAR was evaluated and compared using three different readouts of compound activity – enzymatic inhibition, enzyme thermostabilization, and lysosomal translocation of GC. PMID:21250698

  10. Revisiting the Interaction between the Chaperone Skp and Lipopolysaccharide

    PubMed Central

    Burmann, Björn M.; Holdbrook, Daniel A.; Callon, Morgane; Bond, Peter J.; Hiller, Sebastian

    2015-01-01

    The bacterial outer membrane comprises two main classes of components, lipids and membrane proteins. These nonsoluble compounds are conveyed across the aqueous periplasm along specific molecular transport routes: the lipid lipopolysaccharide (LPS) is shuttled by the Lpt system, whereas outer membrane proteins (Omps) are transported by chaperones, including the periplasmic Skp. In this study, we revisit the specificity of the chaperone-lipid interaction of Skp and LPS. High-resolution NMR spectroscopy measurements indicate that LPS interacts with Skp nonspecifically, accompanied by destabilization of the Skp trimer and similar to denaturation by the nonnatural detergent lauryldimethylamine-N-oxide (LDAO). Bioinformatic analysis of amino acid conservation, structural analysis of LPS-binding proteins, and MD simulations further confirm the absence of a specific LPS binding site on Skp, making a biological relevance of the interaction unlikely. Instead, our analysis reveals a highly conserved salt-bridge network, which likely has a role for Skp function. PMID:25809264

  11. Co-chaperones of the mammalian endoplasmic reticulum.

    PubMed

    Melnyk, Armin; Rieger, Heiko; Zimmermann, Richard

    2015-01-01

    In mammalian cells, the rough endoplasmic reticulum or ER plays a central role in the biogenesis of most extracellular plus many organellar proteins and in cellular calcium homeostasis. Therefore, this organelle comprises molecular chaperones that are involved in import, folding/assembly, export, and degradation of polypeptides in millimolar concentrations. In addition, there are calcium channels/pumps and signal transduction components present in the ER membrane that affect and are affected by these processes. The ER lumenal Hsp70, termed immunoglobulin-heavy chain binding protein or BiP, is the central player in all these activities and involves up to seven different co-chaperones, i.e. ER-membrane integrated as well as ER-lumenal Hsp40s, which are termed ERj or ERdj, and two nucleotide exchange factors.

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

    SciTech Connect

    Boal, Amie K.; Rosenzweig, Amy C.

    2010-08-16

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

  13. Generalized iterative annealing model for the action of RNA chaperones

    NASA Astrophysics Data System (ADS)

    Hyeon, Changbong; Thirumalai, D.

    2013-09-01

    As a consequence of the rugged landscape of RNA molecules their folding is described by the kinetic partitioning mechanism according to which only a small fraction (ϕF) reaches the folded state while the remaining fraction of molecules is kinetically trapped in misfolded intermediates. The transition from the misfolded states to the native state can far exceed biologically relevant time. Thus, RNA folding in vivo is often aided by protein cofactors, called RNA chaperones, that can rescue RNAs from a multitude of misfolded structures. We consider two models, based on chemical kinetics and chemical master equation, for describing assisted folding. In the passive model, applicable for class I substrates, transient interactions of misfolded structures with RNA chaperones alone are sufficient to destabilize the misfolded structures, thus entropically lowering the barrier to folding. For this mechanism to be efficient the intermediate ribonucleoprotein complex between collapsed RNA and protein cofactor should have optimal stability. We also introduce an active model (suitable for stringent substrates with small ϕF), which accounts for the recent experimental findings on the action of CYT-19 on the group I intron ribozyme, showing that RNA chaperones do not discriminate between the misfolded and the native states. In the active model, the RNA chaperone system utilizes chemical energy of adenosine triphosphate hydrolysis to repeatedly bind and release misfolded and folded RNAs, resulting in substantial increase of yield of the native state. The theory outlined here shows, in accord with experiments, that in the steady state the native state does not form with unit probability.

  14. A histone chaperone, DEK, transcriptionally coactivates a nuclear receptor

    PubMed Central

    Sawatsubashi, Shun; Murata, Takuya; Lim, Jinseon; Fujiki, Ryoji; Ito, Saya; Suzuki, Eriko; Tanabe, Masahiko; Zhao, Yue; Kimura, Shuhei; Fujiyama, Sally; Ueda, Takashi; Umetsu, Daiki; Ito, Takashi; Takeyama, Ken-ichi; Kato, Shigeaki

    2010-01-01

    Chromatin reorganization is essential for transcriptional control by sequence-specific transcription factors. However, the molecular link between transcriptional control and chromatin reconfiguration remains unclear. By colocalization of the nuclear ecdysone receptor (EcR) on the ecdysone-induced puff in the salivary gland, Drosophila DEK (dDEK) was genetically identified as a coactivator of EcR in both insect cells and intact flies. Biochemical purification and characterization of the complexes containing fly and human DEKs revealed that DEKs serve as histone chaperones via phosphorylation by forming complexes with casein kinase 2. Consistent with the preferential association of the DEK complex with histones enriched in active epigenetic marks, dDEK facilitated H3.3 assembly during puff formation. In some human myeloid leukemia patients, DEK was fused to CAN by chromosomal translocation. This mutation significantly reduced formation of the DEK complex, which is required for histone chaperone activity. Thus, the present study suggests that at least one histone chaperone can be categorized as a type of transcriptional coactivator for nuclear receptors. PMID:20040570

  15. Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

    PubMed Central

    Costello, Shawn M.; Plummer, Ashlee M.; Fleming, Patrick J.; Fleming, Karen G.

    2016-01-01

    Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed “Outer Membrane Protein Biogenesis Model” (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway. PMID:27482090

  16. The Chaperone TRAP1 As a Modulator of the Mitochondrial Adaptations in Cancer Cells

    PubMed Central

    Masgras, Ionica; Sanchez-Martin, Carlos; Colombo, Giorgio; Rasola, Andrea

    2017-01-01

    Mitochondria can receive, integrate, and transmit a variety of signals to shape many biochemical activities of the cell. In the process of tumor onset and growth, mitochondria contribute to the capability of cells of escaping death insults, handling changes in ROS levels, rewiring metabolism, and reprograming gene expression. Therefore, mitochondria can tune the bioenergetic and anabolic needs of neoplastic cells in a rapid and flexible way, and these adaptations are required for cell survival and proliferation in the fluctuating environment of a rapidly growing tumor mass. The molecular bases of pro-neoplastic mitochondrial adaptations are complex and only partially understood. Recently, the mitochondrial molecular chaperone TRAP1 (tumor necrosis factor receptor associated protein 1) was identified as a key regulator of mitochondrial bioenergetics in tumor cells, with a profound impact on neoplastic growth. In this review, we analyze these findings and discuss the possibility that targeting TRAP1 constitutes a new antitumor approach.

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

    PubMed Central

    Hatori, Yuta; Lutsenko, Svetlana

    2016-01-01

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

  18. Structure of the Yersinia pestis type III secretion chaperone SycH in complex with a stable fragment of YscM2

    SciTech Connect

    Phan, Jason; Tropea, Joseph E.; Waugh, David S.

    2010-11-16

    Pathogenic Yersinia species use a type III secretion system to inject cytotoxic effector proteins directly into the cytosol of mammalian cells, where they neutralize the innate immune response by interfering with the signal-transduction pathways that control phagocytosis and inflammation. To be exported efficiently, some effectors must transiently associate with cognate cytoplasmic secretion chaperones. SycH is the chaperone for YopH, a potent eukaryotic-like protein tyrosine phosphatase that is essential for virulence. SycH also binds two negative regulators of type III secretion, YscM1 and YscM2, both of which share significant sequence homology with the chaperone-binding domain of YopH. Here, the structure of a complex between SycH and a stable fragment of YscM2 that was designed on the basis of limited proteolysis experiments is presented. The overall fold of SycH is very similar to the structures of other homodimeric secretion chaperones that have been determined to date. YscM2 wraps around SycH in an extended fashion, with some secondary but no tertiary structure, assuming a conformation distinct from the globular fold that it is predicted to adopt in the absence of SycH.

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

    PubMed

    Singh, Meetali; Shah, Varun; Tatu, Utpal

    2014-04-17

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

  20. Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates*

    PubMed Central

    Mattoo, Rayees U. H.; Sharma, Sandeep K.; Priya, Smriti; Finka, Andrija; Goloubinoff, Pierre

    2013-01-01

    Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyze ATP. However, they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyze ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimized in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1 and HSPH2) are bona fide chaperones on their own that collaborate with Hsp40 (DNAJA1 and DNAJB1) to hydrolyze ATP and unfold and thus convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding. PMID:23737532

  1. Methods to study histone chaperone function in nucleosome assembly and chromatin transcription.

    PubMed

    Senapati, Parijat; Sudarshan, Deepthi; Gadad, Shrikanth S; Shandilya, Jayasha; Swaminathan, Venkatesh; Kundu, Tapas K

    2015-01-01

    Histone chaperones are histone interacting proteins that are involved in various stages of histone metabolism in the cell such as histone storage, transport, nucleosome assembly and disassembly. Histone assembly and disassembly are essential processes in certain DNA-templated phenomena such as replication, repair and transcription in eukaryotes. Since the first histone chaperone Nucleoplasmin was discovered in Xenopus, a plethora of histone chaperones have been identified, characterized and their functional significance elucidated in the last 35 years or so. Some of the histone chaperone containing complexes such as FACT have been described to play a significant role in nucleosome disassembly during transcription elongation. We have reported earlier that human Nucleophosmin (NPM1), a histone chaperone belonging to the Nucleoplasmin family, is a co-activator of transcription. In this chapter, we describe several methods that are used to study the histone chaperone activity of proteins and their role in transcription.

  2. Copper modulates the degradation of copper chaperone for Cu,Zn superoxide dismutase by the 26 S proteosome.

    PubMed

    Bertinato, Jesse; L'Abbé, Mary R

    2003-09-12

    Copper chaperones are copper-binding proteins that directly insert copper into specific targets, preventing the accumulation of free copper ions that can be toxic to the cell. Despite considerable advances in the understanding of copper transfer from copper chaperones to their target, to date, there is no information regarding how the activity of these proteins is regulated in higher eukaryotes. The insertion of copper into the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) depends on the copper chaperone for SOD1 (CCS). We have recently reported that CCS protein is increased in tissues of rats fed copper-deficient diets suggesting that copper may regulate CCS expression. Here we show that whereas copper deficiency increased CCS protein in rats, mRNA level was unaffected. Rodent and human cell lines cultured in the presence of the specific copper chelator 2,3,2-tetraamine displayed a dose-dependent increase in CCS protein that could be reversed with the addition of copper but not iron or zinc to the cells. Switching cells from copper-deficient to copper-rich medium promoted the rapid degradation of CCS, which could be blocked by the proteosome inhibitors MG132 and lactacystin but not a cysteine protease inhibitor or inhibitors of the lysosomal degradation pathway. In addition, CCS degradation was slower in copper-deficient cells than in cells cultured in copper-rich medium. Together, these data show that copper regulates CCS expression by modulating its degradation by the 26 S proteosome and suggest a novel role for CCS in prioritizing the utilization of copper when it is scarce.

  3. Reactivation of Aggregated Proteins by the ClpB/DnaK Bi-chaperone System

    PubMed Central

    Zolkiewski, Michal; Chesnokova, Liudmila S.; Witt, Stephan N.

    2016-01-01

    Protein aggregation is a common problem in protein biochemistry and is linked to many cellular pathologies and human diseases. The molecular chaperone ClpB can resolubilize and reactivate aggregated proteins. This unit describes the procedure for following reactivation of an aggregated enzyme glucose-6-phosphate dehydrogenase mediated by ClpB from Escherichia coli in cooperation with another molecular chaperone DnaK. The procedures for purification of these chaperones are also described. PMID:26836408

  4. Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase.

    PubMed

    Thöny, Beat; Calvo, Ana C; Scherer, Tanja; Svebak, Randi M; Haavik, Jan; Blau, Nenad; Martinez, Aurora

    2008-07-01

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with l-DOPA responsive and non-responsive dystonia and infantile parkinsonism. In this study, we show that both the cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and the feedback inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription-translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH(4) supplementation to mice increased TH activity and protein levels in brain extracts, while the Th-mRNA level was not affected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding-aid effect of BH(4) and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH(4) crosses the blood-brain barrier and therapeutic regimes based on BH(4) supplementation should thus consider the effect on TH. Furthermore, BH(4) supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the human TH isoform 1 mutation L205P.

  5. The sigma-1 receptor chaperone as an inter-organelle signaling modulator

    PubMed Central

    Su, Tsung-Ping; Hayashi, Teruo; Maurice, Tangui; Buch, Shilpa; Ruoho, Arnold E.

    2010-01-01

    Inter-organelle signaling plays important roles in many physiological functions. Endoplasmic reticulum (ER)-mitochondrion signaling affects intra-mitochondrial calcium (Ca2+) homeostasis and cellular bioenergetics. ER-nucleus signaling attenuates ER stress. ER-plasma membrane signaling regulates cytosolic Ca2+ homeostasis, and ER-mitochondrion-plasma membrane signaling regulates hippocampal dendritic spine formation. Here we propose that the sigma-1 receptor (Sig-1R), an ER chaperone protein, acts as an inter-organelle signaling modulator. Sig-1Rs normally reside at the ER-mitochondrion contact called the MAM (mitochondrion-associated ER membrane), where Sig-1Rs regulate ER-mitochondrion signaling and the ER-nucleus cross-talk. When cells are stimulated by ligands or undergo prolonged stress, Sig-1Rs translocate from the MAM to the ER reticular network and plasmalemma/plasma membrane to regulate a variety of functional proteins, including ion channels, receptors, and kinases. Thus, the Sig-1R serves as an inter-organelle signaling modulator locally at the MAM and remotely at the plasmalemma/plasma membrane. Many pharmacological/physiological effects of Sig-1Rs may relate to this unique action of Sig-1Rs. PMID:20869780

  6. Catapult mechanism renders the chaperone action of Hsp70 unidirectional.

    PubMed

    Gisler, S M; Pierpaoli, E V; Christen, P

    1998-06-19

    Molecular chaperones of the Hsp70 type promote the folding and membrane translocation of proteins. The interaction of Hsp70s with polypeptides is linked to ATP binding and hydrolysis. We formed complexes of seven different fluorescence-labeled peptides with DnaK, the Hsp70 homolog of Escherichia coli, and determined the rate of peptide release under two different sets of conditions. (1) Upon addition of ATP to nucleotide-free peptide.DnaK complexes, all tested peptides were released with similar rate constants (2.2 s-1 to 6.7 s-1). (2) In the binding equilibrium of peptide and ATP-liganded DnaK, the dissociation followed one or two-step reactions, depending on the amino acid sequence of the peptide. For the monophasic reactions, the dissociation rate constants diverged by four orders of magnitude from 0.0004 s-1 to 5.7 s-1; for the biphasic reactions, the rate constants of the second, slower isomerization step were in the range from 0.3 s-1 to 0.0005 s-1. The release of the different peptides in case (1) is 1.4 to 14,000 times faster than in case (2). Apparently, binding of ATP induces a transient state of the chaperone which ejects target peptides before the final state of ATP-liganded DnaK is reached. This "catapult" mechanism provides the chaperone cycle with a mode of peptide release that does not correspond with the reverse of peptide binding. By allowing the conformation of the outgoing polypeptide to differ from that of the incoming polypeptide, a futile cycle with respect to conformational work exerted on the target protein is obviated.

  7. A chemical chaperone induces inhomogeneous conformational changes in flexible proteins.

    PubMed

    Hamdane, Djemel; Velours, Christophe; Cornu, David; Nicaise, Magali; Lombard, Murielle; Fontecave, Marc

    2016-07-27

    Organic osmolytes also known as chemical chaperones are major cellular compounds that favor, by an unclear mechanism, protein's compaction and stabilization of the native state. Here, we have examined the chaperone effect of the naturally occurring trimethylamine N-oxide (TMAO) osmolyte on a loosely packed protein (LPP), known to be a highly flexible form, using an apoprotein mutant of the flavin-dependent RNA methyltransferase as a model. Thermal and chemical denaturation experiments showed that TMAO stabilizes the structural integrity of the apoprotein dramatically. The denaturation reaction is irreversible indicating that the stability of the apoprotein is under kinetic control. This result implies that the stabilization is due to a TMAO-induced reconfiguration of the flexible LPP state, which leads to conformational limitations of the apoprotein likely driven by favorable entropic contribution. Evidence for the conformational perturbation of the apoprotein had been obtained through several biophysical approaches notably analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, labelling experiments and proteolysis coupled to mass spectrometry. Unexpectedly, TMAO promotes an overall elongation or asymmetrical changes of the hydrodynamic shape of the apoprotein without alteration of the secondary structure. The modulation of the hydrodynamic properties of the protein is associated with diverse inhomogenous conformational changes: loss of the solvent accessible cavities resulting in a dried protein matrix; some side-chain residues initially buried become solvent exposed while some others become hidden. Consequently, the TMAO-induced protein state exhibits impaired capability in the flavin binding process. Our study suggests that the nature of protein conformational changes induced by the chemical chaperones may be specific to protein packing and plasticity. This could be an efficient mechanism by which the cell controls and finely tunes the

  8. Chaperone proteins and brain tumors: Potential targets and possible therapeutics1

    PubMed Central

    Graner, Michael W.; Bigner, Darell D.

    2005-01-01

    Chaperone proteins are most notable for the proteo- and cyotoprotective capacities they afford during cellular stress. Under conditions of cellular normalcy, chaperones still play integral roles in the folding of nascent polypeptides into functional entities, in assisting in intracellular/intraorganellar transport, in assembly and maintenance of multi-subunit protein complexes, and in aiding and abetting the degradation of senescent proteins. Tumors frequently have relatively enhanced needs for chaperone number and activity because of the stresses of rapid proliferation, increased metabolism, and overall genetic instability. Thus, it may be possible to take advantage of this reliance that tumor cells have on chaperones by pharmacologic and biologic means. Certain chaperones are abundant in the brain, which implies important roles for them. While it is presumed that the requirements of brain tumors for chaperone proteins are similar to those of any other cell type, tumor or otherwise, very little inquiry has been directed at the possibility of using chaperone proteins as therapeutic targets or even as therapeutic agents against central nervous system malignancies. This review highlights some of the research on the functions of chaperone proteins, on what can be done to modify those functions, and on the physiological responses that tumors and organisms can have to chaperone-targeted or chaperone-based therapies. In particular, this review will also underscore areas of research where brain tumors have been part of the field, although in general those instances are few and far between. This relative dearth of research devoted to chaperone protein targets and therapeutics in brain tumors reveals much untrodden turf to explore for potential treatments of these dreadfully refractive diseases. PMID:16053701

  9. Thanks for asking: Adolescent attitudes and preferences regarding the use of chaperones during physical examinations

    PubMed Central

    Morgan, Renee; Katzman, Debra K; Kaufman, Miriam; Goldberg, Eudice; Toulany, Alene

    2016-01-01

    BACKGROUND: There is no uniformity as to how and when chaperones should be used for general and intimate (genitalia and/or breasts) physical examinations of adolescents. OBJECTIVE: To explore adolescents’ attitudes and preferences regarding the use of medical chaperones during physical examinations. METHODS: The present analysis was a cross-sectional descriptive study performed as part of a quality improvement project in the Adolescent Medicine Clinics at The Hospital for Sick Children (Toronto, Ontario) between January 1 and April 30, 2011. Adolescents 13 to 18 years of age completed an anonymous 10-item, self-administered questionnaire regarding their thoughts on chaperones during physical examinations. Demographic and descriptive data were collected. RESULTS: A total of 127 adolescents participated in the present study. The mean (± SD) age was 16.3±1.5 years and the majority (93.7%) were female. More than one-half (61%) of female adolescents had previous experience with an intimate examination; however, a chaperone was present only 36% of the time. Seventy percent of female adolescents wanted the choice of a chaperone for a general examination compared with 61% for an intimate examination. Among female adolescents with past chaperone experience, 78% wanted the choice of a chaperone for subsequent intimate examinations, compared with 55% among those with no previous chaperone experience. Only 21% believed they would ask for a chaperone if one were not offered. CONCLUSIONS: Although there was variation in adolescents’ attitudes and preferences regarding the use of chaperones, many females indicated a desire to discuss the option of a chaperone for all types of examinations. PMID:27429571

  10. Role of Chaperone-Mediated Autophagy Dysfunctions in the Pathogenesis of Parkinson’s Disease

    PubMed Central

    Sala, Gessica; Marinig, Daniele; Arosio, Alessandro; Ferrarese, Carlo

    2016-01-01

    Chaperone-mediated autophagy (CMA) represents a selective form of autophagy involved in the degradation of specific soluble proteins containing a pentapeptide motif that is recognized by a cytosolic chaperone able to deliver proteins to the lysosomes for degradation. Physiologically, CMA contributes to maintain crucial cellular functions including energetic balance and protein quality control. Dysfunctions in CMA have been associated to the pathogenesis of several neurodegenerative diseases characterized by accumulation and aggregation of proteins identified as CMA substrates. In particular, increasing evidence highlights the existence of a strong relationship between CMA defects and Parkinson’s disease (PD). Several mutations associated with familial forms of PD (SNCA, LRRK2, UCHL1 and DJ-1) have been demonstrated to block or reduce the activity of CMA, the main catabolic pathway for alpha-synuclein (asyn). CMA dysfunctions also leads to a mislocalization and inactivation of the transcription factor MEF2D that plays a key-role in the survival of dopaminergic neurons. Furthermore, reduced levels of CMA markers have been observed in post mortem brain samples from PD patients. The aim of this review article is to provide an organic revision of evidence for the involvement of CMA dysfunctions in the pathogenesis of PD. Updated findings obtained in patient’s specimens will be resumed, and results deriving from in vivo and in vitro studies will be discussed to evidence the current knowledge on the molecular mechanisms underlying CMA alterations in PD. Finally, the possibility of up-regulating CMA pathway as promising neuroprotective strategy will be considered. PMID:28066181

  11. Sulfonylurea receptor 1 mutations that cause opposite insulin secretion defects with chemical chaperone exposure.

    PubMed

    Pratt, Emily B; Yan, Fei-Fei; Gay, Joel W; Stanley, Charles A; Shyng, Show-Ling

    2009-03-20

    The beta-cell ATP-sensitive potassium (K(ATP)) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for K(ATP) channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6- and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants.

  12. Sulfonylurea Receptor 1 Mutations That Cause Opposite Insulin Secretion Defects with Chemical Chaperone Exposure*S⃞

    PubMed Central

    Pratt, Emily B.; Yan, Fei-Fei; Gay, Joel W.; Stanley, Charles A.; Shyng, Show-Ling

    2009-01-01

    The β-cell ATP-sensitive potassium (KATP) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for KATP channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6- and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants. PMID:19151370

  13. The co-chaperone p23 promotes prostate cancer motility and metastasis

    PubMed Central

    Querol Cano, Laia; Lavery, Derek N.; Sin, Soraya; Spanjaard, Emma; Brooke, Greg N.; Tilman, Jessica D.; Abroaf, Ahmed; Gaughan, Luke; Robson, Craig N.; Heer, Rakesh; Mauri, Francesco; de Rooij, Johan; Driouch, Keltouma; Bevan, Charlotte L.

    2015-01-01

    Prostate cancer is an androgen receptor (AR)-dependent malignancy at initiation and progression, therefore hormone therapy is the primary line of systemic treatment. Despite initial disease regression, tumours inevitably recur and progress to an advanced castration-resistant state a major feature of which is metastasis to the bone. Up-regulation of AR cofactors and chaperones that overcome low hormone conditions to maintain basal AR activity has been postulated as a mechanism of therapy relapse. p23, an essential component of the apo-AR complex, acts also after ligand binding to increase AR transcriptional activity and target gene expression, partly by increasing chromatin-loaded holo-receptor-complexes. Immunohistochemical studies have demonstrated increased p23 expression in advanced prostate cancer. Here, we further characterise p23 roles in AR signalling and show that it modulates cytosolic AR levels in the absence of hormone, confirming a chaperoning function in the aporeceptor complex and suggesting p23 upregulates AR signalling at multiple stages. Moreover, p23 protein levels significantly increased upon treatment with not only androgen but also clinically relevant anti-androgens. This was in contrast to the HSP90 inhibitor 17-AAG, which did not modulate expression of the cochaperone – important given the HSP90-independent roles we and others have previously described for p23. Further, we demonstrate p23 is implicated in prostate cancer cell motility and in acquisition of invasiveness capacity through the expression of specific genes known to participate in cancer progression. This may drive metastatic processes in vivo since analysis of prostate tumour biopsies revealed that high nuclear p23 significantly correlated with shorter survival times and with development of metastases in patients with lower grade tumours. We propose that increased p23 expression may allow cells to acquire a more aggressive phenotype, contributing to disease progression, and

  14. The Molecular Chaperone DnaK Is a Source of Mutational Robustness.

    PubMed

    Aguilar-Rodríguez, José; Sabater-Muñoz, Beatriz; Montagud-Martínez, Roser; Berlanga, Víctor; Alvarez-Ponce, David; Wagner, Andreas; Fares, Mario A

    2016-10-05

    Molecular chaperones, also known as heat-shock proteins, refold misfolded proteins and help other proteins reach their native conformation. Thanks to these abilities, some chaperones, such as the Hsp90 protein or the chaperonin GroEL, can buffer the deleterious phenotypic effects of mutations that alter protein structure and function. Hsp70 chaperones use a chaperoning mechanism different from that of Hsp90 and GroEL, and it is not known whether they can also buffer mutations. Here, we show that they can. To this end, we performed a mutation accumulation experiment in Escherichia coli, followed by whole-genome resequencing. Overexpression of the Hsp70 chaperone DnaK helps cells cope with mutational load and completely avoid the extinctions we observe in lineages evolving without chaperone overproduction. Additionally, our sequence data show that DnaK overexpression increases mutational robustness, the tolerance of its clients to nonsynonymous nucleotide substitutions. We also show that this elevated mutational buffering translates into differences in evolutionary rates on intermediate and long evolutionary time scales. Specifically, we studied the evolutionary rates of DnaK clients using the genomes of E. coli, Salmonella enterica, and 83 other gamma-proteobacteria. We find that clients that interact strongly with DnaK evolve faster than weakly interacting clients. Our results imply that all three major chaperone classes can buffer mutations and affect protein evolution. They illustrate how an individual protein like a chaperone can have a disproportionate effect on the evolution of a proteome.

  15. Information encoded in non-native states drives substrate-chaperone pairing.

    PubMed

    Mapa, Koyeli; Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik

    2012-09-05

    Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones.

  16. Type III chaperones & Co in bacterial plant pathogens: a set of specialized bodyguards mediating effector delivery.

    PubMed

    Lohou, David; Lonjon, Fabien; Genin, Stéphane; Vailleau, Fabienne

    2013-11-22

    Gram-negative plant pathogenic bacteria possess a type III secretion system (T3SS) to inject bacterial proteins, called type III effectors (T3Es), into host cells through a specialized syringe structure. T3Es are virulence factors that can suppress plant immunity but they can also conversely be recognized by the plant and trigger specific resistance mechanisms. The T3SS and injected T3Es play a central role in determining the outcome of a host-pathogen interaction. Still little is known in plant pathogens on the assembly of the T3SS and the regulatory mechanisms involved in the temporal control of its biosynthesis and T3E translocation. However, recent insights point out the role of several proteins as prime candidates in the role of regulators of the type III secretion (T3S) process. In this review we report on the most recent advances on the regulation of the T3S by focusing on protein players involved in secretion/translocation regulations, including type III chaperones (T3Cs), type III secretion substrate specificity switch (T3S4) proteins and other T3S orchestrators.

  17. NAP-1, Nucleosome assembly protein 1, a histone chaperone involved in Drosophila telomeres.

    PubMed

    López-Panadès, Elisenda; Casacuberta, Elena

    2016-03-01

    Telomere elongation is a function that all eukaryote cells must accomplish in order to guarantee, first, the stability of the end of the chromosomes and second, to protect the genetic information from the inevitable terminal erosion. The targeted transposition of the telomere transposons HeT-A, TART and TAHRE perform this function in Drosophila, while the telomerase mechanism elongates the telomeres in most eukaryotes. In order to integrate telomere maintenance together with cell cycle and metabolism, different components of the cell interact, regulate, and control the proteins involved in telomere elongation. Different partners of the telomerase mechanism have already been described, but in contrast, very few proteins have been related with assisting the telomere transposons of Drosophila. Here, we describe for the first time, the implication of NAP-1 (Nucleosome assembly protein 1), a histone chaperone that has been involved in nuclear transport, transcription regulation, and chromatin remodeling, in telomere biology. We find that Nap-1 and HeT-A Gag, one of the major components of the Drosophila telomeres, are part of the same protein complex. We also demonstrate that their close interaction is necessary to guarantee telomere stability in dividing cells. We further show that NAP-1 regulates the transcription of the HeT-A retrotransposon, pointing to a positive regulatory role of NAP-1 in telomere expression. All these results facilitate the understanding of the transposon telomere maintenance mechanism, as well as the integration of telomere biology with the rest of the cell metabolism.

  18. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium

    PubMed Central

    Sittka, Alexandra; Pfeiffer, Verena; Tedin, Karsten; Vogel, Jörg

    2007-01-01

    The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Qβ RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression. PMID:17163975

  19. Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival.

    PubMed

    Mori, Tomohisa; Hayashi, Teruo; Hayashi, Eri; Su, Tsung-Ping

    2013-01-01

    The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.

  20. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

    PubMed

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-02-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients.

  1. Azasugar inhibitors as pharmacological chaperones for Krabbe disease

    SciTech Connect

    Hill, Chris H.; Viuff, Agnete H.; Spratley, Samantha J.; Salamone, Stéphane; Christensen, Stig H.; Read, Randy J.; Moriarty, Nigel W.; Jensen, Henrik H.; Deane, Janet E.

    2015-03-23

    Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe a new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.

  2. Azasugar inhibitors as pharmacological chaperones for Krabbe disease

    DOE PAGES

    Hill, Chris H.; Viuff, Agnete H.; Spratley, Samantha J.; ...

    2015-03-23

    Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe amore » new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.« less

  3. Chaperones in Polyglutamine Aggregation: Beyond the Q-Stretch

    PubMed Central

    Kuiper, E. F. E.; de Mattos, Eduardo P.; Jardim, Laura B.; Kampinga, Harm H.; Bergink, Steven

    2017-01-01

    Expanded polyglutamine (polyQ) stretches in at least nine unrelated proteins lead to inherited neuronal dysfunction and degeneration. The expansion size in all diseases correlates with age at onset (AO) of disease and with polyQ protein aggregation, indicating that the expanded polyQ stretch is the main driving force for the disease onset. Interestingly, there is marked interpatient variability in expansion thresholds for a given disease. Between different polyQ diseases the repeat length vs. AO also indicates the existence of modulatory effects on aggregation of the upstream and downstream amino acid sequences flanking the Q expansion. This can be either due to intrinsic modulation of aggregation by the flanking regions, or due to differential interaction with other proteins, such as the components of the cellular protein quality control network. Indeed, several lines of evidence suggest that molecular chaperones have impact on the handling of different polyQ proteins. Here, we review factors differentially influencing polyQ aggregation: the Q-stretch itself, modulatory flanking sequences, interaction partners, cleavage of polyQ-containing proteins, and post-translational modifications, with a special focus on the role of molecular chaperones. By discussing typical examples of how these factors influence aggregation, we provide more insight on the variability of AO between different diseases as well as within the same polyQ disorder, on the molecular level. PMID:28386214

  4. Structural Basis of Pharmacological Chaperoning for Human β-Galactosidase*

    PubMed Central

    Suzuki, Hironori; Ohto, Umeharu; Higaki, Katsumi; Mena-Barragán, Teresa; Aguilar-Moncayo, Matilde; Ortiz Mellet, Carmen; Nanba, Eiji; Garcia Fernandez, Jose M.; Suzuki, Yoshiyuki; Shimizu, Toshiyuki

    2014-01-01

    GM1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal β-galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human β-GalWT and two representative mutations in GM1 gangliosidosis Japanese patients, β-GalR201C and β-GalI51T. We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of β-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds. PMID:24737316

  5. Synthetic cation-selective nanotube: Permeant cations chaperoned by anions

    NASA Astrophysics Data System (ADS)

    Hilder, Tamsyn A.; Gordon, Dan; Chung, Shin-Ho

    2011-01-01

    The ability to design ion-selective, synthetic nanotubes which mimic biological ion channels may have significant implications for the future treatment of bacteria, diseases, and as ultrasensitive biosensors. We present the design of a synthetic nanotube made from carbon atoms that selectively allows monovalent cations to move across and rejects all anions. The cation-selective nanotube mimics some of the salient properties of biological ion channels. Before practical nanodevices are successfully fabricated it is vital that proof-of-concept computational studies are performed. With this in mind we use molecular and stochastic dynamics simulations to characterize the dynamics of ion permeation across a single-walled (10, 10), 36 Å long, carbon nanotube terminated with carboxylic acid with an effective radius of 5.08 Å. Although cations encounter a high energy barrier of 7 kT, its height is drastically reduced by a chloride ion in the nanotube. The presence of a chloride ion near the pore entrance thus enables a cation to enter the pore and, once in the pore, it is chaperoned by the resident counterion across the narrow pore. The moment the chaperoned cation transits the pore, the counterion moves back to the entrance to ferry another ion. The synthetic nanotube has a high sodium conductance of 124 pS and shows linear current-voltage and current-concentration profiles. The cation-anion selectivity ratio ranges from 8 to 25, depending on the ionic concentrations in the reservoirs.

  6. Anticancer Gold(III) Porphyrins Target Mitochondrial Chaperone Hsp60.

    PubMed

    Hu, Di; Liu, Yungen; Lai, Yau-Tsz; Tong, Ka-Chung; Fung, Yi-Man; Lok, Chun-Nam; Che, Chi-Ming

    2016-01-22

    Identification of the molecular target(s) of anticancer metal complexes is a formidable challenge since most of them are unstable toward ligand exchange reaction(s) or biological reduction under physiological conditions. Gold(III) meso-tetraphenylporphyrin (gold-1 a) is notable for its high stability in biological milieux and potent in vitro and in vivo anticancer activities. Herein, extensive chemical biology approaches employing photo-affinity labeling, click chemistry, chemical proteomics, cellular thermal shift, saturation-transfer difference NMR, protein fluorescence quenching, and protein chaperone assays were used to provide compelling evidence that heat-shock protein 60 (Hsp60), a mitochondrial chaperone and potential anticancer target, is a direct target of gold-1 a in vitro and in cells. Structure-activity studies with a panel of non-porphyrin gold(III) complexes and other metalloporphyrins revealed that Hsp60 inhibition is specifically dependent on both the gold(III) ion and the porphyrin ligand.

  7. Pharmacological chaperone approaches for rescuing GPCR mutants: Current state, challenges, and screening strategies.

    PubMed

    Beerepoot, Pieter; Nazari, Reza; Salahpour, Ali

    2017-03-01

    A substantial number of G-protein coupled receptors (GPCRs) genetic disorders are due to mutations that cause misfolding or dysfunction of the receptor product. Pharmacological chaperoning approaches can rescue such mutant receptors by stabilizing protein conformations that behave similar to the wild type protein. For example, this can be achieved by improving folding efficiency and/or interaction with chaperone proteins. Although efficacy of pharmacological chaperones has been demonstrated in vitro for a variety of GPCRs, translation to clinical use has been limited. In this paper we discuss the history of pharmacological chaperones of GPCR's and other membrane proteins, the challenges in translation to the clinic, and the use of different assays for pharmacological chaperone discovery.

  8. Unfolding the Therapeutic Potential of Chemical Chaperones for Age-related Macular Degeneration

    PubMed Central

    Sauer, Theodor; Patel, Mrinali; Chan, Chi-Chao; Tuo, Jingsheng

    2008-01-01

    SUMMARY Recent studies suggest that pathological processes involved in age-related macular degeneration (AMD) might induce endoplasmic reticulum (ER) stress. Growing evidence demonstrates the ability of chemical chaperones to decrease ER stress and ameliorate ER stress-related disease phenotypes, suggesting that the field of chaperone therapy might hold novel treatments for AMD. In this review, we examine the evidence suggesting a role for ER stress in AMD. Furthermore, we discuss the use of chaperone therapy for the treatment of ER stress-associated diseases, including other neurodegenerative diseases and retinopathies. Finally, we examine strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone activity in in vitro and in vivo models of human disease. PMID:18528533

  9. Effect of the Surface Charge of Artificial Chaperones on the Refolding of Thermally Denatured Lysozymes.

    PubMed

    Huang, Fan; Shen, Liangliang; Wang, Jianzu; Qu, Aoting; Yang, Huiru; Zhang, Zhenkun; An, Yingli; Shi, Linqi

    2016-02-17

    Artificial chaperones are of great interest in fighting protein misfolding and aggregation for the protection of protein bioactivity. A comprehensive understanding of the interaction between artificial chaperones and proteins is critical for the effective utilization of these materials in biomedicine. In this work, we fabricated three kinds of artificial chaperones with different surface charges based on mixed-shell polymeric micelles (MSPMs), and investigated their protective effect for lysozymes under thermal stress. It was found that MSPMs with different surface charges showed distinct chaperone-like behavior, and the neutral MSPM with PEG shell and PMEO2MA hydrophobic domain at high temperature is superior to the negatively and positively charged one, because of the excessive electrostatic interactions between the protein and charged MSPMs. The results may benefit to optimize this kind of artificial chaperone with enhanced properties and expand their application in the future.

  10. Targeting the molecular chaperone SlyD to inhibit bacterial growth with a small molecule

    PubMed Central

    Kumar, Amit; Balbach, Jochen

    2017-01-01

    Molecular chaperones are essential molecules for cell growth, whereby they maintain protein homeostasis. Because of their central cellular function, bacterial chaperones might be potential candidates for drug targets. Antimicrobial resistance is currently one of the greatest threats to human health, with gram-negative bacteria being of major concern. We found that a Cu2+ complex readily crosses the bacterial cell wall and inhibits SlyD, which is a molecular chaperone, cis/trans peptidyl prolyl isomerise (PPIase) and involved in various other metabolic pathways. The Cu2+ complex binds to the active sites of SlyD, which suppresses its PPIase and chaperone activities. Significant cell growth retardation could be observed for pathogenic bacteria (e.g., Staphylococcus aureus and Pseudomonas aeruginosa). We anticipate that rational development of drugs targeting molecular chaperones might help in future control of pathogenic bacterial growth, in an era of rapidly increasing antibiotic resistance. PMID:28176839

  11. Endoplasmic Reticulum Chaperones and Their Roles in the Immunogenicity of Cancer Vaccines

    PubMed Central

    Graner, Michael W.; Lillehei, Kevin O.; Katsanis, Emmanuel

    2015-01-01

    The endoplasmic reticulum (ER) is a major site of passage for proteins en route to other organelles, to the cell surface, and to the extracellular space. It is also the transport route for peptides generated in the cytosol by the proteasome into the ER for loading onto major histocompatibility complex class I (MHC I) molecules for eventual antigen presentation at the cell surface. Chaperones within the ER are critical for many of these processes; however, outside the ER certain of those chaperones may play important and direct roles in immune responses. In some cases, particular ER chaperones have been utilized as vaccines against tumors or infectious disease pathogens when purified from tumor tissue or recombinantly generated and loaded with antigen. In other cases, the cell surface location of ER chaperones has implications for immune responses as well as possible tumor resistance. We have produced heat-shock protein/chaperone protein-based cancer vaccines called “chaperone-rich cell lysate” (CRCL) that are conglomerates of chaperones enriched from solid tumors by an isoelectric focusing technique. These preparations have been effective against numerous murine tumors, as well as in a canine with an advanced lung carcinoma treated with autologous CRCL. We also published extensive proteomic analyses of CRCL prepared from human surgically resected tumor samples. Of note, these preparations contained at least 10 ER chaperones and a number of other residents, along with many other chaperones/heat-shock proteins. Gene ontology and network analyses utilizing these proteins essentially recapitulate the antigen presentation pathways and interconnections. In conjunction with our current knowledge of cell surface/extracellular ER chaperones, these data collectively suggest that a systems-level view may provide insight into the potent immune stimulatory activities of CRCL with an emphasis on the roles of ER components in those processes. PMID:25610811

  12. Effects of ligand binding on the stability of aldo–keto reductases: Implications for stabilizer or destabilizer chaperones

    PubMed Central

    Kabir, Aurangazeb; Honda, Ryo P.; Kamatari, Yuji O.; Endo, Satoshi; Fukuoka, Mayuko

    2016-01-01

    Abstract Ligands such as enzyme inhibitors stabilize the native conformation of a protein upon binding to the native state, but some compounds destabilize the native conformation upon binding to the non‐native state. The former ligands are termed “stabilizer chaperones” and the latter ones “destabilizer chaperones.” Because the stabilization effects are essential for the medical chaperone (MC) hypothesis, here we have formulated a thermodynamic system consisting of a ligand and a protein in its native‐ and non‐native state. Using the differential scanning fluorimetry and the circular dichroism varying the urea concentration and temperature, we found that when the coenzyme NADP+ was absent, inhibitors such as isolithocholic acid stabilized the aldo–keto reductase AKR1A1 upon binding, which showed actually the three‐state folding, but destabilized AKR1B10. In contrast, in the presence of NADP+, they destabilized AKR1A1 and stabilized AKR1B10. To explain these phenomena, we decomposed the free energy of stabilization (ΔΔG) into its enthalpy (ΔΔH) and entropy (ΔΔS) components. Then we found that in a relatively unstable protein showing the three‐state folding, native conformation was stabilized by the negative ΔΔH in association with the negative ΔΔS, suggesting that the stabilizer chaperon decreases the conformational fluctuation of the target protein or increase its hydration. However, in other cases, ΔΔG was essentially determined by the delicate balance between ΔΔH and ΔΔS. The proposed thermodynamic formalism is applicable to the system including multiple ligands with allosteric interactions. These findings would promote the development of screening strategies for MCs to regulate the target conformations. PMID:27595938

  13. Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA

    PubMed Central

    Wang, Ping; Eshaq, Randa S.; Meshul, Charles K.; Moore, Cynthia; Hood, Rebecca L.; Leidenheimer, Nancy J.

    2015-01-01

    GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors. PMID

  14. Inhibition of HSP70 and a Collagen-Specific Molecular Chaperone (HSP47) Expression in Rat Osteoblasts by Microgravity

    NASA Technical Reports Server (NTRS)

    Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Kei'ichi; Akiyama, Hideo; Hirano, Masahiko; Sams, Clarence F.; Whitson, Peggy A.

    2003-01-01

    Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

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

    PubMed

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

    2010-08-06

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

  16. More than one way to control hair growth: regulatory mechanisms in enterobacteria that affect fimbriae assembled by the chaperone/usher pathway.

    PubMed

    Clegg, Steven; Wilson, Janet; Johnson, Jeremiah

    2011-05-01

    Many gram-negative enterobacteria produce surface-associated fimbriae that facilitate attachment and adherence to eucaryotic cells and tissues. These organelles are believed to play an important role during infection by enabling bacteria to colonize specific niches within their hosts. One class of these fimbriae is assembled using a periplasmic chaperone and membrane-associated scaffolding protein that has been referred to as an usher because of its function in fimbrial biogenesis. The presence of multiple types of fimbriae assembled by the chaperone/usher pathway can be found both within a single bacterial species and also among different genera. One way of controlling fimbrial assembly in these bacteria is at the genetic level by positively or negatively regulating fimbrial gene expression. This minireview considers the mechanisms that have been described to control fimbrial gene expression and uses specific examples to demonstrate both unique and shared properties of such regulatory mechanisms.

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

    PubMed Central

    Bouthier-de-la-Tour, Claire; Coureux, Pierre-Damien; Ithurbide, Solenne; Vannier, Françoise; Guerin, Philippe P.; Dulberger, Charles L.; Satyshur, Kenneth A.; Keck, James L.; Armengaud, Jean; Cox, Michael M.; Sommer, Suzanne

    2013-01-01

    The bacterium Deinococcus radiodurans exhibits an extreme resistance to ionizing radiation. A small subset of Deinococcus genus-specific genes were shown to be up-regulated upon exposure to ionizing radiation and to play a role in genome reconstitution. These genes include an SSB-like protein called DdrB. Here, we identified a novel protein encoded by the dr1245 gene as an interacting partner of DdrB. A strain devoid of the DR1245 protein is impaired in growth, exhibiting a generation time approximately threefold that of the wild type strain while radioresistance is not affected. We determined the three-dimensional structure of DR1245, revealing a relationship with type III secretion system chaperones and YbjN family proteins. Thus, DR1245 may display some chaperone activity towards DdrB and possibly other substrates. PMID:23441204

  18. Chaperone Hsp47 Drives Malignant Growth and Invasion by Modulating an ECM Gene Network.

    PubMed

    Zhu, Jieqing; Xiong, Gaofeng; Fu, Hanjiang; Evers, B Mark; Zhou, Binhua P; Xu, Ren

    2015-04-15

    The extracellular matrix (ECM) is a determining factor in the tumor microenvironment that restrains or promotes malignant growth. In this report, we show how the molecular chaperone protein Hsp47 functions as a nodal hub in regulating an ECM gene transcription network. A transcription network analysis showed that Hsp47 expression was activated during breast cancer development and progression. Hsp47 silencing reprogrammed human breast cancer cells to form growth-arrested and/or noninvasive structures in 3D cultures, and to limit tumor growth in xenograft assays by reducing deposition of collagen and fibronectin. Coexpression network analysis also showed that levels of microRNA(miR)-29b and -29c were inversely correlated with expression of Hsp47 and ECM network genes in human breast cancer tissues. We found that miR-29 repressed expression of Hsp47 along with multiple ECM network genes. Ectopic expression of miR-29b suppressed malignant phenotypes of breast cancer cells in 3D culture. Clinically, increased expression of Hsp47 and reduced levels of miR-29b and -29c were associated with poor survival outcomes in breast cancer patients. Our results show that Hsp47 is regulated by miR-29 during breast cancer development and progression, and that increased Hsp47 expression promotes cancer progression in part by enhancing deposition of ECM proteins.

  19. Crystal structure of the nucleotide-binding domain of mortalin, the mitochondrial Hsp70 chaperone.

    PubMed

    Amick, Joseph; Schlanger, Simon E; Wachnowsky, Christine; Moseng, Mitchell A; Emerson, Corey C; Dare, Michelle; Luo, Wen-I; Ithychanda, Sujay S; Nix, Jay C; Cowan, J A; Page, Richard C; Misra, Saurav

    2014-06-01

    Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077. Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and a substrate-binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide-binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease-associated mutation is located on the Mortalin-NBD surface and may contribute to Mortalin aggregation. We present structure-based models for how the Mortalin-NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT-077. Our structure may contribute to the understanding of disease-associated Mortalin mutations and to improved Mortalin-targeting antitumor compounds.

  20. Transcellular chaperone signaling: an organismal strategy for integrated cell stress responses

    PubMed Central

    van Oosten-Hawle, Patricija; Morimoto, Richard I.

    2014-01-01

    The ability of each cell within a metazoan to adapt to and survive environmental and physiological stress requires cellular stress-response mechanisms, such as the heat shock response (HSR). Recent advances reveal that cellular proteostasis and stress responses in metazoans are regulated by multiple layers of intercellular communication. This ensures that an imbalance of proteostasis that occurs within any single tissue ‘at risk’ is protected by a compensatory activation of a stress response in adjacent tissues that confers a community protective response. While each cell expresses the machinery for heat shock (HS) gene expression, the HSR is regulated cell non-autonomously in multicellular organisms, by neuronal signaling to the somatic tissues, and by transcellular chaperone signaling between somatic tissues and from somatic tissues to neurons. These cell non-autonomous processes ensure that the organismal HSR is orchestrated across multiple tissues and that transmission of stress signals between tissues can also override the neuronal control to reset cell- and tissue-specific proteostasis. Here, we discuss emerging concepts and insights into the complex cell non-autonomous mechanisms that control stress responses in metazoans and highlight the importance of intercellular communication for proteostasis maintenance in multicellular organisms. PMID:24353212

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

    PubMed Central

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

    2016-01-01

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

  2. The Hsc66-Hsc20 Chaperone System in Escherichia coli: Chaperone Activity and Interactions with the DnaK-DnaJ-GrpE System

    PubMed Central

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

    1998-01-01

    Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in Escherichia coli. Because endogenous substrates for the Hsc66-Hsc20 system have not yet been identified, we investigated chaperone-like activities of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and luciferase. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hsp70-type chaperones. Differences in the activities of Hsc66 and DnaK, however, suggest that these chaperones have dissimilar substrate specificity profiles. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone proteins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did not stimulate the ATPase activity of Hsc66 and thus appears to function specifically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together these results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular functions. PMID:9852006

  3. Mitochondrial metabolic regulation by GRP78.

    PubMed

    Prasad, Manoj; Pawlak, Kevin J; Burak, William E; Perry, Elizabeth E; Marshall, Brendan; Whittal, Randy M; Bose, Himangshu S

    2017-02-01

    Steroids, essential for mammalian survival, are initiated by cholesterol transport by steroidogenic acute regulatory protein (StAR). Appropriate protein folding is an essential requirement of activity. Endoplasmic reticulum (ER) chaperones assist in folding of cytoplasmic proteins, whereas mitochondrial chaperones fold only mitochondrial proteins. We show that glucose regulatory protein 78 (GRP78), a master ER chaperone, is also present at the mitochondria-associated ER membrane (MAM), where it folds StAR for delivery to the outer mitochondrial membrane. StAR expression and activity are drastically reduced following GRP78 knockdown. StAR folding starts at the MAM region; thus, its cholesterol fostering capacity is regulated by GRP78 long before StAR reaches the mitochondria. In summary, GRP78 is an acute regulator of steroidogenesis at the MAM, regulating the intermediate folding of StAR that is crucial for its activity.

  4. Mitochondrial metabolic regulation by GRP78

    PubMed Central

    Prasad, Manoj; Pawlak, Kevin J.; Burak, William E.; Perry, Elizabeth E.; Marshall, Brendan; Whittal, Randy M.; Bose, Himangshu S.

    2017-01-01

    Steroids, essential for mammalian survival, are initiated by cholesterol transport by steroidogenic acute regulatory protein (StAR). Appropriate protein folding is an essential requirement of activity. Endoplasmic reticulum (ER) chaperones assist in folding of cytoplasmic proteins, whereas mitochondrial chaperones fold only mitochondrial proteins. We show that glucose regulatory protein 78 (GRP78), a master ER chaperone, is also present at the mitochondria-associated ER membrane (MAM), where it folds StAR for delivery to the outer mitochondrial membrane. StAR expression and activity are drastically reduced following GRP78 knockdown. StAR folding starts at the MAM region; thus, its cholesterol fostering capacity is regulated by GRP78 long before StAR reaches the mitochondria. In summary, GRP78 is an acute regulator of steroidogenesis at the MAM, regulating the intermediate folding of StAR that is crucial for its activity. PMID:28275724

  5. M1 of Murine Gamma-Herpesvirus 68 Induces Endoplasmic Reticulum Chaperone Production

    PubMed Central

    Feng, Jiaying; Gong, Danyang; Fu, Xudong; Wu, Ting-ting; Wang, Jane; Chang, Jennifer; Zhou, Jingting; Lu, Gang; Wang, Yibin; Sun, Ren

    2015-01-01

    Viruses rely on host chaperone network to support their infection. In particular, the endoplasmic reticulum (ER) resident chaperones play key roles in synthesizing and processing viral proteins. Influx of a large amount of foreign proteins exhausts the folding capacity in ER and triggers the unfolded protein response (UPR). A fully-executed UPR comprises signaling pathways that induce ER folding chaperones, increase protein degradation, block new protein synthesis and may eventually activate apoptosis, presenting both opportunities and threats to the virus. Here, we define a role of the MHV-68M1 gene in differential modulation of UPR pathways to enhance ER chaperone production. Ectopic expression of M1 markedly induces ER chaperone genes and expansion of ER. The M1 protein accumulates in ER during infection and this localization is indispensable for its function, suggesting M1 acts from the ER. We found that M1 protein selectively induces the chaperon-producing pathways (IRE1, ATF6) while, interestingly, sparing the translation-blocking arm (PERK). We identified, for the first time, a viral factor capable of selectively intervening the initiation of ER stress signaling to induce chaperon production. This finding provides a unique opportunity of using viral protein as a tool to define the activation mechanisms of individual UPR pathways. PMID:26615759

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

    PubMed Central

    Kinoshita, Miki; Yamane, Midori; Matsunami, Hideyuki; Minamino, Tohru; Namba, Keiichi; Imada, Katsumi

    2009-01-01

    The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD4C2 complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3121 or P3221 and diffracted to 3.2 Å resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination. PMID:19652350

  7. Flagellin Polymerisation Control by a Cytosolic Export Chaperone

    PubMed Central

    Auvray, Frédéric; Thomas, Joanne; Fraser, Gillian M.; Hughes, Colin

    2008-01-01

    Assembly of the long helical filament of the bacterial flagellum requires polymerisation of ca 20,000 flagellin (FliC) monomeric subunits into the growing structure extending from the cell surface. Here, we show that export of Salmonella flagellin is facilitated specifically by a cytosolic protein, FliS, and that FliS binds to the FliC C-terminal helical domain, which contributes to stabilisation of flagellin subunit interactions during polymerisation. Stable complexes of FliS with flagellin were assembled efficiently in vitro, apparently by FliS homodimers binding to FliC monomers. The data suggest that FliS acts as a substrate-specific chaperone, preventing premature interaction of newly synthesised flagellin subunits in the cytosol. Compatible with this view, FliS was able to prevent in vitro polymerisation of FliC into filaments. PMID:11327763

  8. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

    PubMed Central

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio

    2013-01-01

    Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999

  9. Epiplakin attenuates experimental mouse liver injury by chaperoning keratin reorganization

    PubMed Central

    Szabo, Sandra; Wögenstein, Karl L.; Österreicher, Christoph H.; Guldiken, Nurdan; Chen, Yu; Doler, Carina; Wiche, Gerhard; Boor, Peter; Haybaeck, Johannes; Strnad, Pavel; Fuchs, Peter

    2015-01-01

    Background & Aims Epiplakin is a member of the plakin protein family and exclusively expressed in epithelial tissues where it binds to keratins. Epiplakin-deficient (Eppk1−/−) mice displayed no obvious spontaneous phenotype, but their keratinocytes showed a faster keratin network breakdown in response to stress. The role of epiplakin in the stressed liver remained to be elucidated. Methods Wild-type (WT) and Eppk1−/− mice were subjected to common bile duct ligation (CBDL) or fed with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet. The importance of epiplakin during keratin reorganization was assessed in primary hepatocytes. Results Our experiments revealed that epiplakin is expressed in hepatocytes and cholangiocytes, and binds to keratin 8 (K8) and K18 via multiple domains. In several liver stress models epiplakin and K8 genes displayed identical expression patterns and transgenic K8 overexpression resulted in elevated hepatic epiplakin levels. After CBDL and DDC treatment, Eppk1−/− mice developed a more pronounced liver injury and their livers contained larger amounts of hepatocellular keratin granules, indicating impaired disease-induced keratin network reorganization. In line with these findings, primary Eppk1−/− hepatocytes showed increased formation of keratin aggregates after treatment with the phosphatase inhibitor okadaic acid, a phenotype which was rescued by the chemical chaperone trimethylamine N-oxide (TMAO). Finally, transfection experiments revealed that Eppk1−/− primary hepatocytes were less able to tolerate forced K8 overexpression and that TMAO treatment rescued this phenotype. Conclusion Our data indicate that epiplakin plays a protective role during experimental liver injuries by chaperoning disease-induced keratin reorganization. PMID:25617501

  10. Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide

    PubMed Central

    Macri, Christophe; Wang, Fengjuan; Tasset, Inmaculada; Schall, Nicolas; Page, Nicolas; Briand, Jean-Paul; Cuervo, Ana Maria; Muller, Sylviane

    2015-01-01

    The P140 peptide, a 21-mer linear peptide (sequence 131–151) generated from the spliceosomal SNRNP70/U1–70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired. PMID:25719862

  11. Antarctic Krill 454 Pyrosequencing Reveals Chaperone and Stress Transcriptome

    PubMed Central

    Clark, Melody S.; Thorne, Michael A. S.; Toullec, Jean-Yves; Meng, Yan; Guan, Le Luo; Peck, Lloyd S.; Moore, Stephen

    2011-01-01

    Background The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models. Methodology/Principal Findings The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp). In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS), providing a resource for population and also gene function studies. Conclusions This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical “stress proteins”, such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of “stress” genes for studies understanding marine

  12. Histone chaperone ASF1B promotes human β-cell proliferation via recruitment of histone H3.3.

    PubMed

    Paul, Pradyut K; Rabaglia, Mary E; Wang, Chen-Yu; Stapleton, Donald S; Leng, Ning; Kendziorski, Christina; Lewis, Peter W; Keller, Mark P; Attie, Alan D

    2016-12-01

    Anti-silencing function 1 (ASF1) is a histone H3-H4 chaperone involved in DNA replication and repair, and transcriptional regulation. Here, we identify ASF1B, the mammalian paralog to ASF1, as a proliferation-inducing histone chaperone in human β-cells. Overexpression of ASF1B led to distinct transcriptional signatures consistent with increased cellular proliferation and reduced cellular death. Using multiple methods of monitoring proliferation and mitotic progression, we show that overexpression of ASF1B is sufficient to induce human β-cell proliferation. Co-expression of histone H3.3 further augmented β-cell proliferation, whereas suppression of endogenous H3.3 attenuated the stimulatory effect of ASF1B. Using the histone binding-deficient mutant of ASF1B (V94R), we show that histone binding to ASF1B is required for the induction of β-cell proliferation. In contrast to H3.3, overexpression of histone H3 variants H3.1 and H3.2 did not have an impact on ASF1B-mediated induction of proliferation. Our findings reveal a novel role of ASF1B in human β-cell replication and show that ASF1B and histone H3.3A synergistically stimulate human β-cell proliferation.

  13. Adenosine diphosphate restricts the protein remodeling activity of the Hsp104 chaperone to Hsp70 assisted disaggregation

    PubMed Central

    Kłosowska, Agnieszka; Chamera, Tomasz; Liberek, Krzysztof

    2016-01-01

    Hsp104 disaggregase provides thermotolerance in yeast by recovering proteins from aggregates in cooperation with the Hsp70 chaperone. Protein disaggregation involves polypeptide extraction from aggregates and its translocation through the central channel of the Hsp104 hexamer. This process relies on adenosine triphosphate (ATP) hydrolysis. Considering that Hsp104 is characterized by low affinity towards ATP and is strongly inhibited by adenosine diphosphate (ADP), we asked how Hsp104 functions at the physiological levels of adenine nucleotides. We demonstrate that physiological levels of ADP highly limit Hsp104 activity. This inhibition, however, is moderated by the Hsp70 chaperone, which allows efficient disaggregation by supporting Hsp104 binding to aggregates but not to non-aggregated, disordered protein substrates. Our results point to an additional level of Hsp104 regulation by Hsp70, which restricts the potentially toxic protein unfolding activity of Hsp104 to the disaggregation process, providing the yeast protein-recovery system with substrate specificity and efficiency in ATP consumption. DOI: http://dx.doi.org/10.7554/eLife.15159.001 PMID:27223323

  14. Hsc70 chaperone activity underlies Trio GEF function in axon growth and guidance induced by netrin-1

    PubMed Central

    DeGeer, Jonathan; Kaplan, Andrew; Mattar, Pierre; Morabito, Morgane; Stochaj, Ursula; Kennedy, Timothy E.; Debant, Anne; Cayouette, Michel; Fournier, Alyson E.

    2015-01-01

    During development, netrin-1 is both an attractive and repulsive axon guidance cue and mediates its attractive function through the receptor Deleted in Colorectal Cancer (DCC). The activation of Rho guanosine triphosphatases within the extending growth cone facilitates the dynamic reorganization of the cytoskeleton required to drive axon extension. The Rac1 guanine nucleotide exchange factor (GEF) Trio is essential for netrin-1–induced axon outgrowth and guidance. Here, we identify the molecular chaperone heat shock cognate protein 70 (Hsc70) as a novel Trio regulator. Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio. Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase–deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1–induced Rac1 activation. Hsc70 was required for netrin-1–mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex. These findings demonstrate that Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance. PMID:26323693

  15. Hsp70 chaperone rescues C6 rat glioblastoma cells from oxidative stress by sequestration of aggregating GAPDH.

    PubMed

    Lazarev, Vladimir F; Nikotina, Alina D; Mikhaylova, Elena R; Nudler, Evgeny; Polonik, Sergey G; Guzhova, Irina V; Margulis, Boris A

    2016-02-12

    The Hsp70 chaperone is known to elicit cytoprotective activity and this protection has a negative impact in anti-tumor therapy. In cancer cells subjected to oxidative stress Hsp70 may bind damaged polypeptides and proteins involved in apoptosis signaling. Since one of the important targets of oxidative stress is glyceraldehyde-3-phospate dehydrogenase (GAPDH) we suggested that Hsp70 might elicit its protective effect by binding GAPDH. Microscopy data show that in C6 rat glioma cells subjected to hydrogen peroxide treatment a considerable proportion of the GAPDH molecules are denatured and according to dot ultrafiltration data they form SDS-insoluble aggregates. Using two newly developed assays we show that Hsp70 can bind oxidized GAPDH in an ATP-dependent manner. Pharmacological up- or down-regulation of Hsp70 with the aid of U133 echinochrome or triptolide, respectively, reduced or increased the number of C6 glioma cells containing GAPDH aggregates and dying due to treatment with hydrogen peroxide. Using immunoprecipitation we found that Hsp70 is able to sequester aggregation-prone GAPDH and this may explain the anti-oxidative power of the chaperone. The results of this study led us to conclude that in cancer cells constantly exposed to conditions of oxidative stress, the protective power of Hsp70 should be abolished by specific inhibitors of Hsp70 expression.

  16. The Influence of Chemical Chaperones on Enzymatic Activity under Thermal and Chemical Stresses: Common Features and Variation among Diverse Chemical Families

    PubMed Central

    Feibish, Nir; Sharon, Noa; Schnaider, Lee; Shmul, Guy; Amir, Yaniv; Buzhansky, Ludmila; Gazit, Ehud

    2014-01-01

    Molecular and chemical chaperones are key components of the two main mechanisms that ensure structural stability and activity under environmental stresses. Yet, chemical chaperones are often regarded only as osmolytes and their role beyond osmotic regulation is not fully understood. Here, we systematically studied a large group of chemical chaperones, representatives of diverse chemical families, for their protective influence under either thermal or chemical stresses. Consistent with previous studies, we observed that in spite of the structural similarity between sugars and sugar alcohols, they have an apparent difference in their protective potential. Our results support the notion that the protective activity is mediated by the solvent and the presence of water is essential. In the current work we revealed that i) polyols and sugars have a completely different profile of protective activity toward trifluoroethanol and thermal stress; ii) minor changes in solvent composition that do not affect enzyme activity, yet have a great effect on the ability of osmolytes to act as protectants and iii) increasing the number of active groups of carbohydrates makes them better protectants while increasing the number of active groups of methylamines does not, as revealed by attempts to synthesize de novo designed methylamines with multiple functional groups. PMID:24520396

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

    PubMed Central

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

    2016-01-01

    Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from 1H-15N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell. PMID:26673727

  18. The influence of chemical chaperones on enzymatic activity under thermal and chemical stresses: common features and variation among diverse chemical families.

    PubMed

    Levy-Sakin, Michal; Berger, Or; Feibish, Nir; Sharon, Noa; Schnaider, Lee; Shmul, Guy; Amir, Yaniv; Buzhansky, Ludmila; Gazit, Ehud

    2014-01-01

    Molecular and chemical chaperones are key components of the two main mechanisms that ensure structural stability and activity under environmental stresses. Yet, chemical chaperones are often regarded only as osmolytes and their role beyond osmotic regulation is not fully understood. Here, we systematically studied a large group of chemical chaperones, representatives of diverse chemical families, for their protective influence under either thermal or chemical stresses. Consistent with previous studies, we observed that in spite of the structural similarity between sugars and sugar alcohols, they have an apparent difference in their protective potential. Our results support the notion that the protective activity is mediated by the solvent and the presence of water is essential. In the current work we revealed that i) polyols and sugars have a completely different profile of protective activity toward trifluoroethanol and thermal stress; ii) minor changes in solvent composition that do not affect enzyme activity, yet have a great effect on the ability of osmolytes to act as protectants and iii) increasing the number of active groups of carbohydrates makes them better protectants while increasing the number of active groups of methylamines does not, as revealed by attempts to synthesize de novo designed methylamines with multiple functional groups.

  19. Universal Stress Protein Exhibits a Redox-Dependent Chaperone Function in Arabidopsis and Enhances Plant Tolerance to Heat Shock and Oxidative Stress

    PubMed Central

    Jung, Young Jun; Melencion, Sarah Mae Boyles; Lee, Eun Seon; Park, Joung Hun; Alinapon, Cresilda Vergara; Oh, Hun Taek; Yun, Dae-Jin; Chi, Yong Hun; Lee, Sang Yeol

    2015-01-01

    Although a wide range of physiological information on Universal Stress Proteins (USPs) is available from many organisms, their biochemical, and molecular functions remain unidentified. The biochemical function of AtUSP (At3g53990) from Arabidopsis thaliana was therefore investigated. Plants over-expressing AtUSP showed a strong resistance to heat shock and oxidative stress, compared with wild-type and Atusp knock-out plants, confirming the crucial role of AtUSP in stress tolerance. AtUSP was present in a variety of structures including monomers, dimers, trimers, and oligomeric complexes, and switched in response to external stresses from low molecular weight (LMW) species to high molecular weight (HMW) complexes. AtUSP exhibited a strong chaperone function under stress conditions in particular, and this activity was significantly increased by heat treatment. Chaperone activity of AtUSP was critically regulated by the redox status of cells and accompanied by structural changes to the protein. Over-expression of AtUSP conferred a strong tolerance to heat shock and oxidative stress upon Arabidopsis, primarily via its chaperone function. PMID:26734042

  20. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    DOE PAGES

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; ...

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  1. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    SciTech Connect

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; Tainer, John A.

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  2. A Common Structural Motif in the Binding of Virulence Factors to Bacterial Secretion Chaperones

    SciTech Connect

    Lilic,M.; Vujanac, M.; Stebbins, C.

    2006-01-01

    Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

  3. Histone chaperones FACT and Spt6 prevent histone variants from turning into histone deviants.

    PubMed

    Jeronimo, Célia; Robert, François

    2016-05-01

    Histone variants are specialized histones which replace their canonical counterparts in specific nucleosomes. Together with histone post-translational modifications and DNA methylation, they contribute to the epigenome. Histone variants are incorporated at specific locations by the concerted action of histone chaperones and ATP-dependent chromatin remodelers. Recent studies have shown that the histone chaperone FACT plays key roles in preventing pervasive incorporation of two histone variants: H2A.Z and CenH3/CENP-A. In addition, Spt6, another histone chaperone, was also shown to be important for appropriate H2A.Z localization. FACT and Spt6 are both associated with elongating RNA polymerase II. Based on these two examples, we propose that the establishment and maintenance of histone variant genomic distributions depend on a transcription-coupled epigenome editing (or surveillance) function of histone chaperones.

  4. Evolution of the Chaperone/Usher Assembly Pathway: Fimbrial Classification Goes Greek†

    PubMed Central

    Nuccio, Sean-Paul; Bäumler, Andreas J.

    2007-01-01

    Summary: Many Proteobacteria use the chaperone/usher pathway to assemble proteinaceous filaments on the bacterial surface. These filaments can curl into fimbrial or nonfimbrial surface structures (e.g., a capsule or spore coat). This article reviews the phylogeny of operons belonging to the chaperone/usher assembly class to explore the utility of establishing a scheme for subdividing them into clades of phylogenetically related gene clusters. Based on usher amino acid sequence comparisons, our analysis shows that the chaperone/usher assembly class is subdivided into six major phylogenetic clades, which we have termed α-, β-, γ-, κ-, π-, and σ-fimbriae. Members of each clade share related operon structures and encode fimbrial subunits with similar protein domains. The proposed classification system offers a simple and convenient method for assigning newly discovered chaperone/usher systems to one of the six major phylogenetic groups. PMID:18063717

  5. Cooperative Subunit Refolding of a Light-Harvesting Protein through a Self-Chaperone Mechanism.

    PubMed

    Laos, Alistair J; Dean, Jacob C; Toa, Zi S D; Wilk, Krystyna E; Scholes, Gregory D; Curmi, Paul M G; Thordarson, Pall

    2017-01-27

    The fold of a protein is encoded by its amino acid sequence, but how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear. Here we show that two structurally different phycobiliproteins refold and reassemble in a cooperative manner from their unfolded polypeptide subunits, without biological chaperones. Refolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopy to probe internal chromophores as a marker of quaternary structure. Our results demonstrate a cooperative, self-chaperone refolding mechanism, whereby the β-subunits independently refold, thereby templating the folding of the α-subunits, which then chaperone the assembly of the native complex, quantitatively returning all coherences. Our results indicate that subunit self-chaperoning is a robust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems.

  6. FGL chaperone-assembled fimbrial polyadhesins: anti-immune armament of Gram-negative bacterial pathogens.

    PubMed

    Zavialov, Anton; Zav'yalova, Galina; Korpela, Timo; Zav'yalov, Vladimir

    2007-07-01

    This review summarizes the current knowledge on the structure, function, assembly, and biomedical applications of the family of adhesive fimbrial organelles assembled on the surface of Gram-negative pathogens via the FGL chaperone/usher pathway. Recent studies revealed the unique structural and functional properties of these organelles, distinguishing them from a related family, FGS chaperone-assembled adhesive pili. The FGL chaperone-assembled organelles consist of linear polymers of one or two types of protein subunits, each possessing one or two independent adhesive sites specific to different host cell receptors. This structural organization enables these fimbrial organelles to function as polyadhesins. Fimbrial polyadhesins may ensure polyvalent fastening of bacteria to the host cells, aggregating their receptors and triggering subversive signals that allow pathogens to evade immune defense. The FGL chaperone-assembled fimbrial polyadhesins are attractive targets for vaccine and drug design.

  7. Dissecting functional similarities of ribosome-associated chaperones from Saccharomyces cerevisiae and Escherichia coli.

    PubMed

    Rauch, Thomas; Hundley, Heather A; Pfund, Chris; Wegrzyn, Renee D; Walter, William; Kramer, Günter; Kim, So-Young; Craig, Elizabeth A; Deuerling, Elke

    2005-07-01

    Ribosome-tethered chaperones that interact with nascent polypeptide chains have been identified in both prokaryotic and eukaryotic systems. However, these ribosome-associated chaperones share no sequence similarity: bacterial trigger factors (TF) form an independent protein family while the yeast machinery is Hsp70-based. The absence of any component of the yeast machinery results in slow growth at low temperatures and sensitivity to aminoglycoside protein synthesis inhibitors. After establishing that yeast ribosomal protein Rpl25 is able to recruit TF to ribosomes when expressed in place of its Escherichia coli homologue L23, the ribosomal TF tether, we tested whether such divergent ribosome-associated chaperones are functionally interchangeable. E. coli TF was expressed in yeast cells that lacked the endogenous ribosome-bound machinery. TF associated with yeast ribosomes, cross-linked to yeast nascent polypeptides and partially complemented the aminoglycoside sensitivity, demonstrating that ribosome-associated chaperones from divergent organisms share common functions, despite their lack of sequence similarity.

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

    DTIC Science & Technology

    2012-02-01

    Award Number: W81XWH-06-1-0285 TITLE: The Role of the Co-Chaperone, CHIP, in Androgen-Independent Prostate Cancer ...AND SUBTITLE 5a. CONTRACT NUMBER The Role of the Co-Chaperone, CHIP, in Androgen Independent Prostate Cancer 5b. GRANT NUMBER W81XWH-06-1...ADT), is the mainstay of treatment for patients with locally advanced or metastatic prostate cancer . This therapy is only temporizing, however

  9. Ectopic Expression of Plant RNA Chaperone Offering Multiple Stress Tolerance in E. coli.

    PubMed

    Jabeen, Bushra; Naqvi, S M Saqlan; Mahmood, Tariq; Sultana, Tasawar; Arif, Madiha; Khan, Fariha

    2017-03-01

    Members of the plant glycine-rich RNA-binding proteins (GR-RBPs) family have been reported in flowering, development, circadian rhythms, biotic and abiotic stresses. Particularly, GR-RBPs are reported to function as RNA chaperones, promoting growth and acclimation during cold shock. It is indispensable to further question the efficacy and mechanism of GR-RBPs under various environmental strains. Monitoring the expression of stress-regulated proteins under stress conditions has been a beneficial strategy to study their functional roles. In an effort to elucidate the NtGR-RBP1 function, stress markers such as salinity, drought, low temperature and heat stresses were studied. The NtGR-RBP1 gene was expressed in E. coli followed by the exposure to stress conditions. Recombinant E. coli expressing NtGR-RBP1 were more tolerant to stresses, e.g., salinity, drought, cold and heat shock. Recombinants exhibited higher growth rates compared to control in spot assays. The tolerance was further confirmed by monitoring the growth in liquid culture assays. Cells expressing NtGR-RBP1 under salt (500 mM NaCl), drought (20% PEG), cold (4 and 20 °C) and heat stresses (50 °C) had enhanced growing ability and better endurance. Our study supports the notion that the protective role of NtGR-RBP1 may contribute to growth and survival during diverse environmental stresses.

  10. Structure of the Escherichia coli ProQ RNA chaperone protein.

    PubMed

    Gonzalez, Grecia; Hardwick, Steven; Maslen, Sarah L; Skehel, J Mark; Holmqvist, Erik; Vogel, Jörg; Bateman, Alex; Luisi, Ben; Broadhurst, R William

    2017-02-13

    The protein ProQ has recently been identified as a global RNA chaperone in Salmonella, and a similar role is anticipated for its numerous homologues in divergent bacterial species. We report the solution structure of Escherichia coli ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor-domain fold commonly found in eukaryotes, and an elongated bridging intra-domain linker that is flexible but nonetheless incompressible. Structure based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA binding surfaces on all three domains of ProQ and modelled the protein's conformation in the apo and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor and linker domains of ProQ cooperate to recognise complex RNA structures and serve to promote RNA-mediated regulation.

  11. The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq.

    PubMed

    Heidrich, Nadja; Bauriedl, Saskia; Barquist, Lars; Li, Lei; Schoen, Christoph; Vogel, Jörg

    2017-03-17

    Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of -35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.

  12. Silencing of natural transformation by an RNA chaperone and a multitarget small RNA

    PubMed Central

    Attaiech, Laetitia; Boughammoura, Aïda; Brochier-Armanet, Céline; Allatif, Omran; Peillard-Fiorente, Flora; Edwards, Ross A.; Omar, Ayat R.; MacMillan, Andrew M.; Glover, Mark; Charpentier, Xavier

    2016-01-01

    A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogen Legionella pneumophila. We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterized trans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assist trans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species. PMID:27432973

  13. Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide.

    PubMed

    Grahl, Sabine; Maillard, Julien; Spronk, Chris A E M; Vuister, Geerten W; Sargent, Frank

    2012-03-01

    The twin-arginine translocation (Tat) pathway is a protein targeting system present in many prokaryotes. The physiological role of the Tat pathway is the transmembrane translocation of fully-folded proteins, which are targeted by N-terminal signal peptides bearing conserved SRRxFLK 'twin-arginine' amino acid motifs. In Escherichia coli the majority of Tat targeted proteins bind redox cofactors and it is important that only mature, cofactor-loaded precursors are presented for export. Cellular processes have been unearthed that sequence these events, for example the signal peptide of the periplasmic nitrate reductase (NapA) is bound by a cytoplasmic chaperone (NapD) that is thought to regulate assembly and export of the enzyme. In this work, genetic, biophysical and structural approaches were taken to dissect the interaction between NapD and the NapA signal peptide. A NapD binding epitope was identified towards the N-terminus of the signal peptide, which overlapped significantly with the twin-arginine targeting motif. NMR spectroscopy revealed that the signal peptide adopted a α-helical conformation when bound by NapD, and substitution of single residues within the NapA signal peptide was sufficient to disrupt the interaction. This work provides an increased level of understanding of signal peptide function on the bacterial Tat pathway.

  14. Cold shock protein 1 chaperones mRNAs during translation in Arabidopsis thaliana.

    PubMed

    Juntawong, Piyada; Sorenson, Reed; Bailey-Serres, Julia

    2013-06-01

    RNA binding proteins (RBPs) function post-transcriptionally to fine-tune gene regulation. Arabidopsis thaliana has four Gly-rich, zinc finger-containing RBPs called cold shock proteins 1-4 (CSP1-CSP4), that possess an evolutionary conserved cold shock domain. Here, we determined that CSP1 associates with polyribosomes (polysomes) via an RNA-mediated interaction. Both the abundance and polysomal co-fractionation of CSP1 was enhanced in the cold (4°C), but did not influence global levels of polysomes, which were minimally perturbed by above freezing cold temperatures. Using a polyclonal antiserum, CSP1 was co-immunopurified with several hundred transcripts from rosettes of plants cultivated at 23°C or transferred to 4°C for 12 h. CSP1-associated mRNAs were characterized by G+C-rich 5' untranslated regions and gene ontologies related to cellular respiration, mRNA binding and translation. The majority of the CSP1-associated mRNAs were constitutively expressed and stable in the cold. CSP1 abundance was correlated with improved translation of ribosomal protein mRNAs during cold stress and improved maintenance of homeostasis and translation of mRNAs under water-deficit stress. In summary, CSP1 selectively chaperones mRNAs, providing translational enhancement during stress.

  15. Automodification switches PARP-1 function from chromatin architectural protein to histone chaperone.

    PubMed

    Muthurajan, Uma M; Hepler, Maggie R D; Hieb, Aaron R; Clark, Nicholas J; Kramer, Michael; Yao, Tingting; Luger, Karolin

    2014-09-02

    Poly [ADP-ribose] polymerase 1 (PARP-1) is a highly abundant chromatin-associated enzyme. It catalyzes the NAD(+)-dependent polymerization of long chains of poly-ADP ribose (PAR) onto itself in response to DNA damage and other cues. More recently, the enzymatic activity of PARP-1 has also been implicated in the regulation of gene expression. The molecular basis for the functional switch from chromatin architectural protein to transcription factor and DNA damage responder, triggered by PARP-1 automodification, is unknown. Here, we show that unmodified PARP-1 engages in at least two high-affinity binding modes with chromatin, one of which does not involve free DNA ends, consistent with its role as a chromatin architectural protein. Automodification reduces PARP-1 affinity for intact chromatin but not for nucleosomes with exposed DNA ends. Automodified (AM) PARP-1 has the ability to sequester histones (both in vitro and in cells) and to assemble nucleosomes efficiently in vitro. This unanticipated nucleosome assembly activity of AM-PARP-1, coupled with the fast turnover of the modification, suggests a model in which DNA damage or transcription events trigger transient histone chaperone activity.

  16. Substrate-Activated Conformational Switch on Chaperones Encodes aTargeting Signal in Type III Secretion

    PubMed Central

    Chen, Li; Ai, Xuanjun; Portaliou, Athina G.; Minetti, Conceicao A.S.A.; Remeta, David P.; Economou, Anastassios; Kalodimos, Charalampos G.

    2013-01-01

    SUMMARY Targeting of type III secretion proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone CesAB to the EscN ATPase of the enteropathogenic E. coli (EPEC). We show that EscN selectively engages the EspA-loaded CesAB, but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon binding of its physiological substrate, EspA. Abrogation of the interaction between the CesAB–EspA complex and EscN resulted in severe secretion and infection defects. We further show that the targeting and secretion signals are distinct and the two processes are likely regulated by different mechanisms. PMID:23523349

  17. Gigaxonin controls vimentin organization through a tubulin chaperone-independent pathway

    PubMed Central

    Cleveland, Don W.; Yamanaka, Koji; Bomont, Pascale

    2009-01-01

    Gigaxonin mutations cause the fatal human neurodegenerative disorder giant axonal neuropathy (GAN). Broad deterioration of the nervous system in GAN patients is accompanied by massive disorganization of intermediate filaments (IFs) both in neurons and many non-neuronal cells. With newly developed antibodies, gigaxonin is now shown to be expressed at extremely low levels throughout the nervous system. In lymphoblast cell lines derived from severe and mild forms of GAN, mutations in gigaxonin are shown to yield highly unstable proteins, thereby permitting a rapid diagnostic test for the spectrum of GAN mutations as an alternative to invasive nerve biopsy or systematic sequencing of the GAN gene. Gigaxonin has been proposed as a substrate adaptor for an E3 ubiquitin ligase, which affects proteasome-dependent degradation of microtubule-related proteins including MAP1B, MAP8 and the tubulin folding chaperone TBCB. We demonstrate that, unlike its counterpart TBCE, TBCB only moderately destabilizes microtubules. Neither TBCB abundance nor microtubule organization or densities are altered in GAN mutant fibroblasts, thus demonstrating that altered TBCB levels are not primary determinants of IF disorganization in GAN. Characteristic GAN mutant-induced ovoid aggregates of vimentin are not produced in normal fibroblasts after disrupting microtubule assembly, either by TBCE overexpression or depolymerizing drugs. Thus, IF disorganization in GAN fibroblasts is independent of TBCB and microtubule loss and must be regulated by a yet unidentified mechanism. PMID:19168853

  18. Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae.

    PubMed

    Valaviciute, Monika; Norkiene, Milda; Goda, Karolis; Slibinskas, Rimantas; Gedvilaite, Alma

    2016-07-01

    A number of viruses utilize molecular chaperones during various stages of their life cycle. It has been shown that members of the heat-shock protein 70 (Hsp70) chaperone family assist polyomavirus capsids during infection. However, the molecular chaperones that assist the formation of recombinant capsid viral protein 1 (VP1)-derived virus-like particles (VLPs) in yeast remain unclear. A panel of yeast strains with single chaperone gene deletions were used to evaluate the chaperones required for biosynthesis of recombinant hamster polyomavirus capsid protein VP1. The impact of deletion or mild overexpression of chaperone genes was determined in live cells by flow cytometry using enhanced green fluorescent protein (EGFP) fused with VP1. Targeted genetic analysis demonstrated that VP1-EGFP fusion protein levels were significantly higher in yeast strains in which the SSZ1 or ZUO1 genes encoding ribosome-associated complex components were deleted. The results confirmed the participation of cytosolic Hsp70 chaperones and suggested the potential involvement of the Ydj1 and Caj1 co-chaperones and the endoplasmic reticulum chaperones in the biosynthesis of VP1 VLPs in yeast. Likewise, the markedly reduced levels of VP1-EGFP in Δhsc82 and Δhsp82 yeast strains indicated that both Hsp70 and Hsp90 chaperones might assist VP1 VLPs during protein biosynthesis.

  19. A NAP-Family Histone Chaperone Functions in Abiotic Stress Response and Adaptation1[OPEN

    PubMed Central

    Pareek, Ashwani; Singla-Pareek, Sneh Lata

    2016-01-01

    Modulation of gene expression is one of the most significant molecular mechanisms of abiotic stress response in plants. Via altering DNA accessibility, histone chaperones affect the transcriptional competence of genomic loci. However, in contrast to other factors affecting chromatin dynamics, the role of plant histone chaperones in abiotic stress response and adaptation remains elusive. Here, we studied the physiological function of a stress-responsive putative rice (Oryza sativa) histone chaperone of the NAP superfamily: OsNAPL6. We show that OsNAPL6 is a nuclear-localized H3/H4 histone chaperone capable of assembling a nucleosome-like structure. Utilizing overexpression and knockdown approaches, we found a positive correlation between OsNAPL6 expression levels and adaptation to multiple abiotic stresses. Results of comparative transcriptome profiling and promoter-recruitment studies indicate that OsNAPL6 functions during stress response via modulation of expression of various genes involved in diverse functions. For instance, we show that OsNAPL6 is recruited to OsRad51 promoter, activating its expression and leading to more efficient DNA repair and abrogation of programmed cell death under salinity and genotoxic stress conditions. These results suggest that the histone chaperone OsNAPL6 may serve a regulatory role in abiotic stress physiology possibly via modulating nucleosome dynamics at various stress-associated genomic loci. Taken together, our findings establish a hitherto unknown link between histone chaperones and abiotic stress response in plants. PMID:27342307

  20. Heterogeneous binding of the SH3 client protein to the DnaK molecular chaperone

    PubMed Central

    Lee, Jung Ho; Zhang, Dongyu; Hughes, Christopher; Okuno, Yusuke; Sekhar, Ashok; Cavagnero, Silvia

    2015-01-01

    The molecular chaperone heat shock protein 70 (Hsp70) plays a vital role in cellular processes, including protein folding and assembly, and helps prevent aggregation under physiological and stress-related conditions. Although the structural changes undergone by full-length client proteins upon interaction with DnaK (i.e., Escherichia coli Hsp70) are fundamental to understand chaperone-mediated protein folding, these changes are still largely unexplored. Here, we show that multiple conformations of the SRC homology 3 domain (SH3) client protein interact with the ADP-bound form of the DnaK chaperone. Chaperone-bound SH3 is largely unstructured yet distinct from the unfolded state in the absence of DnaK. The bound client protein shares a highly flexible N terminus and multiple slowly interconverting conformations in different parts of the sequence. In all, there is significant structural and dynamical heterogeneity in the DnaK-bound client protein, revealing that proteins may undergo some conformational sampling while chaperone-bound. This result is important because it shows that the surface of the Hsp70 chaperone provides an aggregation-free environment able to support part of the search for the native state. PMID:26195753

  1. Amyloid-β oligomers are sequestered by both intracellular and extracellular chaperones

    PubMed Central

    Narayan, Priyanka; Meehan, Sarah; Carver, John A.; Wilson, Mark R.; Dobson, Christopher M.; Klenerman, David

    2016-01-01

    The aberrant aggregation of the amyloid-β peptide into β-sheet rich, fibrillar structures proceeds via a heterogeneous ensemble of oligomeric intermediates that have been associated with neurotoxicity in Alzheimer’s disease (AD). Of particular interest in this context are the mechanisms by which molecular chaperones, part of the primary biological defenses against protein misfolding, influence Aβ aggregation. We have used single-molecule fluorescence techniques to compare the interactions between distinct aggregation states (monomers, oligomers, amyloid fibrils) of the AD-associated amyloid-β(1-40) peptide, and two molecular chaperones, both of which are upregulated in the brains of patients with AD and have been found colocalized with Aβ in senile plaques. One of the chaperones, αB-crystallin, is primarily found inside cells while the other, clusterin, is predominantly located in the extracellular environment. We find that both chaperones bind to misfolded oligomeric species and form long-lived complexes thereby preventing both their further growth into fibrils and their dissociation. From these studies, we conclude that these chaperones have a common mechanism of action based on sequestering Aβ oligomers. This conclusion suggests that these chaperones, both of which are ATP-independent, are able to inhibit potentially pathogenic Aβ oligomer-associated processes whether they occur in the extracellular or intracellular environment. PMID:23106396

  2. Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

    PubMed Central

    Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R.; Li, Melody M. H.; Rice, Charles M.

    2016-01-01

    polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development. PMID:26739057

  3. Photoreceptor IFT complexes containing chaperones, guanylyl cyclase 1 and rhodopsin.

    PubMed

    Bhowmick, Reshma; Li, Mei; Sun, Jun; Baker, Sheila A; Insinna, Christine; Besharse, Joseph C

    2009-06-01

    Intraflagellar transport (IFT) provides a mechanism for the transport of cilium-specific proteins, but the mechanisms for linkage of cargo and IFT proteins have not been identified. Using the sensory outer segments (OS) of photoreceptors, which are derived from sensory cilia, we have identified IFT-cargo complexes containing IFT proteins, kinesin 2 family proteins, two photoreceptor-specific membrane proteins, guanylyl cyclase 1 (GC1, Gucy2e) and rhodopsin (RHO), and the chaperones, mammalian relative of DNAJ, DnajB6 (MRJ), and HSC70 (Hspa8). Analysis of these complexes leads to a model in which MRJ through its binding to IFT88 and GC1 plays a critical role in formation or stabilization of the IFT-cargo complexes. Consistent with the function of MRJ in the activation of HSC70 ATPase activity, Mg-ATP enhances the co-IP of GC1, RHO, and MRJ with IFT proteins. Furthermore, RNAi knockdown of MRJ in IMCD3 cells expressing GC1-green fluorescent protein (GFP) reduces cilium membrane targeting of GC1-GFP without apparent effect on cilium elongation.

  4. Activation of a bacterial lipase by its chaperone.

    PubMed Central

    Hobson, A H; Buckley, C M; Aamand, J L; Jørgensen, S T; Diderichsen, B; McConnell, D J

    1993-01-01

    The gene lipA of Pseudomonas cepacia DSM 3959 encodes a prelipase from which a signal peptide is cleaved during secretion, producing a mature extracellular lipase. Expression of lipase in several heterologous hosts depends on the presence of another gene, limA, in cis or in trans. Lipase protein has been overproduced in Escherichia coli in the presence and absence of the lipase modulator gene limA. Therefore, limA is not required for the transcription of lipA or for the translation of the lipA mRNA. However, no lipase activity is observed in the absence of limA. limA has been overexpressed and encodes a 33-kDa protein, Lim. If lipase protein is denatured in 8 M urea and the urea is removed by dialysis, lipase activity is quantitatively recovered provided Lim protein is present during renaturation. Lip and Lim proteins form a complex precipitable either by an anti-lipase or anti-Lim antibody. The Lim protein has therefore the properties of a chaperone. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7685908

  5. A Novel Function of Molecular Chaperone HSP70

    PubMed Central

    Halasi, Marianna; Váraljai, Renáta; Benevolenskaya, Elizaveta; Gartel, Andrei L.

    2016-01-01

    The oncogenic transcription factor FOXM1 is overexpressed in the majority of human cancers, and it is a potential target for anticancer therapy. We identified proteasome inhibitors as the first type of drugs that target FOXM1 in cancer cells. Here we found that HSP90 inhibitor PF-4942847 and heat shock also suppress FOXM1. The common effector, which was induced after treatment with proteasome and HSP90 inhibitors or heat shock, was the molecular chaperone HSP70. We show that HSP70 binds to FOXM1 following proteotoxic stress and that HSP70 inhibits FOXM1 DNA-binding ability. Inhibition of FOXM1 transcriptional autoregulation by HSP70 leads to the suppression of FOXM1 protein expression. In addition, HSP70 suppression elevates FOXM1 expression, and simultaneous inhibition of FOXM1 and HSP70 increases the sensitivity of human cancer cells to anticancer drug-induced apoptosis. Overall, we determined the unique and novel mechanism of FOXM1 suppression by proteasome inhibitors. PMID:26559972

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

    PubMed

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

    2016-01-01

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

  7. Bacterial Discrimination by FISH using Molecular Chaperon GroE

    NASA Astrophysics Data System (ADS)

    Nakamura, T.; Maruyama, A.; Kurusu, Y.

    2004-12-01

    FISH(Fluorescence In Situ hybridization) is a powerful method for the analysis of the phylogenetic classification of microorganism in the environment. In many cases, 16s rRNA sequences of microorganisms are employed as target probe. Here we showed that novel probe was used in FISH in order to discriminate among the bacteria including psychrophile, mesophile, and thermophile. Molecular Chaperon GroE is a best characterized protein based on Escherichia coli and essential for bacterial proliferation. In E. coli, the amount of GroEL protein per cell reaches to about 5% of total cellualr protein at heat-shock response. This response occurred at transcription levels, the amount of groEL mRNA increases at about 10-fold per cell, reaches to 0.4% of total synthesized RNA. Therefore, we considered that groEL gene was employed FISH analysis as a target probe. Moreover, we found that Gly-Gly-Met (GGM) repeats in the carboxy-terminal of GroEL strongly conserved among psychrophile and mesophile, but not thermophile. In this report, we attempted to discriminate among the bacteria including psychrophile, mesophile, and thermophile by FISH using the specific sequence of GroEL as a probe. Furthermore, we proposed the novel phylogenetic trees based on the amino acids sequences of carboxy-terminal of GroEL for bacterial evolution by temperature adaptation.

  8. Pharmacological Chaperoning: A Potential Treatment for PMM2-CDG.

    PubMed

    Yuste-Checa, Patricia; Brasil, Sandra; Gámez, Alejandra; Underhaug, Jarl; Desviat, Lourdes R; Ugarte, Magdalena; Pérez-Cerdá, Celia; Martinez, Aurora; Pérez, Belén

    2017-02-01

    The congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG), the most common N-glycosylation disorder, is a multisystem disease for which no effective treatment is available. The recent functional characterization of disease-causing mutations described in patients with PMM2-CDG led to the idea of a therapeutic strategy involving pharmacological chaperones (PC) to rescue PMM2 loss-of-function mutations. The present work describes the high-throughput screening, by differential scanning fluorimetry, of 10,000 low-molecular-weight compounds from a commercial library, to search for possible PCs for the enzyme PMM2. This exercise identified eight compounds that increased the thermal stability of PMM2. Of these, four compounds functioned as potential PCs that significantly increased the stability of several destabilizing and oligomerization mutants and also increased PMM activity in a disease model of cells overexpressing PMM2 mutations. Structural analysis revealed one of these compounds to provide an excellent starting point for chemical optimization since it passed tests based on a number of pharmacochemical quality filters. The present results provide the first proof-of-concept of a possible treatment for PMM2-CDG and describe a promising chemical structure as a starting point for the development of new therapeutic agents for this severe orphan disease.

  9. Withaferin A Analogs That Target the AAA+ Chaperone p97

    PubMed Central

    Wijeratne, E. M. Kithsiri; Xu, Ya-ming; Kang, MinJin; Wu, Tongde; Lau, Eric C.; Mesa, Celestina; Mason, Damian J.; Brown, Robert V.; Clair, James J. La; Gunatilaka, A. A. Leslie; Zhang, Donna D.; Chapman, Eli

    2015-01-01

    Understanding the mode of action (MOA) of many natural products can be puzzling with mechanistic clues that seem to lack a common thread. One such puzzle lies in the evaluation of the antitumor properties of the natural product withaferin A (WFA). A variety of seemingly unrelated pathways have been identified to explain its activity, suggesting a lack of selectivity. We now show that WFA acts as an inhibitor of the chaperone, p97, both in vitro and in cell models in addition to inhibiting the proteasome in vitro. Through medicinal chemistry, we have refined the activity of WFA toward p97 and away from the proteasome. Subsequent studies indicated that these WFA analogs retained p97 activity and cytostatic activity in cell models, suggesting that the modes of action reported for WFA could be connected by proteostasis modulation. Through this endeavor, we highlight how the parallel integration of medicinal chemistry with chemical biology offers a potent solution to one of natures’ intriguing molecular puzzles. PMID:26006219

  10. Sulphur shuttling across a chaperone during molybdenum cofactor maturation.

    PubMed

    Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, Marina I; Toci, René; Mendel, Ralf R; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne

    2015-02-04

    Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

  11. Proteins with RNA Chaperone Activity: A World of Diverse Proteins with a Common Task—Impediment of RNA Misfolding

    PubMed Central

    Semrad, Katharina

    2011-01-01

    Proteins with RNA chaperone activity are ubiquitous proteins that play important roles in cellular mechanisms. They prevent RNA from misfolding by loosening misfolded structures without ATP consumption. RNA chaperone activity is studied in vitro and in vivo using oligonucleotide- or ribozyme-based assays. Due to their functional as well as structural diversity, a common chaperoning mechanism or universal motif has not yet been identified. A growing database of proteins with RNA chaperone activity has been established based on evaluation of chaperone activity via the described assays. Although the exact mechanism is not yet understood, it is more and more believed that disordered regions within proteins play an important role. This possible mechanism and which proteins were found to possess RNA chaperone activity are discussed here. PMID:21234377

  12. Chaperones are the target in aloe-emodin-induced human lung nonsmall carcinoma H460 cell apoptosis.

    PubMed

    Lai, Miao-Ying; Hour, Mann-Jen; Wing-Cheung Leung, Henry; Yang, Wen-Hui; Lee, Hong-Zin

    2007-11-14

    Our previous study has demonstrated that aloe-emodin induced a significant change in the expression of apoptosis-related proteins in H460 cells. However, the molecular mechanisms underlying the biological effects of aloe-emodin still remain unknown. The present study applied 2D electrophoresis (pH range 4-7) to the proteins involved in aloe-emodin (40 muM)-induced H460 cell apoptosis. Eleven proteins were found to markedly change. These altered proteins were identified as ATP synthase, vimentin, HSP60, HSP70 and protein disulfide isomerase. Aloe-emodin caused a time-dependent decrease in intracellular ATP levels, which might be related to direct inhibition of ATP synthase. We also observed that the activity of mitochondria was injured by aloe-emodin. These data clearly demonstrated that mitochondria may play a critical role in aloe-emodin-induced H460 cell death. Many reports emphasize that chaperones have a complex role in apoptosis. The present study suggested that the increasing protein expression of HSP60, HSP70, 150 kDa oxygen-regulated protein and protein disulfide isomerase is involved in aloe-emodin-induced H460 cell apoptosis. HSP70, 150 kDa oxygen-regulated protein and protein disulfide isomerase are endoplasmic reticulum chaperone. Therefore, we hypothesized that the increasing endoplasmic reticulum stress serves to promote H460 cell apoptosis after treatment with aloe-emodin. We also demonstrated aloe-emodin-induced H460 cell death through caspase-3 apoptotic pathway, but not apoptosis-inducing factor apoptotic pathway.

  13. The Chaperone ClpX Stimulates Expression of Staphylococcus aureus Protein A by Rot Dependent and Independent Pathways

    PubMed Central

    Jelsbak, Lotte; Ingmer, Hanne; Valihrach, Lukás; Cohn, Marianne Thorup; Christiansen, Mie H. G.; Kallipolitis, Birgitte H.; Frees, Dorte

    2010-01-01

    The Clp ATPases (Hsp100) constitute a family of closely related proteins that have protein reactivating and remodelling activities typical of molecular chaperones. In Staphylococcus aureus the ClpX chaperone is essential for virulence and for transcription of spa encoding Protein A. The present study was undertaken to elucidate the mechanism by which ClpX stimulates expression of Protein A. For this purpose, we prepared antibodies directed against Rot, an activator of spa transcription, and demonstrated that cells devoid of ClpX contain three-fold less Rot than wild-type cells. By varying Rot expression from an inducible promoter we showed that expression of Protein A requires a threshold level of Rot. In the absence of ClpX the Rot content is reduced below this threshold level, hence, explaining the substantially reduced Protein A expression in the clpX mutant. Experiments addressed at pinpointing the role of ClpX in Rot synthesis revealed that ClpX is required for translation of Rot. Interestingly, translation of the spa mRNA was, like the rot mRNA, enhanced by ClpX. These data demonstrate that ClpX performs dual roles in regulating Protein A expression, as ClpX stimulates transcription of spa by enhancing translation of Rot, and that ClpX additionally is required for full translation of the spa mRNA. The current findings emphasize that ClpX has a central role in fine-tuning virulence regulation in S. aureus. PMID:20856878

  14. The Posttranslocation Chaperone PrsA2 Contributes to Multiple Facets of Listeria monocytogenes Pathogenesis▿ †

    PubMed Central

    Alonzo, Francis; Port, Gary C.; Cao, Min; Freitag, Nancy E.

    2009-01-01

    Listeria monocytogenes is an intracellular bacterial pathogen whose virulence depends on the regulated expression of numerous secreted bacterial factors. As for other gram-positive bacteria, many proteins secreted by L. monocytogenes are translocated across the bacterial membrane in an unfolded state to the compartment existing between the membrane and the cell wall. This compartment presents a challenging environment for protein folding due to its high density of negative charge, high concentrations of cations, and low pH. We recently identified PrsA2 as a gene product required for L. monocytogenes virulence. PrsA2 was identified based on its increased secretion by strains containing a mutationally activated form of prfA, the key regulator of L. monocytogenes virulence gene expression. The prsA2 gene product is one of at least two predicted peptidyl-prolyl cis/trans-isomerases encoded by L. monocytogenes; these proteins function as posttranslocation protein chaperones and/or foldases. In this study, we demonstrate that PrsA2 plays a unique and important role in L. monocytogenes pathogenesis by promoting the activity and stability of at least two critical secreted virulence factors: listeriolysin O (LLO) and a broad-specificity phospholipase. Loss of PrsA2 activity severely attenuated virulence in mice and impaired bacterial cell-to-cell spread in host cells. In contrast, mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth and cell-to-cell spread as well as virulence in mice. PrsA2 is thus distinct from PrsA1 in its unique requirement for the stability and full activity of L. monocytogenes-secreted factors that contribute to host infection. PMID:19451247

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

    PubMed

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

    2016-09-01

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

  16. The RNA Chaperone Hfq Promotes Fitness of Actinobacillus pleuropneumoniae during Porcine Pleuropneumonia

    PubMed Central

    Subashchandrabose, Sargurunathan; Leveque, Rhiannon M.; Kirkwood, Roy N.; Kiupel, Matti

    2013-01-01

    Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, an economically important disease of pigs. The hfq gene in A. pleuropneumoniae, encoding the RNA chaperone and posttranscriptional regulator Hfq, is upregulated during infection of porcine lungs. To investigate the role of this in vivo-induced gene in A. pleuropneumoniae, an hfq mutant strain was constructed. The hfq mutant was defective in biofilm formation on abiotic surfaces. The level of pgaC transcript, encoding the biosynthesis of poly-β-1,6-N-acetylglucosamine (PNAG), a major biofilm matrix component, was lower and PNAG content was 10-fold lower in the hfq mutant than in the wild-type strain. When outer membrane proteins were examined, cysteine synthase, implicated in resistance to oxidative stress and tellurite, was not found at detectable levels in the absence of Hfq. The hfq mutant displayed enhanced sensitivity to superoxide generated by methyl viologen and tellurite. These phenotypes were readily reversed by complementation with the hfq gene expressed from its native promoter. The role of Hfq in the fitness of A. pleuropneumoniae was assessed in a natural host infection model. The hfq mutant failed to colonize porcine lungs and was outcompeted by the wild-type strain (median competitive index of 2 × 10−5). Our data demonstrate that the in vivo-induced gene hfq is involved in the regulation of PNAG-dependent biofilm formation, resistance to superoxide stress, and the fitness and virulence of A. pleuropneumoniae in pigs and begin to elucidate the role of an in vivo-induced gene in the pathogenesis of pleuropneumonia. PMID:23732171

  17. The RNA chaperone Hfq promotes fitness of Actinobacillus pleuropneumoniae during porcine pleuropneumonia.

    PubMed

    Subashchandrabose, Sargurunathan; Leveque, Rhiannon M; Kirkwood, Roy N; Kiupel, Matti; Mulks, Martha H

    2013-08-01

    Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, an economically important disease of pigs. The hfq gene in A. pleuropneumoniae, encoding the RNA chaperone and posttranscriptional regulator Hfq, is upregulated during infection of porcine lungs. To investigate the role of this in vivo-induced gene in A. pleuropneumoniae, an hfq mutant strain was constructed. The hfq mutant was defective in biofilm formation on abiotic surfaces. The level of pgaC transcript, encoding the biosynthesis of poly-β-1,6-N-acetylglucosamine (PNAG), a major biofilm matrix component, was lower and PNAG content was 10-fold lower in the hfq mutant than in the wild-type strain. When outer membrane proteins were examined, cysteine synthase, implicated in resistance to oxidative stress and tellurite, was not found at detectable levels in the absence of Hfq. The hfq mutant displayed enhanced sensitivity to superoxide generated by methyl viologen and tellurite. These phenotypes were readily reversed by complementation with the hfq gene expressed from its native promoter. The role of Hfq in the fitness of A. pleuropneumoniae was assessed in a natural host infection model. The hfq mutant failed to colonize porcine lungs and was outcompeted by the wild-type strain (median competitive index of 2 × 10(-5)). Our data demonstrate that the in vivo-induced gene hfq is involved in the regulation of PNAG-dependent biofilm formation, resistance to superoxide stress, and the fitness and virulence of A. pleuropneumoniae in pigs and begin to elucidate the role of an in vivo-induced gene in the pathogenesis of pleuropneumonia.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-01

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

  20. Ribosomal crystallography: peptide bond formation, chaperone assistance and antibiotics activity.

    PubMed

    Yonath, Ada

    2005-08-31

    The peptidyl transferase center (PTC) is located in a protein free environment, thus confirming that the ribosome is a ribozyme. This arched void has dimensions suitable for accommodating the 3' ends of the A-and the P-site tRNAs, and is situated within a universal sizable symmetry-related region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between the elaborate PTC architecture and the A-site tRNA position revealed that the A- to P-site passage of the tRNA 3' end is performed by a rotatory motion, which leads to stereochemistry suitable for peptide bond formation and for substrate mediated catalysis, thus suggesting that the PTC evolved by gene-fusion. Adjacent to the PTC is the entrance of the protein exit tunnel, shown to play active roles in sequence-specific gating of nascent chains and in responding to cellular signals. This tunnel also provides a site that may be exploited for local co-translational folding and seems to assist in nascent chain trafficking into the hydrophobic space formed by the first bacterial chaperone, the trigger factor. Many antibiotics target ribosomes. Although the ribosome is highly conserved, subtle sequence and/or conformational variations enable drug selectivity, thus facilitating clinical usage. Comparisons of high-resolution structures of complexes of antibiotics bound to ribosomes from eubacteria resembling pathogens, to an archaeon that shares properties with eukaryotes and to its mutant that allows antibiotics binding, demonstrated the unambiguous difference between mere binding and therapeutical effectiveness. The observed variability in antibiotics inhibitory modes, accompanied by the elucidation of the structural basis to antibiotics mechanism justifies expectations for structural based improved properties of existing compounds as well as for the development of novel drugs.

  1. The expanding proteome of the molecular chaperone HSP90

    PubMed Central

    Samant, Rahul S; Clarke, Paul A

    2012-01-01

    The molecular chaperone HSP90 maintains the activity and stability of a diverse set of “client” proteins that play key roles in normal and disease biology. Around 20 HSP90 inhibitors that deplete the oncogenic clientele have entered clinical trials for cancer. However, the full extent of the HSP90-dependent proteome, which encompasses not only clients but also proteins modulated by downstream transcriptional responses, is still incompletely characterized and poorly understood. Earlier large-scale efforts to define the HSP90 proteome have been valuable but are incomplete because of limited technical sensitivity. Here, we discuss previous large-scale surveys of proteome perturbations induced by HSP90 inhibitors in light of a significant new study using state-of-the-art stable isotope labeling by amino acids (SILAC) technology combined with more sensitive high-resolution mass spectrometry (MS) that extends the catalog of proteomic changes in inhibitor-treated cancer cells. Among wide-ranging changes, major functional responses include downregulation of protein kinase activity and the DNA damage response alongside upregulation of the protein degradation machinery. Despite this improved proteomic coverage, there was surprisingly little overlap with previous studies. This may be due in part to technical issues but is likely also due to the variability of the HSP90 proteome with the inhibitor conditions used, the cancer cell type and the genetic status of client proteins. We suggest future proteomic studies to address these factors, to help distinguish client protein components from indirect transcriptional components and to address other key questions in fundamental and translational HSP90 research. Such studies should also reveal new biomarkers for patient selection and novel targets for therapeutic intervention. PMID:22421145

  2. Biologic activities of molecular chaperones and pharmacologic chaperone imidazole-containing dipeptide-based compounds: natural skin care help and the ultimate challenge: implication for adaptive responses in the skin.

    PubMed

    Babizhayev, Mark A; Nikolayev, Gennady M; Nikolayeva, Juliana G; Yegorov, Yegor E

    2012-03-01

    Accumulation of molecular damage and increased molecular heterogeneity are hallmarks of photoaged skin and pathogenesis of human cutaneous disease. Growing evidence demonstrates the ability of molecular chaperone proteins and of pharmacologic chaperones to decrease the environmental stress and ameliorate the oxidation stress-related and glycation disease phenotypes, suggesting that the field of chaperone therapy might hold novel treatments for skin diseases and aging. In this review, we examine the evidence suggesting a role for molecular chaperone proteins in the skin and their inducer and protecting agents: pharmacologic chaperone imidazole dipeptide-based agents (carcinine and related compounds) in cosmetics and dermatology. Furthermore, we discuss the use of chaperone therapy for the treatment of skin photoaging diseases and other skin pathologies that have a component of increased glycation and/or free radical-induced oxidation in their genesis. We examine biologic activities of molecular and pharmacologic chaperones, including strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone activity in in vitro and in vivo models of human skin disease. This allows the protein to function and traffic to the appropriate location in the skin, thereby increasing protein activity and cellular function and reducing stress on skin cells. The benefits of imidazole dipeptide antioxidants with transglycating activity (such as carcinine) in skin care are that they help protect and repair cell membrane damage and help retain youthful, younger-looking skin. All skin types will benefit from daily, topical application of pharmacologic chaperone antioxidants, anti-irritants, in combination with water-binding protein agents that work to mimic the structure and function of healthy skin. General strategies are presented addressing ground techniques to improve absorption of usually active chaperone proteins and dipeptide compounds, include

  3. Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis.

    PubMed

    Chow, Ari M; Steel, Rohan; Anderson, Robin L

    2009-05-01

    In addition to its role as a molecular chaperone, heat shock protein 72 (Hsp72) protects cells against a wide range of apoptosis inducing stresses. However, it is unclear if these two roles are functionally related or whether Hsp72 inhibits apoptosis by a mechanism independent of chaperone activity. The N-terminal adenosine triphosphatase domain, substrate-binding domain and the C-terminal EEVD regulatory motif of Hsp72 are all essential for chaperone activity. In this study, we show that Hsp72 mutants with a functional substrate-binding domain but lacking chaperone activity retain their ability to protect cells against apoptosis induced by heat and tumor necrosis factor alpha. In contrast, a deletion mutant lacking a functional substrate-binding domain has no protective capacity. The ability of the Hsp72 substrate-binding domain to inhibit apoptosis independent of the regulatory effects of the adenosine triphosphate-binding domain indicates that the inhibition of apoptosis may involve a stable binding interaction with a regulatory substrate rather than Hsp72 chaperone activity.

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

    PubMed Central

    Requena, Jose M.; Montalvo, Ana M.; Fraga, Jorge

    2015-01-01

    Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges) for drug discovery and improving of current treatments against leishmaniasis. PMID:26167482

  5. Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis

    SciTech Connect

    Blaby-Haas, Crysten E.; Padilla-Benavides, Teresita; Stübe, Roland; Argüello, José M.; Merchant, Sabeeha S.

    2014-12-02

    Metallochaperones traffic copper (Cu+) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. In this paper, we present a previously unidentified Cu+ chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu+ chaperone delivers Cu+ with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu+ import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu+-delivery mechanism and provides a unique snapshot into the evolution of a Cu+ distribution pathway. Finally, we also provide evidence for an interaction between PAA2, the Cu+-ATPase in thylakoids, and the Cu+-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu+ network that has evolved to fine-tune Cu+ distribution.

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

    PubMed

    Masison, Daniel C; Reidy, Michael

    2015-01-01

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

  7. Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis

    DOE PAGES

    Blaby-Haas, Crysten E.; Padilla-Benavides, Teresita; Stübe, Roland; ...

    2014-12-02

    Metallochaperones traffic copper (Cu+) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. In this paper, we present a previously unidentified Cu+ chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu+ chaperone delivers Cu+ with specificity for PAA1, which is flipped in themore » envelope relative to prototypical bacterial ATPases, compatible with a role in Cu+ import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu+-delivery mechanism and provides a unique snapshot into the evolution of a Cu+ distribution pathway. Finally, we also provide evidence for an interaction between PAA2, the Cu+-ATPase in thylakoids, and the Cu+-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu+ network that has evolved to fine-tune Cu+ distribution.« less

  8. Characteristics analysis of the luzA gene encoding chaperone from Photobacterium leiognathi related to bioluminescence.

    PubMed

    Lin, J W; Lin, B J; Chen, H Y; Weng, S F

    1998-03-27

    Nucleotide sequence of the luzA gene (GenBank accession No. AF039303) from Photobacterium leiognathi ATCC 25521 (NCIMB 2193) has been determined, and the chaperone encoded by the luzA gene was deduced. The LuzA chaperone has a calculated M(r) 26,295 and comprises 230 amino acid residues; the hydrophobic alpha-helix N-terminal 21 amino acid residues MKKTIFALLFMSVFI SYPSFA is the leader peptide, therefore the matured LuzA chaperone has a calculated M(r) 23,871 and comprises 209 amino acid residues only. The periplasmic LuzA chaperone is the protein concerned with the protein folding, assembly and stability. The luzA gene and the related genes are closely linked to the sod gene, that encoding Cu/Zn superoxide dismutase enables to enhance bioluminescence of the lux operon; the gene order of the luzA gene and related genes is -ufo'-luzA-ufoI-ufoII-ter->-R&R'-sod-ufo-- >. In trans complementation bioluminoassays in vivo elicit that the LuzA chaperone might be not directly concerned with bioluminescence of the lux operon from P. leiognathi in E. coli, but might enable to stabilize the proteins related to bioluminescence. The unidentified ufoII gene closely linked to the luzA gene is able to enhance bioluminescence.

  9. Is Catalytic Activity of Chaperones a Selectable Trait for the Emergence of Heat Shock Response?

    PubMed Central

    Çetinbaş, Murat; Shakhnovich, Eugene I.

    2015-01-01

    Although heat shock response is ubiquitous in bacterial cells, the underlying physical chemistry behind heat shock response remains poorly understood. To study the response of cell populations to heat shock we employ a physics-based ab initio model of living cells where protein biophysics (i.e., folding and protein-protein interactions in crowded cellular environments) and important aspects of proteins homeostasis are coupled with realistic population dynamics simulations. By postulating a genotype-phenotype relationship we define a cell division rate in terms of functional concentrations of proteins and protein complexes, whose Boltzmann stabilities of folding and strengths of their functional interactions are exactly evaluated from their sequence information. We compare and contrast evolutionary dynamics for two models of chaperon action. In the active model, foldase chaperones function as nonequilibrium machines to accelerate the rate of protein folding. In the passive model, holdase chaperones form reversible complexes with proteins in their misfolded conformations to maintain their solubility. We find that only cells expressing foldase chaperones are capable of genuine heat shock response to the increase in the amount of unfolded proteins at elevated temperatures. In response to heat shock, cells’ limited resources are redistributed differently for active and passive models. For the active model, foldase chaperones are overexpressed at the expense of downregulation of high abundance proteins, whereas for the passive model; cells react to heat shock by downregulating their high abundance proteins, as their low abundance proteins are upregulated. PMID:25606691

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

    PubMed

    Requena, Jose M; Montalvo, Ana M; Fraga, Jorge

    2015-01-01

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

  11. Recognition and targeting mechanisms by chaperones in flagellum assembly and operation.

    PubMed

    Khanra, Nandish; Rossi, Paolo; Economou, Anastassios; Kalodimos, Charalampos G

    2016-08-30

    The flagellum is a complex bacterial nanomachine that requires the proper assembly of several different proteins for its function. Dedicated chaperones are central in preventing aggregation or undesired interactions of flagellar proteins, including their targeting to the export gate. FliT is a key flagellar chaperone that binds to several flagellar proteins in the cytoplasm, including its cognate filament-capping protein FliD. We have determined the solution structure of the FliT chaperone in the free state and in complex with FliD and the flagellar ATPase FliI. FliT adopts a four-helix bundle and uses a hydrophobic surface formed by the first three helices to recognize its substrate proteins. We show that the fourth helix constitutes the binding site for FlhA, a membrane protein at the export gate. In the absence of a substrate protein FliT adopts an autoinhibited structure wherein both the binding sites for substrates and FlhA are occluded. Substrate binding to FliT activates the complex for FlhA binding and thus targeting of the chaperone-substrate complex to the export gate. The activation and targeting mechanisms reported for FliT appear to be shared among the other flagellar chaperones.

  12. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

  13. Effect of hesperetin on chaperone activity in selenite-induced cataract

    PubMed Central

    Oka, Mikako; Tamura, Hiroomi; Takehana, Makoto

    2016-01-01

    Abstract Background. Chaperone activity of α-crystallin in the lens works to prevent protein aggregation and is important to maintain the lens transparency. This study evaluated the effect of hesperetin on lens chaperone activity in selenite-induced cataracts. Methodology. Thirteen-day-old rats were divided into four groups. Animals were given hesperetin (groups G2 and G4) or vehicle (G1 and G3) on Days 0, 1, and 2. Rats in G3 and G4 were administered selenite subcutaneously 4 hours after the first hesperetin injection. On Days 2, 4, and 6, cataract grades were evaluated using slit-lamp biomicroscopy. The amount of a-crystallin and chaperone activity in water-soluble fraction were measured after animals sacrificed. Results. G3 on day 4 had developed significant cataract, as an average cataract grading of 4.6 ± 0.2. In contrast, G4 had less severe central opacities and lower stage cataracts than G3, as an average cataract grading of 2.4 ± 0.4. The a-crystallin levels in G3 lenses were lower than in G1, but the same as G4. Additionally, chaperone activity was weaker in G3 lenses than G1, but the same as in G4. Conclusions. Our results suggest that hesperetin can prevent the decreasing lens chaperone activity and a-crystallin water solubility by administered of selenite. PMID:28352791

  14. Functional adaptations of the bacterial chaperone trigger factor to extreme environmental temperatures.

    PubMed

    Godin-Roulling, Amandine; Schmidpeter, Philipp A M; Schmid, Franz X; Feller, Georges

    2015-07-01

    Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

  15. Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis

    PubMed Central

    Chow, Ari M.; Steel, Rohan

    2008-01-01

    In addition to its role as a molecular chaperone, heat shock protein 72 (Hsp72) protects cells against a wide range of apoptosis inducing stresses. However, it is unclear if these two roles are functionally related or whether Hsp72 inhibits apoptosis by a mechanism independent of chaperone activity. The N-terminal adenosine triphosphatase domain, substrate-binding domain and the C-terminal EEVD regulatory motif of Hsp72 are all essential for chaperone activity. In this study, we show that Hsp72 mutants with a functional substrate-binding domain but lacking chaperone activity retain their ability to protect cells against apoptosis induced by heat and tumor necrosis factor alpha. In contrast, a deletion mutant lacking a functional substrate-binding domain has no protective capacity. The ability of the Hsp72 substrate-binding domain to inhibit apoptosis independent of the regulatory effects of the adenosine triphosphate-binding domain indicates that the inhibition of apoptosis may involve a stable binding interaction with a regulatory substrate rather than Hsp72 chaperone activity. PMID:18819021

  16. Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity

    PubMed Central

    Mack, Korrie L.; Shorter, James

    2016-01-01

    Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702

  17. Is catalytic activity of chaperones a selectable trait for the emergence of heat shock response?

    PubMed

    Çetinbaş, Murat; Shakhnovich, Eugene I

    2015-01-20

    Although heat shock response is ubiquitous in bacterial cells, the underlying physical chemistry behind heat shock response remains poorly understood. To study the response of cell populations to heat shock we employ a physics-based ab initio model of living cells where protein biophysics (i.e., folding and protein-protein interactions in crowded cellular environments) and important aspects of proteins homeostasis are coupled with realistic population dynamics simulations. By postulating a genotype-phenotype relationship we define a cell division rate in terms of functional concentrations of proteins and protein complexes, whose Boltzmann stabilities of folding and strengths of their functional interactions are exactly evaluated from their sequence information. We compare and contrast evolutionary dynamics for two models of chaperon action. In the active model, foldase chaperones function as nonequilibrium machines to accelerate the rate of protein folding. In the passive model, holdase chaperones form reversible complexes with proteins in their misfolded conformations to maintain their solubility. We find that only cells expressing foldase chaperones are capable of genuine heat shock response to the increase in the amount of unfolded proteins at elevated temperatures. In response to heat shock, cells' limited resources are redistributed differently for active and passive models. For the active model, foldase chaperones are overexpressed at the expense of downregulation of high abundance proteins, whereas for the passive model; cells react to heat shock by downregulating their high abundance proteins, as their low abundance proteins are upregulated.

  18. Histone H2A/H2B chaperones: from molecules to chromatin-based functions in plant growth and development.

    PubMed

    Zhou, Wangbin; Zhu, Yan; Dong, Aiwu; Shen, Wen-Hui

    2015-07-01

    Nucleosomal core histones (H2A, H2B, H3 and H4) must be assembled, replaced or exchanged to preserve or modify chromatin organization and function according to cellular needs. Histone chaperones escort histones, and play key functions during nucleosome assembly/disassembly and in nucleosome structure configuration. Because of their location at the periphery of nucleosome, histone H2A-H2B dimers are remarkably dynamic. Here we focus on plant histone H2A/H2B chaperones, particularly members of the NUCLEOSOME ASSEMBLY PROTEIN-1 (NAP1) and FACILITATES CHROMATIN TRANSCRIPTION (FACT) families, discussing their molecular features, properties, regulation and function. Covalent histone modifications (e.g. ubiquitination, phosphorylation, methylation, acetylation) and H2A variants (H2A.Z, H2A.X and H2A.W) are also discussed in view of their crucial importance in modulating nucleosome organization and function. We further discuss roles of NAP1 and FACT in chromatin-based processes, such as transcription, DNA replication and repair. Specific functions of NAP1 and FACT are evident when their roles are considered with respect to regulation of plant growth and development and in plant responses to environmental stresses. Future major challenges remain in order to define in more detail the overlapping and specific roles of various members of the NAP1 family as well as differences and similarities between NAP1 and FACT family members, and to identify and characterize their partners as well as new families of chaperones to understand histone variant incorporation and chromatin target specificity.

  19. Efficient Activation of Pathogenic ΔPhe501 Mutation in Monocarboxylate Transporter 8 by Chemical and Pharmacological Chaperones.

    PubMed

    Braun, Doreen; Schweizer, Ulrich

    2015-12-01

    Monocarboxylate transporter 8 (MCT8) is a thyroid hormone transmembrane transporter expressed in many cell types, including neurons. Mutations that inactivate transport activity of MCT8 cause severe X-linked psychomotor retardation in male patients, a syndrome originally described as the Allan-Herndon-Dudley syndrome. Treatment options currently explored the focus on finding thyroid hormone-like compounds that bypass MCT8 and enter cells through different transporters. Because MCT8 is a multipass transmembrane protein, some pathogenic mutations affect membrane trafficking while potentially retaining some transporter activity. We explore here the effects of chemical and pharmacological chaperones on the expression and transport activity of the MCT8 mutant ΔPhe501. Dimethylsulfoxide, 4-phenylbutyric acid as well as its sodium salt, and the isoflavone genistein increase T3 uptake into MDCK1 cells stably transfected with mutant MCT8-ΔPhe501. We show that ΔPhe501 represents a temperature-sensitive mutant protein that is stabilized by the proteasome inhibitor MG132. 4-Phenylbutyrate has been used to stabilize ΔPhe508 mutant cystic fibrosis transmembrane conductance regulator protein and is in clinical use in patients with urea cycle defects. Genistein is enriched in soy and available as a nutritional supplement. It is effective in stabilizing MCT8-ΔPhe501 at 100 nM concentration. Expression of the L471P mutant is increased in response to phenylbutyrate, but T3 uptake activity is not induced, supporting the notion that the chaperone specifically increases membrane expression. Our findings suggest that certain pathogenic MCT8 mutants may be responsive to (co-)treatment with readily available compounds, which increase endogenous protein function.

  20. Restored mutant receptor:Corticoid binding in chaperone complexes by trimethylamine N-oxide

    PubMed Central

    Miller, Aaron L.; Elam, W. Austin; Johnson, Betty H.; Khan, Shagufta H.; Kumar, Raj; Thompson, E. Brad

    2017-01-01

    Without a glucocorticoid (GC) ligand, the transcription factor glucocorticoid receptor (GR) is largely cytoplasmic, with its GC-binding domain held in high affinity conformation by a cluster of chaperones. Binding a GC causes serial dis- and re-associations with chaperones, translocation of the GR to the nucleus, where it binds to DNA sites and associates with coregulatory proteins and basic transcription complexes. Herein, we describe the effects of a potent protective osmolyte, trimethylamine N-oxide (TMAO), on a conditions-dependent “activation-labile” mutant GR (GRact/l), which under GR-activating conditions cannot bind GCs in cells or in cell cytosols. In both cells and cytosols, TMAO restores binding to GRact/l by stabilizing it in complex with chaperones. Cells bathed in much lower concentrations of TMAO than those required in vitro show restoration of GC binding, presumably due to intracellular molecular crowding effects. PMID:28301576

  1. Structural insights on two hypothetical secretion chaperones from Xanthomonas axonopodis pv. citri.

    PubMed

    Fattori, Juliana; Prando, Alessandra; Assis, Leandro H P; Aparicio, Ricardo; Tasic, Ljubica

    2011-06-01

    Several Gram-negative bacterial pathogens have developed type III secretion systems (T3SSs) to deliver virulence proteins directly into eukaryotic cells in a process essential for many diseases. The type III secretion processes require customized chaperones with high specificity for binding partners, thus providing the secretion to occur. Due to the very low sequence similarities among secretion chaperones, annotation and discrimination of a great majority of them is extremely difficult and a task with low scores even if genes are encountered that codify for small (<20 kDa) proteins with low pI and a tendency to dimerise. Concerning about this, herein, we present structural features on two hypothetical T3SSs chaperones belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest how low resolution models based on Small Angle X-ray Scattering patterns can provide new structural insights that could be very helpful in their analysis and posterior classification.

  2. Chaperone Proteins in the Central Nervous System and Peripheral Nervous System after Nerve Injury.

    PubMed

    Ousman, Shalina S; Frederick, Ariana; Lim, Erin-Mai F

    2017-01-01

    Injury to axons of the central nervous system (CNS) and the peripheral nervous system (PNS) is accompanied by the upregulation and downregulation of numerous molecules that are involved in mediating nerve repair, or in augmentation of the original damage. Promoting the functions of beneficial factors while reducing the properties of injurious agents determines whether regeneration and functional recovery ensues. A number of chaperone proteins display reduced or increased expression following CNS and PNS damage (crush, transection, contusion) where their roles have generally been found to be protective. For example, chaperones are involved in mediating survival of damaged neurons, promoting axon regeneration and remyelination and, improving behavioral outcomes. We review here the various chaperone proteins that are involved after nervous system axonal damage, the functions that they impact in the CNS and PNS, and the possible mechanisms by which they act.

  3. Crystal structure of archaeal homolog of proteasome-assembly chaperone PbaA.

    PubMed

    Sikdar, Arunima; Satoh, Tadashi; Kawasaki, Masato; Kato, Koichi

    2014-10-24

    Formation of the eukaryotic proteasome is not a spontaneous process but a highly ordered process assisted by several assembly chaperones. In contrast, archaeal proteasome subunits can spontaneously assemble into an active form. Recent bioinformatic analysis identified the proteasome-assembly chaperone-like proteins, PbaA and PbaB, in archaea. Our previous study showed that the PbaB homotetramer functions as a proteasome activator through its tentacle-like C-terminal segments. However, a functional role of the other homolog PbaA has remained elusive. Here we determined the 2.25-Å resolution structure of PbaA, illustrating its disparate tertiary and quaternary structures compared with PbaB. PbaA forms a homopentamer in which the C-terminal segments, with a putative proteasome-activating motif, are packed against the core. These findings offer deeper insights into the molecular evolution relationships between the proteasome-assembly chaperones and the proteasome activators.

  4. Structural Basis for Protein anti-Aggregation Activity of the Trigger Factor Chaperone*

    PubMed Central

    Saio, Tomohide; Guan, Xiao; Rossi, Paolo; Economou, Anastassios; Kalodimos, Charalampos G.

    2014-01-01

    Molecular chaperones prevent aggregation and misfolding of proteins but scarcity of structural data has impeded an understanding of the recognition and anti-aggregation mechanisms. Here we report the solution structure, dynamics and energetics of three Trigger Factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. NMR relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion but as the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and how by acting as unfoldases and holdases prevent the aggregation and premature (mis)folding of unfolded proteins. PMID:24812405

  5. Chaperone Proteins in the Central Nervous System and Peripheral Nervous System after Nerve Injury

    PubMed Central

    Ousman, Shalina S.; Frederick, Ariana; Lim, Erin-Mai F.

    2017-01-01

    Injury to axons of the central nervous system (CNS) and the peripheral nervous system (PNS) is accompanied by the upregulation and downregulation of numerous molecules that are involved in mediating nerve repair, or in augmentation of the original damage. Promoting the functions of beneficial factors while reducing the properties of injurious agents determines whether regeneration and functional recovery ensues. A number of chaperone proteins display reduced or increased expression following CNS and PNS damage (crush, transection, contusion) where their roles have generally been found to be protective. For example, chaperones are involved in mediating survival of damaged neurons, promoting axon regeneration and remyelination and, improving behavioral outcomes. We review here the various chaperone proteins that are involved after nervous system axonal damage, the functions that they impact in the CNS and PNS, and the possible mechanisms by which they act. PMID:28270745

  6. Roles of histone chaperone CIA/Asf1 in nascent DNA elongation during nucleosome replication.

    PubMed

    Ishikawa, Katsuyuki; Ohsumi, Tatsuya; Tada, Shusuke; Natsume, Ryo; Kundu, Lena Rani; Nozaki, Naohito; Senda, Toshiya; Enomoto, Takemi; Horikoshi, Masami; Seki, Masayuki

    2011-10-01

    The nucleosome, which is composed of DNA wrapped around a histone octamer, is a fundamental unit of chromatin and is duplicated during the eukaryotic DNA replication process. The evolutionarily conserved histone chaperone cell cycle gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is involved in histone transfer and nucleosome reassembly during DNA replication. CIA/Asf1 has been reported to split the histone (H3-H4)(2) tetramer into histone H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication, CIA/Asf1 is involved in nucleosome disassembly and the promotion of semi-conservative histone H3-H4 dimer deposition onto each daughter strand in vivo. Despite numerous studies on the functional roles of CIA/Asf1, its mechanistic role(s) remains elusive because of lack of biochemical analyses. The biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks histone (H3-H4)(2) tetramer splitting activity, does not form efficiently a quaternary complex with histones H3-H4 and the minichromosome maintenance 2 (Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in histone transfer during DNA replication, negatively regulates the progression of the replication fork.

  7. The RNA Chaperone Hfq Impacts Growth, Metabolism and Production of Virulence Factors in Yersinia enterocolitica

    PubMed Central

    Kakoschke, Tamara; Kakoschke, Sara; Magistro, Giuseppe; Schubert, Sören; Borath, Marc; Heesemann, Jürgen; Rossier, Ombeline

    2014-01-01

    To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs) which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin. PMID:24454955

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  9. Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].

    PubMed

    Palumbo, Valeria; Pellacani, Claudia; Heesom, Kate J; Rogala, Kacper B; Deane, Charlotte M; Mottier-Pavie, Violaine; Gatti, Maurizio; Bonaccorsi, Silvia; Wakefield, James G

    2015-06-29

    Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.

  10. Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits.

    PubMed

    Chan, Puiyee; Thomas, Celestine J; Sprang, Stephen R; Tall, Gregory G

    2013-03-05

    We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein α (Gα) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during Gα subunit translation and protein folding. Gα translation rates and overall produced protein amounts were equivalent in mock and Ric-8A-immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF4(-)-bound Gαi, Gαq, Gα13, and Gαs produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. Gαi, Gαq, and Gα13, but not Gαs produced from Ric-8A-depleted RRL were not protected from trypsinization and therefore not folded correctly. Addition of recombinant Ric-8A to the Ric-8A-depleted RRL enhanced GDP-AlF4(-)-bound Gα subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated Gαq was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of Gαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS to Ric-8A-supplemented WGE Gαq translation resulted in dissociation of the Ric-8A:Gαq heterodimer and production of functional Gαq-GTPγS monomer. Excess Gβγ supplementation of WGE did not support functional Gαq production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein α subunits.

  11. Epigenetic silencing of the chaperone Cosmc in human leukocytes expressing tn antigen.

    PubMed

    Mi, Rongjuan; Song, Lina; Wang, Yingchun; Ding, Xiaokun; Zeng, Junwei; Lehoux, Sylvain; Aryal, Rajindra P; Wang, Jianmei; Crew, Vanja K; van Die, Irma; Chapman, Arlene B; Cummings, Richard D; Ju, Tongzhong

    2012-11-30

    Cosmc is the specific molecular chaperone in the endoplasmic reticulum for T-synthase, a Golgi β3-galactosyltransferase that generates the core 1 O-glycan, Galβ1-3GalNAcα-Ser/Thr, in glycoproteins. Dysfunctional Cosmc results in the formation of inactive T-synthase and consequent expression of the Tn antigen (GalNAcα1-Ser/Thr), which is associated with several human diseases. However, the molecular regulation of expression of Cosmc, which is encoded by a single gene on Xq24, is poorly understood. Here we show that epigenetic silencing of Cosmc through hypermethylation of its promoter leads to loss of Cosmc transcripts in Tn4 cells, an immortalized B cell line from a male patient with a Tn-syndrome-like phenotype. These cells lack T-synthase activity and express the Tn antigen. Treatment of cells with 5-aza-2'-deoxycytidine causes restoration of Cosmc transcripts, restores T-synthase activity, and reduces Tn antigen expression. Bisulfite sequencing shows that CG dinucleotides in the Cosmc core promoter are hypermethylated. Interestingly, several other X-linked genes associated with glycosylation are not silenced in Tn4 cells, and we observed no correlation of a particular DNA methyltransferase to aberrant methylation of Cosmc in these cells. Thus, hypermethylation of the Cosmc promoter in Tn4 cells is relatively specific. Epigenetic silencing of Cosmc provides another mechanism underlying the abnormal expression of the Tn antigen, which may be important in understanding aberrant Tn antigen expression in human diseases, including IgA nephropathy and cancer.

  12. Epigenetic Silencing of the Chaperone Cosmc in Human Leukocytes Expressing Tn Antigen*

    PubMed Central

    Mi, Rongjuan; Song, Lina; Wang, Yingchun; Ding, Xiaokun; Zeng, Junwei; Lehoux, Sylvain; Aryal, Rajindra P.; Wang, Jianmei; Crew, Vanja K.; van Die, Irma; Chapman, Arlene B.; Cummings, Richard D.; Ju, Tongzhong

    2012-01-01

    Cosmc is the specific molecular chaperone in the endoplasmic reticulum for T-synthase, a Golgi β3-galactosyltransferase that generates the core 1 O-glycan, Galβ1–3GalNAcα-Ser/Thr, in glycoproteins. Dysfunctional Cosmc results in the formation of inactive T-synthase and consequent expression of the Tn antigen (GalNAcα1-Ser/Thr), which is associated with several human diseases. However, the molecular regulation of expression of Cosmc, which is encoded by a single gene on Xq24, is poorly understood. Here we show that epigenetic silencing of Cosmc through hypermethylation of its promoter leads to loss of Cosmc transcripts in Tn4 cells, an immortalized B cell line from a male patient with a Tn-syndrome-like phenotype. These cells lack T-synthase activity and express the Tn antigen. Treatment of cells with 5-aza-2′-deoxycytidine causes restoration of Cosmc transcripts, restores T-synthase activity, and reduces Tn antigen expression. Bisulfite sequencing shows that CG dinucleotides in the Cosmc core promoter are hypermethylated. Interestingly, several other X-linked genes associated with glycosylation are not silenced in Tn4 cells, and we observed no correlation of a particular DNA methyltransferase to aberrant methylation of Cosmc in these cells. Thus, hypermethylation of the Cosmc promoter in Tn4 cells is relatively specific. Epigenetic silencing of Cosmc provides another mechanism underlying the abnormal expression of the Tn antigen, which may be important in understanding aberrant Tn antigen expression in human diseases, including IgA nephropathy and cancer. PMID:23035125

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

    PubMed

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

    2015-07-01

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

  14. Trapping and Identification of Cellular Substrates of the Staphylococcus aureus ClpC Chaperone

    PubMed Central

    Graham, Justin W.; Lei, Mei G.

    2013-01-01

    ClpC is an ATP-dependent Hsp100/Clp chaperone involved in protein quality control in low-GC Gram-positive bacteria. Previously, we found that ClpC affected the expression of a large number of genes, including capsule genes in Staphylococcus aureus. Here we constructed a His-tagged ClpC variant (ClpCtrap) with mutations within the Walker B motifs to identify the direct substrates of ClpC by copurification with ClpCtrap followed by gel electrophoresis combined with liquid chromatography-tandem mass spectrometry proteomics. We identified a total of 103 proteins that are potential substrates of ClpC in strain Newman. The direct protein-protein interaction of ClpC with a subset of the captured proteins was verified in a bacterial two-hybrid system. The captured proteins could be grouped into various functional categories, but most were related to proteins involved in the stress response. Several known ClpC substrates were captured, including ClpP, TrfA/MecA, ClpB, DnaK, DnaJ, GroL, RecA, and CodY, supporting the validity of our approach. Our results also revealed many new ClpC substrates, including AgrA, CcpA, RsbW, MurG, FtsA, SrtA, Rex, Atl, ClfA, and SbcC. Analysis of capsule production showed that three of the captured proteins, which were not previously known to be transcriptional regulators, did affect capsule production. PMID:23913326

  15. Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

    PubMed Central

    Nagaraj, Ram H.; Nahomi, Rooban B.; Shanthakumar, Shilpa; Linetsky, Mikhail; Padmanabha, Smitha; Pasupuleti, Nagarekha; Wang, Benlian; Santhoshkumar, Puttur; Panda, Alok Kumar; Biswas, Ashis

    2011-01-01

    α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens.To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a Nε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it could affect the chaperone function of αA-crystallin. PMID:22120592

  16. Quantification of interaction strengths between chaperones and tetratricopeptide repeat domain-containing membrane proteins.

    PubMed

    Schweiger, Regina; Soll, Jürgen; Jung, Kirsten; Heermann, Ralf; Schwenkert, Serena

    2013-10-18

    The three tetratricopeptide repeat domain-containing docking proteins Toc64, OM64, and AtTPR7 reside in the chloroplast, mitochondrion, and endoplasmic reticulum of Arabidopsis thaliana, respectively. They are suggested to act during post-translational protein import by association with chaperone-bound preprotein complexes. Here, we performed a detailed biochemical, biophysical, and computational analysis of the interaction between Toc64, OM64, and AtTPR7 and the five cytosolic chaperones HSP70.1, HSP90.1, HSP90.2, HSP90.3, and HSP90.4. We used surface plasmon resonance spectroscopy in combination with Interaction Map® analysis to distinguish between chaperone oligomerization and docking protein-chaperone interactions and to calculate binding affinities for all tested interactions. Complementary to this, we applied pulldown assays as well as microscale thermophoresis as surface immobilization independent techniques. The data revealed that OM64 prefers HSP70 over HSP90, whereas Toc64 binds all chaperones with comparable affinities. We could further show that AtTPR7 is able to bind HSP90 in addition to HSP70. Moreover, differences between the HSP90 isoforms were detected and revealed a weaker binding for HSP90.1 to AtTPR7 and OM64, showing that slight differences in the amino acid composition or structure of the chaperones influence binding to the tetratricopeptide repeat domain. The combinatory approach of several methods provided a powerful toolkit to determine binding affinities of similar interaction partners in a highly quantitative manner.

  17. Analysis of the potency of various low molecular weight chemical chaperones to prevent protein aggregation.

    PubMed

    Upagupta, Chandak; Carlisle, Rachel E; Dickhout, Jeffrey G

    2017-04-22

    Newly translated proteins must undergo proper folding to ensure their function. To enter a low energy state, misfolded proteins form aggregates, which are associated with many degenerative diseases, such as Huntington's disease and chronic kidney disease (CKD). Recent studies have shown the use of low molecular weight chemical chaperones to be an effective method of reducing protein aggregation in various cell types. This study demonstrates a novel non-biased assay to assess the molecular efficacy of these compounds at preventing protein misfolding and/or aggregation. This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells. The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum (ER) stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress. A total of five different compounds were selected: 4-phenylbutyrate (4-PBA), docosahexaenoic acid (DHA), tauroursodeoxycholic acid, trehalose, and glycerol. The dose-dependent effects of these compounds at reducing thapsigargin-induced ER stress was then analyzed, and used to determine their EC50 values. Of the chaperones, 4-PBA and DHA provided the greatest reduction of ER stress and did so at relatively low concentrations. Upon analyzing the efficiency of these compounds and their corresponding structures, it was determined that chaperones with a localized hydrophilic, polar end followed by a long hydrophobic chain, such as 4-PBA and DHA, were most effective at reducing ER stress. This study provides some insight into the use of low molecular weight chemical chaperones and may serve as the first step towards developing new chaperones of greater potency thereby providing potential treatments for diseases caused by protein aggregation.

  18. Regulatory Role of RNA Chaperone TDP-43 for RNA Misfolding and Repeat-Associated Translation in SCA31.

    PubMed

    Ishiguro, Taro; Sato, Nozomu; Ueyama, Morio; Fujikake, Nobuhiro; Sellier, Chantal; Kanegami, Akemi; Tokuda, Eiichi; Zamiri, Bita; Gall-Duncan, Terence; Mirceta, Mila; Furukawa, Yoshiaki; Yokota, Takanori; Wada, Keiji; Taylor, J Paul; Pearson, Christopher E; Charlet-Berguerand, Nicolas; Mizusawa, Hidehiro; Nagai, Yoshitaka; Ishikawa, Kinya

    2017-04-05

    Microsatellite expansion disorders are pathologically characterized by RNA foci formation and repeat-associated non-AUG (RAN) translation. However, their underlying pathomechanisms and regulation of RAN translation remain unknown. We report that expression of expanded UGGAA (UGGAAexp) repeats, responsible for spinocerebellar ataxia type 31 (SCA31) in Drosophila, causes neurodegeneration accompanied by accumulation of UGGAAexp RNA foci and translation of repeat-associated pentapeptide repeat (PPR) proteins, consistent with observations in SCA31 patient brains. We revealed that motor-neuron disease (MND)-linked RNA-binding proteins (RBPs), TDP-43, FUS, and hnRNPA2B1, bind to and induce structural alteration of UGGAAexp. These RBPs suppress UGGAAexp-mediated toxicity in Drosophila by functioning as RNA chaperones for proper UGGAAexp folding and regulation of PPR translation. Furthermore, nontoxic short UGGAA repeat RNA suppressed mutated RBP aggregation and toxicity in MND Drosophila models. Thus, functional crosstalk of the RNA/RBP network regulates their own quality and balance, suggesting convergence of pathomechanisms in microsatellite expansion disorders and RBP proteinopathies.

  19. Copy-choice recombination by reverse transcriptases: Reshuffling of genetic markers mediated by RNA chaperones

    PubMed Central

    Negroni, Matteo; Buc, Henri

    2000-01-01

    Copy-choice recombination efficiently reshuffles genetic markers in retroviruses. In vivo, the folding of the genomic RNA is controlled by the nucleocapsid protein (NC). We show that binding of NC onto the acceptor RNA molecule is sufficient to enhance recombination, providing evidence for a mechanism where the structure of the acceptor template determines the template switch. NC as well as another RNA chaperone (StpA) converts recombination into a widespread process no longer restricted to rare hot spots, an effect maximized when both the NC and the reverse transcriptase come from HIV-1. These data suggest that RNA chaperones confer a higher genetic flexibility to retroviruses. PMID:10829081

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

    PubMed Central

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

    2015-01-01

    The molecular chaperone Hsp70 plays a central role in the import of cytoplasmic proteins into organelles, driving their translocation by binding them from the organellar interior. Starting from the experimentally-determined structure of the E. coli Hsp70, we computed, by means of molecular simulations, the effective free-energy profile for substrate translocation upon chaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import process, whose comparison with unassisted translocation highlights the essential role played by Hsp70 in importing cytoplasmic proteins. PMID:25988176

  1. The Second RNA Chaperone, Hfq2, Is Also Required for Survival under Stress and Full Virulence of Burkholderia cenocepacia J2315▿

    PubMed Central

    Ramos, Christian G.; Sousa, Sílvia A.; Grilo, André M.; Feliciano, Joana R.; Leitão, Jorge H.

    2011-01-01

    Burkholderia cenocepacia J2315 is a highly virulent and epidemic clinical isolate of the B. cepacia complex (Bcc), a group of bacteria that have emerged as important pathogens to cystic fibrosis patients. This bacterium, together with all Bcc strains and a few other prokaryotes, is unusual for encoding in its genome two distinct and functional Hfq-like proteins. In this work, we show results indicating that the 188-amino-acid Hfq2 protein is required for the full virulence and stress resistance of B. cenocepacia J2315, despite the presence on its genome of the functional 79-amino-acid Hfq protein encoded by the hfq gene. Similar to other Hfq proteins, Hfq2 is able to bind RNA. However, Hfq2 is unique in its ability to apparently form trimers in vitro. Maximal transcription of hfq was observed in B. cenocepacia J2315 cells in the early exponential phase of growth. In contrast, hfq2 transcription reached maximal levels in cells in the stationary phase, depending on the CepR quorum-sensing regulator. These results suggest that tight regulation of the expression of these two RNA chaperones is required to maximize the fitness and virulence of this bacterium. In addition, the ability of Hfq2 to bind DNA, not observed for Hfq, suggests that Hfq2 might play additional roles besides acting as an RNA chaperone. PMID:21278292

  2. Hold on to your friends: Dedicated chaperones of ribosomal proteins: Dedicated chaperones mediate the safe transfer of ribosomal proteins to their site of pre-ribosome incorporation.

    PubMed

    Pillet, Benjamin; Mitterer, Valentin; Kressler, Dieter; Pertschy, Brigitte

    2017-01-01

    Eukaryotic ribosomes are assembled from their components, the ribosomal RNAs and ribosomal proteins, in a tremendously complex, multi-step process, which primarily takes place in the nuclear compartment. Therefore, most ribosomal proteins have to travel from the cytoplasm to their incorporation site on pre-ribosomes within the nucleus. However, due to their particular characteristics, such as a highly basic amino acid composition and the presence of unstructured extensions, ribosomal proteins are especially prone to aggregation and degradation in their unassembled state, hence specific mechanisms must operate to ensure their safe delivery. Recent studies have uncovered a group of proteins, termed dedicated chaperones, specialized in accompanying and guarding individual ribosomal proteins. In this essay, we review how these dedicated chaperones utilize different folds to interact with their ribosomal protein clients and how they ensure their soluble expression and interconnect their intracellular transport with their efficient assembly into pre-ribosomes.

  3. Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20

    PubMed Central

    Maio, N.; Rouault, T. A.

    2017-01-01

    Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery. PMID:27714045

  4. A [Cu]rious Ribosomal Profiling Pattern Leads to the Discovery of Ribosomal Frameshifting in the Synthesis of a Copper Chaperone.

    PubMed

    Atkins, John F; Loughran, Gary; Baranov, Pavel V

    2017-01-19

    In many bacteria, separate genes encode a copper binding chaperone and a copper efflux pump, but in some the chaperone encoding gene has been elusive. In this issue of Molecular Cell, Meydan et al. (2017) report that ribosomes translating the ORF that encodes the copper pump frequently frameshift and terminate to produce the copper chaperone.

  5. Secretion Chaperones PrsA2 and HtrA Are Required for Listeria monocytogenes Replication following Intracellular Induction of Virulence Factor Secretion

    PubMed Central

    Ahmed, Jana K.

    2016-01-01

    The Gram-positive bacterium Listeria monocytogenes transitions from an environmental organism to an intracellular pathogen following its ingestion by susceptible mammalian hosts. Bacterial replication within the cytosol of infected cells requires activation of the central virulence regulator PrfA followed by a PrfA-dependent induction of secreted virulence factors. The PrfA-induced secreted chaperone PrsA2 and the chaperone/protease HtrA contribute to the folding and stability of select proteins translocated across the bacterial membrane. L. monocytogenes strains that lack both prsA2 and htrA exhibit near-normal patterns of growth in broth culture but are severely attenuated in vivo. We hypothesized that, in the absence of PrsA2 and HtrA, the increase in PrfA-dependent protein secretion that occurs following bacterial entry into the cytosol results in misfolded proteins accumulating at the bacterial membrane with a subsequent reduction in intracellular bacterial viability. Consistent with this hypothesis, the introduction of a constitutively activated allele of prfA (prfA*) into ΔprsA2 ΔhtrA strains was found to essentially inhibit bacterial growth at 37°C in broth culture. ΔprsA2 ΔhtrA strains were additionally found to be defective for cell invasion and vacuole escape in selected cell types, steps that precede full PrfA activation. These data establish the essential requirement for PrsA2 and HtrA in maintaining bacterial growth under conditions of PrfA activation. In addition, chaperone function is required for efficient bacterial invasion and rapid vacuole lysis within select host cell types, indicating roles for PrsA2/HtrA prior to cytosolic PrfA activation and the subsequent induction of virulence factor secretion. PMID:27481256

  6. Protein aggregation can inhibit clathrin-mediated endocytosis by chaperone competition

    PubMed Central

    Yu, Anan; Shibata, Yoko; Shah, Bijal; Calamini, Barbara; Lo, Donald C.; Morimoto, Richard I.

    2014-01-01

    Protein conformational diseases exhibit complex pathologies linked to numerous molecular defects. Aggregation of a disease-associated protein causes the misfolding and aggregation of other proteins, but how this interferes with diverse cellular pathways is unclear. Here, we show that aggregation of neurodegenerative disease-related proteins (polyglutamine, huntingtin, ataxin-1, and superoxide dismutase-1) inhibits clathrin-mediated endocytosis (CME) in mammalian cells by aggregate-driven sequestration of the major molecular chaperone heat shock cognate protein 70 (HSC70), which is required to drive multiple steps of CME. CME suppression was also phenocopied by HSC70 RNAi depletion and could be restored by conditionally increasing HSC70 abundance. Aggregation caused dysregulated AMPA receptor internalization and also inhibited CME in primary neurons expressing mutant huntingtin, showing direct relevance of our findings to the pathology in neurodegenerative diseases. We propose that aggregate-associated chaperone competition leads to both gain-of-function and loss-of-function phenotypes as chaperones become functionally depleted from multiple clients, leading to the decline of multiple cellular processes. The inherent properties of chaperones place them at risk, contributing to the complex pathologies of protein conformational diseases. PMID:24706768

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

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

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

  8. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

    PubMed Central

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388

  9. Chaperone Activity of Small Heat Shock Proteins Underlies Therapeutic Efficacy in Experimental Autoimmune Encephalomyelitis*

    PubMed Central

    Kurnellas, Michael P.; Brownell, Sara E.; Su, Leon; Malkovskiy, Andrey V.; Rajadas, Jayakumar; Dolganov, Gregory; Chopra, Sidharth; Schoolnik, Gary K.; Sobel, Raymond A.; Webster, Jonathan; Ousman, Shalina S.; Becker, Rachel A.; Steinman, Lawrence; Rothbard, Jonathan B.

    2012-01-01

    To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73–92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions. PMID:22955287

  10. Chaperone-driven polymer translocation through nanopore: Spatial distribution and binding energy.

    PubMed

    Abdolvahab, Rouhollah Haji

    2017-04-01

    Chaperones are binding proteins working as a driving force in biopolymer translocation. They bind to the biopolymer near the pore and prevent its backsliding. Chaperones may have different spatial distributions. Recently, we showed the importance of their spatial distribution in translocation and its effects on the sequence dependency of the translocation time. Here we focus on homopolymers and exponential distribution. Because of the exponential distribution of chaperones, the energy dependency of the translocation time will change. Here we find a minimum in translocation time versus binding effective energy (EBE) curve. The same trend can be seen in the scaling exponent of time versus polymer length, [Formula: see text] ([Formula: see text]), when plotted against EBE. Interestingly in some special cases, e.g. chaperones of size [Formula: see text] and with an exponential distribution rate of [Formula: see text], the minimum even reaches to an amount of less than 1 ([Formula: see text]). We explain the possibility of this rare result. Moreover, based on a theoretical discussion we show that, by taking into account the velocity dependency of the translocation on polymer length, one can truly predict the value of this minimum.

  11. A conserved co-chaperone is required for virulence in fungal plant pathogens.

    PubMed

    Lo Presti, Libera; López Díaz, Cristina; Turrà, David; Di Pietro, Antonio; Hampel, Martin; Heimel, Kai; Kahmann, Regine

    2016-02-01

    The maize pathogenic fungus Ustilago maydis experiences endoplasmic reticulum (ER) stress during plant colonization and relies on the unfolded protein response (UPR) to cope with this stress. We identified the U. maydis co-chaperone, designated Dnj1, as part of this conserved cellular response to ER stress. ∆dnj1 cells are sensitive to the ER stressor tunicamycin and display a severe virulence defect in maize infection assays. A dnj1 mutant allele unable to stimulate the ATPase activity of chaperones phenocopies the null allele. A Dnj1-mCherry fusion protein localizes in the ER and interacts with the luminal chaperone Bip1. The Fusarium oxysporum Dnj1 ortholog contributes to the virulence of this fungal pathogen in tomato plants. Unlike the human ortholog, F. oxysporum Dnj1 partially rescues the virulence defect of the Ustilago dnj1 mutant. By enabling the fungus to restore ER homeostasis and maintain a high secretory activity, Dnj1 contributes to the establishment of a compatible interaction with the host. Dnj1 orthologs are present in many filamentous fungi, but are absent in budding and fission yeasts. We postulate a conserved and essential role during virulence for this class of co-chaperones.

  12. Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity.

    PubMed

    Sastry, M S R; Zhou, Weibin; Baneyx, François

    2009-07-01

    Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging.

  13. The story of stolen chaperones: how overexpression of Q/N proteins cures yeast prions.

    PubMed

    Derkatch, Irina L; Liebman, Susan W

    2013-01-01

    Prions are self-seeding alternate protein conformations. Most yeast prions contain glutamine/asparagine (Q/N)-rich domains that promote the formation of amyloid-like prion aggregates. Chaperones, including Hsp104 and Sis1, are required to continually break these aggregates into smaller "seeds." Decreasing aggregate size and increasing the number of growing aggregate ends facilitates both aggregate transmission and growth. Our previous work showed that overexpression of 11 proteins with Q/N-rich domains facilitates the de novo aggregation of Sup35 into the [PSI(+)] prion, presumably by a cross-seeding mechanism. We now discuss our recent paper, in which we showed that overexpression of most of these same 11 Q/N-rich proteins, including Pin4C and Cyc8, destabilized pre-existing Q/N rich prions. Overexpression of both Pin4C and Cyc8 caused [PSI(+)] aggregates to enlarge. This is incompatible with a previously proposed "capping" model where the overexpressed Q/N-rich protein poisons, or "caps," the growing aggregate ends. Rather the data match what is expected of a reduction in prion severing by chaperones. Indeed, while Pin4C overexpression does not alter chaperone levels, Pin4C aggregates sequester chaperones away from the prion aggregates. Cyc8 overexpression cures [PSI(+)] by inducing an increase in Hsp104 levels, as excess Hsp104 binds to [PSI(+)] aggregates in a way that blocks their shearing.

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

    PubMed

    Sporn, Zachary A; Hines, Justin K

    2015-01-01

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

  15. Histone chaperone activity of Arabidopsis thaliana NRP1 is blocked by cytochrome c.

    PubMed

    González-Arzola, Katiuska; Díaz-Quintana, Antonio; Rivero-Rodríguez, Francisco; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2016-12-06

    Higher-order plants and mammals use similar mechanisms to repair and tolerate oxidative DNA damage. Most studies on the DNA repair process have focused on yeast and mammals, in which histone chaperone-mediated nucleosome disassembly/reassembly is essential for DNA to be accessible to repair machinery. However, little is known about the specific role and modulation of histone chaperones in the context of DNA damage in plants. Here, the histone chaperone NRP1, which is closely related to human SET/TAF-Iβ, was found to exhibit nucleosome assembly activity in vitro and to accumulate in the chromatin of Arabidopsis thaliana after DNA breaks. In addition, this work establishes that NRP1 binds to cytochrome c, thereby preventing the former from binding to histones. Since NRP1 interacts with cytochrome c at its earmuff domain, that is, its histone-binding domain, cytochrome c thus competes with core histones and hampers the activity of NRP1 as a histone chaperone. Altogether, the results obtained indicate that the underlying molecular mechanisms in nucleosome disassembly/reassembly are highly conserved throughout evolution, as inferred from the similar inhibition of plant NRP1 and human SET/TAF-Iβ by cytochrome c during DNA damage response.

  16. Action of the Hsp70 chaperone system observed with single proteins

    NASA Astrophysics Data System (ADS)

    Nunes, João M.; Mayer-Hartl, Manajit; Hartl, F. Ulrich; Müller, Daniel J.

    2015-02-01

    In Escherichia coli, the binding of non-native protein substrates to the Hsp70 chaperone DnaK is mediated by the co-chaperone DnaJ. DnaJ accelerates ATP hydrolysis on DnaK, by closing the peptide-binding cleft of DnaK. GrpE catalysed nucleotide exchange and ATP re-binding then lead to substrate release from DnaK, allowing folding. Here we refold immunoglobulin 27 (I27) to better understand how DnaJ-DnaK-GrpE chaperones cooperate. When DnaJ is present, I27 is less likely to misfold and more likely to fold, whereas the unfolded state remains unaffected. Thus, the ‘holdase’ DnaJ shows foldase behaviour. Misfolding of I27 is fully abrogated when DnaJ cooperates with DnaK, which stabilizes the unfolded state and increases the probability of folding. Addition of GrpE shifts the unfolded fraction of I27 to pre-chaperone levels. These insights reveal synergistic mechanisms within the evolutionary highly conserved Hsp70 system that prevent substrates from misfolding and promote their productive transition to the native state.

  17. A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion

    SciTech Connect

    Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko

    2014-11-06

    Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

  18. The chaperone activity and toxicity of ambroxol on Gaucher cells and normal mice.

    PubMed

    Luan, Zhuo; Li, Linjing; Higaki, Katsumi; Nanba, Eiji; Suzuki, Yoshiyuki; Ohno, Kousaku

    2013-04-01

    Gaucher disease (GD), caused by a defect of acid β-glucosidase (β-Glu), is one of the most common sphingolipidoses. Recently, ambroxol, an FDA-approved drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified as a chemical chaperone for GD. In the present study, we investigated the chaperone activity and toxicity of ambroxol on both cultured GD patient cells and normal mice. We found that ambroxol treatment significantly increased N370S, F213I, N188S/G193W and R120W mutant β-Glu activities in GD fibroblasts with low cytotoxicity. Additionally, we measured the β-Glu activity in the tissues of normal mice which received water containing increasing concentrations of ambroxol ad libitum for one week. No serious adverse effect was observed during this experiment. Ambroxol significantly increased the β-Glu activity in the spleen, heart and cerebellum of the mice. This result showed its oral availability and wide distribution and chaperone activity in the tissues, including the brain, and its lack of acute toxicity. These characteristics of ambroxol would make it a potential therapeutic chaperone in the treatment of GD with neurological manifestations.

  19. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding.

    PubMed

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.

  20. A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion

    DOE PAGES

    Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko

    2014-11-06

    Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components thatmore » form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.« less

  1. Recognition and targeting mechanisms by chaperones in flagellum assembly and operation

    PubMed Central

    Khanra, Nandish; Rossi, Paolo; Economou, Anastassios; Kalodimos, Charalampos G.

    2016-01-01

    The flagellum is a complex bacterial nanomachine that requires the proper assembly of several different proteins for its function. Dedicated chaperones are central in preventing aggregation or undesired interactions of flagellar proteins, including their targeting to the export gate. FliT is a key flagellar chaperone that binds to several flagellar proteins in the cytoplasm, including its cognate filament-capping protein FliD. We have determined the solution structure of the FliT chaperone in the free state and in complex with FliD and the flagellar ATPase FliI. FliT adopts a four-helix bundle and uses a hydrophobic surface formed by the first three helices to recognize its substrate proteins. We show that the fourth helix constitutes the binding site for FlhA, a membrane protein at the export gate. In the absence of a substrate protein FliT adopts an autoinhibited structure wherein both the binding sites for substrates and FlhA are occluded. Substrate binding to FliT activates the complex for FlhA binding and thus targeting of the chaperone–substrate complex to the export gate. The activation and targeting mechanisms reported for FliT appear to be shared among the other flagellar chaperones. PMID:27528687

  2. Malaria heat shock proteins: drug targets that chaperone other drug targets.

    PubMed

    Pesce, E-R; Cockburn, I L; Goble, J L; Stephens, L L; Blatch, G L

    2010-06-01

    Ongoing research into the chaperone systems of malaria parasites, and particularly of Plasmodium falciparum, suggests that heat shock proteins (Hsps) could potentially be an excellent class of drug targets. The P. falciparum genome encodes a vast range and large number of chaperones, including 43 Hsp40, six Hsp70, and three Hsp90 proteins (PfHsp40s, PfHsp70s and PfHsp90s), which are involved in a number of fundamental cellular processes including protein folding and assembly, protein translocation, signal transduction and the cellular stress response. Despite the fact that Hsps are relatively conserved across different species, PfHsps do exhibit a considerable number of unique structural and functional features. One PfHsp90 is thought to be sufficiently different to human Hsp90 to allow for selective targeting. PfHsp70s could potentially be used as drug targets in two ways: either by the specific inhibition of Hsp70s by small molecule modulators, as well as disruption of the interactions between Hsp70s and co-chaperones such as the Hsp70/Hsp90 organising protein (Hop) and Hsp40s. Of the many PfHsp40s present on the parasite, there are certain unique or essential members which are considered to have good potential as drug targets. This review critically evaluates the potential of Hsps as malaria drug targets, as well as the use of chaperones as aids in the heterologous expression of other potential malarial drug targets.

  3. Stimulation of Estrogen Receptor Signaling in Breast Cancer by a Novel Chaperone Synuclein Gamma

    DTIC Science & Technology

    2006-06-01

    AD_________________ Award Number: W81XWH- 04 -1-0569 TITLE: Stimulation of estrogen receptor...Stimulation of estrogen receptor signaling in breast cancer by a novel chaperone 5a. CONTRACT NUMBER synuclein gamma 5b. GRANT NUMBER W81XWH- 04 -1...UNIT NUMBER 7 . PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER North Shore University Hospital

  4. Functional characterization of the chaperon-like protein Cdc48 in cryptogein-induced immune response in tobacco.

    PubMed

    Rosnoblet, Claire; Bègue, Hervé; Blanchard, Cécile; Pichereaux, Carole; Besson-Bard, Angélique; Aimé, Sébastien; Wendehenne, David

    2017-04-01

    Cdc48, a molecular chaperone conserved in different kingdoms, is a member of the AAA+ family contributing to numerous processes in mammals including proteins quality control and degradation, vesicular trafficking, autophagy and immunity. The functions of Cdc48 plant orthologues are less understood. We previously reported that Cdc48 is regulated by S-nitrosylation in tobacco cells undergoing an immune response triggered by cryptogein, an elicitin produced by the oomycete Phytophthora cryptogea. Here, we inv estigated the function of NtCdc48 in cryptogein signalling and induced hypersensitive-like cell death. NtCdc48 was found to accumulate in elicited cells at both the protein and transcript levels. Interestingly, only a small proportion of the overall NtCdc48 population appeared to be S-nitrosylated. Using gel filtration in native conditions, we confirmed that NtCdc48 was present in its hexameric active form. An immunoprecipitation-based strategy following my mass spectrometry analysis led to the identification of about a hundred NtCdc48 partners and underlined its contribution in cellular processes including targeting of ubiquitylated proteins for proteasome-dependent degradation, subcellular trafficking and redox regulation. Finally, the analysis of cryptogein-induced events in NtCdc48-overexpressing cells highlighted a correlation between NtCdc48 expression and hypersensitive cell death. Altogether, this study identified NtCdc48 as a component of cryptogein signalling and plant immunity.

  5. Expression of the histone chaperone SET/TAF-Iβ during the strobilation process of Mesocestoides corti (Platyhelminthes, Cestoda).

    PubMed

    Costa, Caroline B; Monteiro, Karina M; Teichmann, Aline; da Silva, Edileuza D; Lorenzatto, Karina R; Cancela, Martín; Paes, Jéssica A; Benitz, André de N D; Castillo, Estela; Margis, Rogério; Zaha, Arnaldo; Ferreira, Henrique B

    2015-08-01

    The histone chaperone SET/TAF-Iβ is implicated in processes of chromatin remodelling and gene expression regulation. It has been associated with the control of developmental processes, but little is known about its function in helminth parasites. In Mesocestoides corti, a partial cDNA sequence related to SET/TAF-Iβ was isolated in a screening for genes differentially expressed in larvae (tetrathyridia) and adult worms. Here, the full-length coding sequence of the M. corti SET/TAF-Iβ gene was analysed and the encoded protein (McSET/TAF) was compared with orthologous sequences, showing that McSET/TAF can be regarded as a SET/TAF-Iβ family member, with a typical nucleosome-assembly protein (NAP) domain and an acidic tail. The expression patterns of the McSET/TAF gene and protein were investigated during the strobilation process by RT-qPCR, using a set of five reference genes, and by immunoblot and immunofluorescence, using monospecific polyclonal antibodies. A gradual increase in McSET/TAF transcripts and McSET/TAF protein was observed upon development induction by trypsin, demonstrating McSET/TAF differential expression during strobilation. These results provided the first evidence for the involvement of a protein from the NAP family of epigenetic effectors in the regulation of cestode development.

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

    PubMed

    Dennerlein, Sven; Oeljeklaus, Silke; Jans, Daniel; Hellwig, Christin; Bareth, Bettina; Jakobs, Stefan; Deckers, Markus; Warscheid, Bettina; Rehling, Peter

    2015-09-08

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

  7. A dual function for chaperones SSB–RAC and the NAC nascent polypeptide–associated complex on ribosomes

    PubMed Central

    Koplin, Ansgar; Preissler, Steffen; Ilina, Yulia; Koch, Miriam; Scior, Annika; Erhardt, Marc

    2010-01-01

    The yeast Hsp70/40 system SSB–RAC (stress 70 B–ribosome-associated complex) binds to ribosomes and contacts nascent polypeptides to assist cotranslational folding. In this study, we demonstrate that nascent polypeptide–associated complex (NAC), another ribosome-tethered system, is functionally connected to SSB–RAC and the cytosolic Hsp70 network. Simultaneous deletions of genes encoding NAC and SSB caused conditional loss of cell viability under protein-folding stress conditions. Furthermore, NAC mutations revealed genetic interaction with a deletion of Sse1, a nucleotide exchange factor regulating the cytosolic Hsp70 network. Cells lacking SSB or Sse1 showed protein aggregation, which is enhanced by additional loss of NAC; however, these mutants differ in their potential client repertoire. Aggregation of ribosomal proteins and biogenesis factors accompanied by a pronounced deficiency in ribosomal particles and translating ribosomes only occurs in ssbΔ and nacΔssbΔ cells, suggesting that SSB and NAC control ribosome biogenesis. Thus, SSB–RAC and NAC assist protein folding and likewise have important functions for regulation of ribosome levels. These findings emphasize the concept that ribosome production is coordinated with the protein-folding capacity of ribosome-associated chaperones. PMID:20368618

  8. Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis.

    PubMed

    Li, Chao; Yeh, Fang-Ling; Cheung, Alice Y; Duan, Qiaohong; Kita, Daniel; Liu, Ming-Che; Maman, Jacob; Luu, Emily J; Wu, Brendan W; Gates, Laura; Jalal, Methun; Kwong, Amy; Carpenter, Hunter; Wu, Hen-Ming

    2015-06-08

    The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor.

  9. Differential Loss of Prolyl Isomerase or Chaperone Activity of Ran-binding Protein 2 (Ranbp2) Unveils Distinct Physiological Roles of Its Cyclophilin Domain in Proteostasis*

    PubMed Central

    Cho, Kyoung-in; Patil, Hemangi; Senda, Eugene; Wang, Jessica; Yi, Haiqing; Qiu, Sunny; Yoon, Dosuk; Yu, Minzhong; Orry, Andrew; Peachey, Neal S.; Ferreira, Paulo A.

    2014-01-01

    The immunophilins, cyclophilins, catalyze peptidyl cis-trans prolyl-isomerization (PPIase), a rate-limiting step in protein folding and a conformational switch in protein function. Cyclophilins are also chaperones. Noncatalytic mutations affecting the only cyclophilins with known but distinct physiological substrates, the Drosophila NinaA and its mammalian homolog, cyclophilin-B, impair opsin biogenesis and cause osteogenesis imperfecta, respectively. However, the physiological roles and substrates of most cyclophilins remain unknown. It is also unclear if PPIase and chaperone activities reflect distinct cyclophilin properties. To elucidate the physiological idiosyncrasy stemming from potential cyclophilin functions, we generated mice lacking endogenous Ran-binding protein-2 (Ranbp2) and expressing bacterial artificial chromosomes of Ranbp2 with impaired C-terminal chaperone and with (Tg-Ranbp2WT-HA) or without PPIase activities (Tg-Ranbp2R2944A-HA). The transgenic lines exhibit unique effects in proteostasis. Either line presents selective deficits in M-opsin biogenesis with its accumulation and aggregation in cone photoreceptors but without proteostatic impairment of two novel Ranbp2 cyclophilin partners, the cytokine-responsive effectors, STAT3/STAT5. Stress-induced STAT3 activation is also unaffected in Tg-Ranbp2R2944A-HA::Ranbp2−/−. Conversely, proteomic analyses found that the multisystem proteinopathy/amyotrophic lateral sclerosis proteins, heterogeneous nuclear ribonucleoproteins A2/B1, are down-regulated post-transcriptionally only in Tg-Ranbp2R2944A-HA::Ranbp2−/−. This is accompanied by the age- and tissue-dependent reductions of diubiquitin and ubiquitylated proteins, increased deubiquitylation activity, and accumulation of the 26 S proteasome subunits S1 and S5b. These manifestations are absent in another line, Tg-Ranbp2CLDm-HA::Ranbp2−/−, harboring SUMO-1 and S1-binding mutations in the Ranbp2 cyclophilin-like domain. These results unveil

  10. Protein folding rates and thermodynamic stability are key determinants for interaction with the Hsp70 chaperone system

    PubMed Central

    Sekhar, Ashok; Lam, Hon Nam; Cavagnero, Silvia

    2012-01-01

    The Hsp70 family of molecular chaperones participates in vital cellular processes including the heat shock response and protein homeostasis. E. coli's Hsp70, known as DnaK, works in concert with the DnaJ and GrpE co-chaperones (K/J/E chaperone system), and mediates cotranslational and post-translational protein folding in the cytoplasm. While the role of the K/J/E chaperones is well understood in the presence of large substrates unable to fold independently, it is not known if and how K/J/E modulates the folding of smaller proteins able to fold even in the absence of chaperones. Here, we combine experiments and computation to evaluate the significance of kinetic partitioning as a model to describe the interplay between protein folding and binding to the K/J/E chaperone system. First, we target three nonobligatory substrates, that is, proteins that do not require chaperones to fold. The experimentally observed chaperone association of these client proteins during folding is entirely consistent with predictions from kinetic partitioning. Next, we develop and validate a computational model (CHAMP70) that assumes kinetic partitioning of substrates between folding and interaction with K/J/E. CHAMP70 quantitatively predicts the experimentally measured interaction of RNase HD as it refolds in the presence of various chaperones. CHAMP70 shows that substrates are posed to interact with K/J/E only if they are slow-folding proteins with a folding rate constant kf <50 s−1, and/or thermodynamically unstable proteins with a folding free energy ΔG0UN ≥−2 kcal mol−1. Hence, the K/J/E system is tuned to use specific protein folding rates and thermodynamic stabilities as substrate selection criteria. PMID:22886941

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

    PubMed

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

    2016-03-25

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

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

    PubMed Central

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

    2016-01-01

    DnaK is the major bacterial Hsp70, participating in DNA replication, protein folding, and the stress response. DnaK cooperates with the Hsp40 co-chaperone DnaJ and the nucleotide exchange factor GrpE. Under non-stress conditions, DnaK binds to the heat shock transcription factor σ32 and facilitates its degradation. Oxidative stress results in temporary inactivation of DnaK due to depletion of cellular ATP and thiol modifications such as glutathionylation until normal cellular ATP levels and a reducing environment are restored. However, the biological significance of DnaK glutathionylation remains unknown, and the mechanisms by which glutathionylation may regulate the activity of DnaK are also unclear. We investigated the conditions under which Escherichia coli DnaK undergoes S-glutathionylation. We observed glutathionylation of DnaK in lysates of E. coli cells that had been subjected to oxidative stress. We also obtained homogeneously glutathionylated DnaK using purified DnaK in the apo state. We found that glutathionylation of DnaK reversibly changes the secondary structure and tertiary conformation, leading to reduced nucleotide and peptide binding ability. The chaperone activity of DnaK was reversibly down-regulated by glutathionylation, accompanying the structural changes. We found that interaction of DnaK with DnaJ, GrpE, or σ32 becomes weaker when DnaK is glutathionylated, and the interaction is restored upon deglutathionylation. This study confirms that glutathionylation down-regulates the functions of DnaK under oxidizing conditions, and this down-regulation may facilitate release of σ32 from its interaction with DnaK, thus triggering the heat shock response. Such a mechanism provides a link between oxidative stress and the heat shock response in bacteria. PMID:26823468

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

    PubMed

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

    2011-12-09

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

  14. Loss of function mutation in LARP7, chaperone of 7SK ncRNA, causes a syndrome of facial dysmorphism, intellectual disability, and primordial dwarfism.

    PubMed

    Alazami, Anas M; Al-Owain, Mohammad; Alzahrani, Fatema; Shuaib, Taghreed; Al-Shamrani, Hussain; Al-Falki, Yahya H; Al-Qahtani, Saleh M; Alsheddi, Tarfa; Colak, Dilek; Alkuraya, Fowzan S

    2012-10-01

    Primordial dwarfism (PD) is a clinically and genetically heterogeneous condition. Various molecular mechanisms are known to underlie the disease including impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA damage response, defective spliceosomal machinery, and abnormal replication licensing. Here, we describe a syndromic form of PD associated with severe intellectual disability and distinct facial features in a large multiplex Saudi family. Analysis reveals a novel underlying mechanism for PD involving depletion of 7SK, an abundant cellular noncoding RNA (ncRNA), due to mutation of its chaperone LARP7. We show that 7SK levels are tightly linked to LARP7 expression across cell lines, and that this chaperone is ubiquitously expressed in the mouse embryo. The 7SK is known to influence the expression of a wide array of genes through its inhibitory effect on the positive transcription elongation factor b (P-TEFb) as well as its competing role in HMGA1-mediated transcriptional regulation. This study documents a critical role played by ncRNA in human development and adds to the growing list of molecular mechanisms that, when perturbed, converge on the PD phenotype.

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

    PubMed

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

    2015-01-19

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

  16. Chaperone Function in Androgen-Independent Prostate Cancer

    DTIC Science & Technology

    2012-05-01

    molecular mechanism by which FKBP51 regulates AR activity. Using recombi - nant proteins, we show that FKBP51 stimulates recruitment of the cochaperone...organization that includes an N-terminal AF-1 domain, a central DNA -binding domain, a hinge region, and a C-terminal ligand-binding domain (LBD). AR

  17. Drosophila Frataxin: an Iron Chaperone During Cellular [2Fe-2S] Cluster Bioassembly

    SciTech Connect

    Kondapalli,K.; Kok, N.; Dancis, A.; Stemmler, T.

    2008-01-01

    Frataxin, a mitochondrial protein that is directly involved in regulating cellular iron homeostasis, has been suggested to serve as an iron chaperone during cellular Fe-S cluster biosynthesis. In humans, decreased amounts or impaired function of frataxin causes the autosomal recessive neurodegenerative disorder Friedreich's ataxia. Cellular production of Fe-S clusters is accomplished by the Fe cofactor assembly platform enzymes Isu (eukaryotes) and IscU (prokaryotes). In this report, we have characterized the overall stability and iron binding properties of the Drosophila frataxin homologue (Dfh). Dfh is highly folded with secondary structural elements consistent with the structurally characterized frataxin orthologs. While the melting temperature (TM {approx} 59 C) and chemical stability ([urea]50% {approx} 2.4 M) of Drosophila frataxin, measured using circular dichroism (CD) and fluorescence spectroscopy, closely match values determined for the human ortholog, pure Dfh is more stable against autodegradation than both the human and yeast proteins. The ferrous iron binding affinity (Kd {approx} 6.0 {mu}M) and optimal metal to protein stoichiometry (1:1) for Dfh have been measured using isothermal titration calorimetry (ITC). Under anaerobic conditions with salt present, holo-Dfh is a stable iron-loaded protein monomer. Frataxin prevents reactive oxygen species-induced oxidative damage to DNA when presented with both Fe(II) and H2O2. Ferrous iron bound to Dfh is high-spin and held in a partially symmetric Fe-(O/N)6 coordination environment, as determined by X-ray absorption spectroscopy (XAS). Extended X-ray absorption fine structure (EXAFS) simulations indicate the average Fe-O/N bond length in Dfh is 2.13 Angstroms, consistent with a ligand geometry constructed by water and carboxylate oxygens most likely supplied in part by surface-exposed conserved acidic residues located on helix 1 and strand 1 in the structurally characterized frataxin orthologs. The iron

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

    SciTech Connect

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

    2009-05-20

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

  19. Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH<3) in E. coli and Shigella spp. Here we investigated the roles of these two acid chaperones in survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, th...

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

    PubMed

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

    2016-10-01

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

  1. Essential control of mitochondrial morphology and function by chaperone-mediated autophagy through degradation of PARK7

    PubMed Central

    Wang, Bao; Cai, Zhibiao; Tao, Kai; Zeng, Weijun; Lu, Fangfang; Yang, Ruixin; Feng, Dayun; Gao, Guodong; Yang, Qian

    2016-01-01

    ABSTRACT As a selective degradation system, chaperone-mediated autophagy (CMA) is essential for maintaining cellular homeostasis and survival under stress conditions. Increasing evidence points to an important role for the dysfunction of CMA in the pathogenesis of Parkinson disease (PD). However, the mechanisms by which CMA regulates neuronal survival under stress and its role in neurodegenerative diseases are not fully understood. PARK7/DJ-1 is an autosomal recessive familial PD gene. PARK7 plays a critical role in antioxidative response and its dysfunction leads to mitochondrial defects. In the current study, we showed that CMA mediated the lysosome-dependent degradation of PARK7. Importantly, CMA preferentially removed the oxidatively damaged nonfunctional PARK7 protein. Furthermore, CMA protected cells from mitochondrial toxin MPP+-induced changes in mitochondrial morphology and function, and increased cell viability. These protective effects were lost under PARK7-deficiency conditions. Conversely, overexpression of PARK7 significantly attenuated the mitochondrial dysfunction and cell death exacerbated by blocking CMA under oxidative stress. Thus, our findings reveal a mechanism by which CMA protects mitochondrial function by degrading nonfunctional PARK7 and maintaining its homeostasis, and dysregulation of this pathway may contribute to the neuronal stress and death in PD pathogenesis. PMID:27171370

  2. A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation.

    PubMed

    Echeverría, Pablo C; Briand, Pierre-André; Picard, Didier

    2016-04-01

    Hsp90 is the ATP-consuming core component of a very abundant molecular chaperone machine that handles a substantial portion of the cytosolic proteome. Rather than one machine, it is in fact an ensemble of molecular machines, since most mammalian cells express two cytosolic isoforms of Hsp90 and a subset of up to 40 to 50 cochaperones and regulate their interactions and functions by a variety of posttranslational modifications. We demonstrate that the Hsp90 ensemble is fundamentally remodeled during muscle differentiation and that this remodeling is not just a consequence of muscle differentiation but possibly one of the drivers to accompany and to match the vast proteomic changes associated with this process. As myoblasts differentiate into myotubes, Hsp90α disappears and only Hsp90β remains, which is the only isoform capable of interacting with the novel muscle-specific Hsp90 cochaperone Aarsd1L. Artificially maintaining Hsp90α or knocking down Aarsd1L expression interferes with the differentiation of C2C12 myotubes. During muscle differentiation, Aarsd1L replaces the more ubiquitous cochaperone p23 and in doing so dampens the activity of the glucocorticoid receptor, one of the Hsp90 clients relevant to muscle functions. This cochaperone switch protects muscle cells against the inhibitory effects of glucocorticoids and may contribute to preventing muscle wasting induced by excess glucocorticoids.

  3. A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation

    PubMed Central

    Echeverría, Pablo C.; Briand, Pierre-André

    2016-01-01

    Hsp90 is the ATP-consuming core component of a very abundant molecular chaperone machine that handles a substantial portion of the cytosolic proteome. Rather than one machine, it is in fact an ensemble of molecular machines, since most mammalian cells express two cytosolic isoforms of Hsp90 and a subset of up to 40 to 50 cochaperones and regulate their interactions and functions by a variety of posttranslational modifications. We demonstrate that the Hsp90 ensemble is fundamentally remodeled during muscle differentiation and that this remodeling is not just a consequence of muscle differentiation but possibly one of the drivers to accompany and to match the vast proteomic changes associated with this process. As myoblasts differentiate into myotubes, Hsp90α disappears and only Hsp90β remains, which is the only isoform capable of interacting with the novel muscle-specific Hsp90 cochaperone Aarsd1L. Artificially maintaining Hsp90α or knocking down Aarsd1L expression interferes with the differentiation of C2C12 myotubes. During muscle differentiation, Aarsd1L replaces the more ubiquitous cochaperone p23 and in doing so dampens the activity of the glucocorticoid receptor, one of the Hsp90 clients relevant to muscle functions. This cochaperone switch protects muscle cells against the inhibitory effects of glucocorticoids and may contribute to preventing muscle wasting induced by excess glucocorticoids. PMID:26884463

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

    PubMed

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

    2015-01-01

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

  5. Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts

    PubMed Central

    Evans, Margery L.; Jain, Neha; Götheson, Anna; Åden, Jörgen; Chapman, Matthew R.; Almqvist, Fredrik; Wittung-Stafshede, Pernilla

    2015-01-01

    Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another. PMID:26465894

  6. The Endoplasmic Reticulum Chaperone GRP78/BiP Modulates Prion Propagation in vitro and in vivo

    PubMed Central

    Park, Kyung-Won; Eun Kim, Gyoung; Morales, Rodrigo; Moda, Fabio; Moreno-Gonzalez, Ines; Concha-Marambio, Luis; Lee, Amy S.; Hetz, Claudio; Soto, Claudio

    2017-01-01

    Prion diseases are fatal neurodegenerative disorders affecting several mammalian species, characterized by the accumulation of the misfolded form of the prion protein, which is followed by the induction of endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). GRP78, also called BiP, is a master regulator of the UPR, reducing ER stress levels and apoptosis due to an enhancement of the cellular folding capacity. Here, we studied the role of GRP78 in prion diseases using several in vivo and in vitro approaches. Our results show that a reduction in the expression of this molecular chaperone accelerates prion pathogenesis in vivo. In addition, we observed that prion replication in cell culture was inversely related to the levels of expression of GRP78 and that both proteins interact in the cellular context. Finally, incubation of PrPSc with recombinant GRP78 led to the dose-dependent reduction of protease-resistant PrPSc in vitro. Our results uncover a novel role of GRP78 in reducing prion pathogenesis, suggesting that modulating its levels/activity may offer a novel opportunity for designing therapeutic approaches for these diseases. These findings may also have implications for other diseases involving the accumulation of misfolded proteins. PMID:28333162

  7. Prion-Associated Toxicity is Rescued by Elimination of Cotranslational Chaperones

    PubMed Central

    Keefer, Kathryn M.; True, Heather L.

    2016-01-01

    The nascent polypeptide-associated complex (NAC) is a highly conserved but poorly characterized triad of proteins that bind near the ribosome exit tunnel. The NAC is the first cotranslational factor to bind to polypeptides and assist with their proper folding. Surprisingly, we found that deletion of NAC subunits in Saccharomyces cerevisiae rescues toxicity associated with the strong [PSI+] prion. This counterintuitive finding can be explained by changes in chaperone balance and distribution whereby the folding of the prion protein is improved and the prion is rendered nontoxic. In particular, the ribosome-associated Hsp70 Ssb is redistributed away from Sup35 prion aggregates to the nascent chains, leading to an array of aggregation phenotypes that can mimic both overexpression and deletion of Ssb. This toxicity rescue demonstrates that chaperone modification can block key steps of the prion life cycle and has exciting implications for potential treatment of many human protein conformational disorders. PMID:27828954

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

    SciTech Connect

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

    2010-03-08

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

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

    PubMed Central

    Ribeiro, Raquel; Gilberto, Samuel; Gomes, Ricardo A.; Ferreira, António; Mateus, Élia; Barroso, Eduardo; Coelho, Ana V.; Freire, Ana Ponces; Cordeiro, Carlos

    2015-01-01

    Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis. PMID:26147092

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

    SciTech Connect

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

    2005-06-15

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

  11. The Histone Chaperones FACT and Spt6 Restrict H2A.Z from Intragenic Locations

    PubMed Central

    Jeronimo, Célia; Watanabe, Shinya; Kaplan, Craig D.; Peterson, Craig L.; Robert, François

    2015-01-01

    SUMMARY H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of efficient nucleosome reassembly coupled to pervasive incorporation of H2A.Z by mislocalized SWR-C alters chromatin composition and contributes to cryptic initiation. Thus, chaperone-mediated H2A.Z confinement is crucial for restricting the chromatin signature of gene promoters, which otherwise may license or promote cryptic transcription. PMID:25959393

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

    PubMed

    Marcianò, G; Huang, D T

    2016-02-01

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

  13. Amphiphilic polysaccharide nanoballs: a new building block for nanogel biomedical engineering and artificial chaperones.

    PubMed

    Takahashi, Haruko; Sawada, Shin-Ichi; Akiyoshi, Kazunari

    2011-01-25

    Enzymatically synthesized glycogen (ESG), a highly branched (1→4)(1→6)-linked α-glucan, is a new monodisperse spherical hyperbranched nanoparticle (molecular weight, 10(6)-10(7); diameter, 20-30 nm), polysaccharide nanoball. Amphiphilic ESG nanoballs were synthesized by introducing a cholesterol group to enzymatically synthesized glycogen (CHESG). CHESG assembled into a structure containing a few molecules to form cluster nanogels (approximately 35 nm in diameter) in water. The cluster nanogels were dissociated by the addition of cyclodextrin (CD) to form a supramolecular CHESG-CD nanocomplex due to complexation with the cholesterol group and CD. The CHESG nanogel showed high capacity for complexation with proteins, and the CHESG-CD nanocomplex showed high chaperone-like activity for thermal stabilization of enzymes. CHESG has great potential to become a new building block for nanogel biomedical engineering and to act as an artificial chaperone for protein engineering.

  14. Dimeric and trimeric triazole based molecules as a new class of Hsp90 molecular chaperone inhibitors.

    PubMed

    Terracciano, Stefania; Chini, Maria Giovanna; Piaz, Fabrizio Dal; Vassallo, Antonio; Riccio, Raffaele; Bruno, Ines; Bifulco, Giuseppe

    2013-07-01

    In the last decade Hsp90 inhibitors have emerged as attractive candidates for the development of new potent anticancer therapeutics. In order to identify novel agents able to block the chaperone activity, following a structure-based approach, we used in silico screening to direct the synthesis of potential inhibitors bearing the triazole scaffold, a widespread motif in drug-like molecules. Docking results, performed on a larger collection of dimeric and trimeric triazole derivatives, suggested the synthesis of some molecules showing different calculated binding energies and modes. Surface Plasmon Resonance Binding assay, performed on the synthesized compounds, allow to identify a series of molecules able to potently interact with the target enzyme and to disclose an interesting hit: compound 2b showed to interact with the ATP binding site in the N-terminus domain of Hsp90 and to efficiently inhibit the chaperone activity.

  15. Crystal structure of Escherichia coli YidC, a membrane protein chaperone and insertase.

    PubMed

    Kumazaki, Kaoru; Kishimoto, Toshiki; Furukawa, Arata; Mori, Hiroyuki; Tanaka, Yoshiki; Dohmae, Naoshi; Ishitani, Ryuichiro; Tsukazaki, Tomoya; Nureki, Osamu

    2014-12-03

    Bacterial YidC, an evolutionally conserved membrane protein, functions as a membrane protein chaperone in cooperation with the Sec translocon and as an independent insertase for membrane proteins. In Gram-negative bacteria, the transmembrane and periplasmic regions of YidC interact with the Sec proteins, forming a multi-protein complex for Sec-dependent membrane protein integration. Here, we report the crystal structure of full-length Escherichia coli YidC. The structure reveals that a hydrophilic groove, formed by five transmembrane helices, is a conserved structural feature of YidC, as compared to the previous YidC structure from Bacillus halodurans, which lacks a periplasmic domain. Structural mapping of the substrate- or Sec protein-contact sites suggested the importance of the groove for the YidC functions as a chaperone and an insertase, and provided structural insight into the multi-protein complex.

  16. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    SciTech Connect

    Singh, Chingakham R.; Lovell, Scott; Mehzabeen, Nurjahan; Chowdhury, Wasimul Q.; Geanes, Eric S.; Battaile, Kevin P.; Roelofs, Jeroen

    2014-03-25

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

  17. Promiscuous Substrate Recognition in Folding and Assembly Activities of the Trigger Factor Chaperone

    SciTech Connect

    Martinez-Hackert, E.; Hendrickson, W

    2009-01-01

    Trigger factor (TF) is a molecular chaperone that binds to bacterial ribosomes where it contacts emerging nascent chains, but TF is also abundant free in the cytosol where its activity is less well characterized. In vitro studies show that TF promotes protein refolding. We find here that ribosome-free TF stably associates with and rescues from misfolding a large repertoire of full-length proteins. We identify over 170 members of this cytosolic Escherichia coli TF substrate proteome, including ribosomal protein S7. We analyzed the biochemical properties of a TF:S7 complex from Thermotoga maritima and determined its crystal structure. Thereby, we obtained an atomic-level picture of a promiscuous chaperone in complex with a physiological substrate protein. The structure of the complex reveals the molecular basis of substrate recognition by TF, indicates how TF could accelerate protein folding, and suggests a role for TF in the biogenesis of protein complexes.

  18. Chemical chaperone TUDCA prevents apoptosis and improves survival during polymicrobial sepsis in mice

    PubMed Central

    Doerflinger, Marcel; Glab, Jason; Nedeva, Christina; Jose, Irvin; Lin, Ann; O’Reilly, Lorraine; Allison, Cody; Pellegrini, Marc; Hotchkiss, Richard S.; Puthalakath, Hamsa

    2016-01-01

    Sepsis-induced lymphopenia is a major cause of morbidities in intensive care units and in populations with chronic conditions such as renal failure, diabetes, HIV and alcohol abuse. Currently, other than supportive care and antibiotics, there are no treatments for this condition. We developed an in vitro assay to understand the role of the ER-stress-mediated apoptosis process in lymphocyte death during polymicrobial sepsis, which was reproducible in in vivo mouse models. Modulating ER stress using chemical chaperones significantly reduced the induction of the pro-apoptotic protein Bim both in vitro and in mice. Furthermore, in a ‘two-hit’ pneumonia model in mice, we have been able to demonstrate that administration of the chemical chaperone TUDCA helped to maintain lymphocyte homeostasis by significantly reducing lymphocyte apoptosis and this correlated with four-fold improvement in survival. Our results demonstrate a novel therapeutic opportunity for treating sepsis-induced lymphopenia in humans. PMID:27694827

  19. Molecular chaperons and co-chaperons, Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Ito, Makoto; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2015-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt disease. To better understand the molecular mechanisms involved in interaction between Nicotiana benthamiana and R. solanacearum, we focused on Hsp90, RAR1 and SGT1. Appearances of wilt symptom were significantly suppressed in Hsp90, RAR1 and SGT1-silenced plants compared with control plants. In RAR1-silenced plants, population of R. solanacearum increased in a similar manner to control plants. In contrast, multiplication of R. solanacearum was significantly suppressed in Hsp90 and SGT1-silenced plants. In addition, expression of PR genes were increased in Hsp90 and SGT1-silenced plants challenged with R. solanacearum. Therefore, RAR1 might be required for disease development or suppression of disease tolerance. These results also suggested that Hsp90 and/or SGT1 might play an important role in suppression of plant defenses leading to disease susceptibility and disease development.

  20. Dephosphorylation of alpha(s)- and beta-caseins and its effect on chaperone activity: a structural and functional investigation.

    PubMed

    Koudelka, Tomas; Hoffmann, Peter; Carver, John A

    2009-07-08

    Milk casein proteins can act as molecular chaperones: under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. In this study, alpha(s)- and beta-caseins were dephosphorylated using alkaline phosphatase. A structural and functional investigation was undertaken to determine the effect of dephosphorylation on the chaperone activity of alpha(s)- and beta-caseins against two types of protein misfolding, i.e., amorphous aggregation and amyloid fibril assembly. The dephosphorylation of alpha(s)- and beta-caseins resulted in a decrease in the chaperone efficiency against both heat- and reduction-induced amorphously aggregating target proteins. In contrast, dephosphorylation had no effect on the chaperone activity of alpha(s)- and beta-caseins against the amyloid-forming target protein kappa-casein. Circular dichroism and fluorescence spectroscopic data indicated that the loss of negative charge associated with dephosphorylation led to an increase in ordered structure of alpha(s)- and beta-caseins. It is concluded that the flexible, dynamic, and relatively unstructured and amphiphatic nature of alpha(s)- and beta-caseins is important in their chaperone action.

  1. Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP.

    PubMed

    Brinkworth, Amanda J; Malcolm, Denise S; Pedrosa, António T; Roguska, Katarzyna; Shahbazian, Sevanna; Graham, James E; Hayward, Richard D; Carabeo, Rey A

    2011-10-01

    Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion-competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well-characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two-hybrid, co-precipitation, cross-linking and size exclusion chromatography we show that Slc1 (SycE-like chaperone 1; CT043) specifically interacts with a 200-amino-acid residue N-terminal region of TARP (TARP¹⁻²⁰⁰). Slc1 formed homodimers in vitro, as shown in cross-linking and gel filtration experiments. Biochemical analysis of an isolated Slc1-TARP¹⁻²⁰⁰ complex was consistent with a characteristic 2:1 chaperone-effector stoichiometry. Furthermore, Slc1 was co-immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP.

  2. Chaperone activities of bovine and camel beta-caseins: Importance of their surface hydrophobicity in protection against alcohol dehydrogenase aggregation.

    PubMed

    Barzegar, Abolfazl; Yousefi, Reza; Sharifzadeh, Ahmad; Dalgalarrondo, Michèle; Chobert, Jean-Marc; Ganjali, Mohammad Reza; Norouzi, Parviz; Ehsani, Mohammad Reza; Niasari-Naslaji, Amir; Saboury, Ali Akbar; Haertlé, Thomas; Moosavi-Movahedi, Ali Akbar

    2008-05-01

    Beta-casein (beta-CN) showing properties of intrinsically unstructured proteins (IUP) displays many similarities with molecular chaperones and shows anti-aggregation activity in vitro. Chaperone activities of bovine and camel beta-CN were studied using alcohol dehydrogenase (ADH) as a substrate. To obtain an adequate relevant information about the chaperone capacities of studied caseins, three different physical parameters including chaperone constant (k(c), microM(-1)), thermal aggregation constant (k(T), degrees C(-1)) and aggregation rate constant (k(t), min(-1)) were measured. Bovine beta-CN displays greater chaperone activity than camel beta-CN. Fluorescence studies of 8-anilino-1-naphthalenesulfonic acid (ANS) binding demonstrated that bovine beta-CN is doted with larger effective hydrophobic surfaces at all studied temperatures than camel beta-CN. Greater relative hydrophobicity of bovine beta-CN than camel beta-CN may be a factor responsible for stronger interactions of bovine beta-CN with the aggregation-prone pre denatured molecular species of the substrate ADH, which resulted in greater chaperone activity of bovine beta-CN.

  3. Metabolic and Chaperone Gene Loss Marks the Origin of Animals: Evidence for Hsp104 and Hsp78 Chaperones Sharing Mitochondrial Enzymes as Clients

    PubMed Central

    Erives, Albert J.; Fassler, Jan S.

    2015-01-01

    The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i) sixteen distinct biosynthetic functions; (ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii) six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones. PMID:25710177

  4. Identification of Core Segment of Amyloidal Peptide Mediated by Chaperone Molecules by using Scanning Tunneling Microscopy.

    PubMed

    Yu, Yue; Yang, Yanlian; Wang, Chen

    2015-10-05

    We illustrate in this work that pristine assemblies of amyloidal peptides can be obtained by perturbations of reduced scanning bias, and show a broad distribution in peptide length. In contrast, the chaperone-mediated peptide co-assembly presents ordered lamellar structures with a homogeneous distribution in length, which could be attributed to the core segment of the peptide. The efforts are beneficial for gaining insight into the aggregation propensity of peptides and inter-peptide interactions.

  5. Multiscale Modeling of a Conditionally Disordered pH-Sensing Chaperone

    PubMed Central

    Ahlstrom, Logan S.; Law, Sean M.; Dickson, Alex; Brooks, Charles L.

    2015-01-01

    The pH-sensing chaperone HdeA promotes the survival of enteropathogenic bacteria during transit through the harshly acidic environment of the mammalian stomach. At low pH, HdeA transitions from an inactive, folded, dimer to chaperone-active, disordered, monomers to protect against the acid-induced aggregation of periplasmic proteins. Toward achieving a detailed mechanistic understanding of the pH response of HdeA, we develop a multiscale modeling approach to capture its pH-dependent thermodynamics. Our approach combines pKa calculations from all-atom constant pH molecular dynamics simulations with coarse-grained modeling, and yields new, atomic-level, insights into HdeA chaperone function that can be directly tested by experiment. “pH triggers” that significantly destabilize the dimer are each located near the N-terminus of a helix, suggesting that their neutralization at low pH destabilizes the helix macrodipole as a mechanism of monomer disordering. Moreover, we observe a non-monotonic change in the pH-dependent stability of HdeA, with maximal stability of the dimer near pH 5. This affect is attributed to the protonation Glu37, which exhibits an anomalously high pKa value and is located within the hydrophobic dimer interface. Finally, the pH-dependent binding pathway of HdeA comprises a partially unfolded, dimeric intermediate that becomes increasingly stable relative to the native dimer at lower pH values and displays key structural features for chaperone-substrate interaction. We anticipate that the insights from our model will help inform ongoing NMR and biochemical investigations. PMID:25584862

  6. Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process*

    PubMed Central

    Tsai, Chai-jui; Aslam, Kiran; Drendel, Holli M.; Asiago, Josephat M.; Goode, Kourtney M.; Paul, Lake N.; Rochet, Jean-Christophe; Hazbun, Tony R.

    2015-01-01

    The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H2O2, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins. PMID:26306045

  7. Stretched Extracellular Matrix Proteins Turn Fouling and Are Functionally Rescued by the Chaperones Albumin and Casein

    PubMed Central

    2009-01-01

    While evidence is mounting that cells exploit protein unfolding for mechanochemical signal conversion (mechanotransduction), what mechanisms are in place to deal with the unwanted consequences of exposing hydrophobic residues upon force-induced protein unfolding? Here, we show that mechanical chaperones exist that can transiently bind to hydrophobic residues that are freshly exposed by mechanical force. The stretch-upregulated binding of albumin or casein to fibronectin fibers is reversible and does not inhibit fiber contraction once the tension is released. PMID:19743815

  8. Discovery of Benzisoxazoles as Potent Inhibitors of Chaperone Heat Shock Protein 90

    SciTech Connect

    Gopalsamy, Ariamala; Shi, Mengxiao; Golas, Jennifer; Vogan, Erik; Jacob, Jaison; Johnson, Mark; Lee, Frederick; Nilakantan, Ramaswamy; Petersen, Roseann; Svenson, Kristin; Chopra, Rajiv; Tam, May S.; Wen, Yingxia; Ellingboe, John; Arndt, Kim; Boschelli, Frank

    2008-08-11

    Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for activating many signaling proteins and is a promising target in tumor biology. We have identified small-molecule benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic studies show that these compounds bind in the ATP binding pocket interacting with the Asp93. Structure based optimization led to the identification of potent analogues, such as 13, with good biochemical profiles.

  9. The conformational bases for the two functionalities of 2-cysteine peroxiredoxins as peroxidase and chaperone

    PubMed Central

    Dietz, Karl-Josef

    2013-01-01

    2-Cysteine peroxiredoxins (2-CysPrxs) are ubiquitous and highly abundant proteins that serve multiple functions as peroxidases, chaperones, and thiol oxidases and in redox-dependent cell signalling. The chloroplast protein plays a role in seedling development and protection of the photosynthetic apparatus. This study aimed to unequivocally link conformation and function. To this end, a set of non-tagged site-directed mutagenized At2-CysPrx variants was engineered, which mimicked the conformational states and their specific functions: hyperoxidized form (C54D), reduced form (C54S, C176S), oxidized form (C54DC176K), phosphorylated form (T92D), reduced ability for oligomerization by interfering with the dimer–dimer interface (F84R) and a C-terminally truncated form [ΔC (–20 aa)]. These variants were fully or partly fixed in their quaternary structure and function, respectively, and were analysed for their conformational state and peroxidase and chaperone activity, as well as for their sensitivity to hyperoxidation. The presence of a His6-tag strongly influenced the properties of the protein. The ΔC variant became insensitive to hyperoxidation, while T92D and F84R became more sensitive. The C54D variant revealed the highest chaperone activity. The highest peroxidase activity was observed for the F84R and ΔC variants. Efficient interaction with NADP-dependent thioredoxin reductase C depended on the presence of Cys residues and the C-terminal tail. The results suggest that the structural flexibility is important for the switch between peroxidase and chaperone function and that evolution has conserved the functional switch instead of maximizing a single function. These variants are ideal tools for future conformation-specific studies in vivo and in vitro. PMID:23828546

  10. Structural and Functional Characterization of Pseudomonas aeruginosa CupB Chaperones

    PubMed Central

    Cai, Xun; Wang, Rui; Filloux, Alain; Waksman, Gabriel; Meng, Guoyu

    2011-01-01

    Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for ∼10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e.g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 Å crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5. PMID:21304995

  11. Multiscale modeling of a conditionally disordered pH-sensing chaperone.

    PubMed

    Ahlstrom, Logan S; Law, Sean M; Dickson, Alex; Brooks, Charles L

    2015-04-24

    The pH-sensing chaperone HdeA promotes the survival of enteropathogenic bacteria during transit through the harshly acidic environment of the mammalian stomach. At low pH, HdeA transitions from an inactive, folded, dimer to chaperone-active, disordered, monomers to protect against the acid-induced aggregation of periplasmic proteins. Toward achieving a detailed mechanistic understanding of the pH response of HdeA, we develop a multiscale modeling approach to capture its pH-dependent thermodynamics. Our approach combines pK(a) (logarithmic acid dissociation constant) calculations from all-atom constant pH molecular dynamics simulations with coarse-grained modeling and yields new, atomic-level, insights into HdeA chaperone function that can be directly tested by experiment. "pH triggers" that significantly destabilize the dimer are each located near the N-terminus of a helix, suggesting that their neutralization at low pH destabilizes the helix macrodipole as a mechanism of monomer disordering. Moreover, we observe a non-monotonic change in the pH-dependent stability of HdeA, with maximal stability of the dimer near pH5. This affect is attributed to the protonation Glu37, which exhibits an anomalously high pK(a) value and is located within the hydrophobic dimer interface. Finally, the pH-dependent binding pathway of HdeA comprises a partially unfolded, dimeric intermediate that becomes increasingly stable relative to the native dimer at lower pH values and displays key structural features for chaperone-substrate interaction. We anticipate that the insights from our model will help inform ongoing NMR and biochemical investigations.

  12. Conversion of scFv peptide-binding specificity for crystal chaperone development

    SciTech Connect

    Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.

    2012-02-07

    In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 {angstrom} resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a {approx}52 {angstrom} channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.

  13. Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

    PubMed Central

    Milanov, Peter; Abriss, Daniela; Ungerer, Christopher; Quade-Lyssy, Patricia; Simpson, Jeremy C.; Pepperkok, Rainer; Seifried, Erhard; Tonn, Torsten

    2012-01-01

    Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking. PMID:22973456

  14. Hsp70 chaperones are non-equilibrium machines that achieve ultra-affinity by energy consumption.

    PubMed

    De Los Rios, Paolo; Barducci, Alessandro

    2014-05-27

    70-kDa Heat shock proteins are ATP-driven molecular chaperones that perform a myriad of essential cellular tasks. Although structural and biochemical studies have shed some light on their functional mechanism, the fundamental issue of the role of energy consumption, due to ATP-hydrolysis, has remained unaddressed. Here we establish a clear connection between the non-equilibrium nature of Hsp70, due to ATP hydrolysis, and the determining feature of its function, namely its high affinity for its substrates. Energy consumption can indeed decrease the dissociation constant of the chaperone-substrate complex by several orders of magnitude with respect to an equilibrium scenario. We find that the biochemical requirements for observing such ultra-affinity coincide with the physiological conditions in the cell. Our results rationalize several experimental observations and pave the way for further analysis of non-equilibrium effects underlying chaperone functions.DOI: http://dx.doi.org/10.7554/eLife.02218.001.

  15. Procollagen triple helix assembly: an unconventional chaperone-assisted folding paradigm.

    PubMed

    Makareeva, Elena; Leikin, Sergey

    2007-10-10

    Fibers composed of type I collagen triple helices form the organic scaffold of bone and many other tissues, yet the energetically preferred conformation of type I collagen at body temperature is a random coil. In fibers, the triple helix is stabilized by neighbors, but how does it fold? The observations reported here reveal surprising features that may represent a new paradigm for folding of marginally stable proteins. We find that human procollagen triple helix spontaneously folds into its native conformation at 30-34 degrees C but not at higher temperatures, even in an environment emulating Endoplasmic Reticulum (ER). ER-like molecular crowding by nonspecific proteins does not affect triple helix folding or aggregation of unfolded chains. Common ER chaperones may prevent aggregation and misfolding of procollagen C-propeptide in their traditional role of binding unfolded polypeptide chains. However, such binding only further destabilizes the triple helix. We argue that folding of the triple helix requires stabilization by preferential binding of chaperones to its folded, native conformation. Based on the triple helix folding temperature measured here and published binding constants, we deduce that HSP47 is likely to do just that. It takes over 20 HSP47 molecules to stabilize a single triple helix at body temperature. The required 50-200 microM concentration of free HSP47 is not unusual for heat-shock chaperones in ER, but it is 100 times higher than used in reported in vitro experiments, which did not reveal such stabilization.

  16. CCT2 Mutations Evoke Leber Congenital Amaurosis due to Chaperone Complex Instability

    PubMed Central

    Minegishi, Yuriko; Sheng, XunLun; Yoshitake, Kazutoshi; Sergeev, Yuri; Iejima, Daisuke; Shibagaki, Yoshio; Monma, Norikazu; Ikeo, Kazuho; Furuno, Masaaki; Zhuang, Wenjun; Liu, Yani; Rong, Weining; Hattori, Seisuke; Iwata, Takeshi

    2016-01-01

    Leber congenital amaurosis (LCA) is a hereditary early-onset retinal dystrophy that is accompanied by severe macular degeneration. In this study, novel compound heterozygous mutations were identified as LCA-causative in chaperonin-containing TCP-1, subunit 2 (CCT2), a gene that encodes the molecular chaperone protein, CCTβ. The zebrafish mutants of CCTβ are known to exhibit the eye phenotype while its mutation and association with human disease have been unknown. The CCT proteins (CCT α-θ) forms ring complex for its chaperon function. The LCA mutants of CCTβ, T400P and R516H, are biochemically instable and the affinity for the adjacent subunit, CCTγ, was affected distinctly in both mutants. The patient-derived induced pluripotent stem cells (iPSCs), carrying these CCTβ mutants, were less proliferative than the control iPSCs. Decreased proliferation under Cct2 knockdown in 661W cells was significantly rescued by wild-type CCTβ expression. However, the expression of T400P and R516H didn’t exhibit the significant effect. In mouse retina, both CCTβ and CCTγ are expressed in the retinal ganglion cells and connecting cilium of photoreceptor cells. The Cct2 knockdown decreased its major client protein, transducing β1 (Gβ1). Here we report the novel LCA mutations in CCTβ and the impact of chaperon disability by these mutations in cellular biology. PMID:27645772

  17. Efficient antibody production in the methylotrophic yeast Ogataea minuta by overexpression of chaperones.

    PubMed

    Suzuki, Takeshi; Baba, Satoshi; Ono, Minako; Nonaka, Koichi; Ichikawa, Kimihisa; Yabuta, Masayuki; Ito, Rie; Chiba, Yasunori

    2017-03-26

    A production system for a therapeutic monoclonal antibody was developed using the methylotrophic yeast Ogataea minuta IFO10746. The genetically engineered O. minuta secreted a detectable amount of anti-TRAIL receptor antibody into the culture supernatant, and the secreted antibody was purified by multiple column chromatography steps. In the purification process, both fully and partially assembled antibodies were detected and isolated. The fully assembled antibody from O. minuta showed almost the same biological activity as that derived from mammalian cells despite the distinct glycosylation profile, whereas the partially assembled antibody showed no cytotoxic activity. To increase the production of active antibody in O. minuta, we overexpressed selected chaperone proteins (included protein disulfide isomerase (OmPDI1), thiol oxidase (OmERO1), and immunoglobulin heavy chain binding protein (OmKAR2)) known to assist in the proper folding (in the endoplasmic reticulum) of proteins destined for secretion. Each of these chaperones enhanced antibody secretion, and together these three factors yielded 16-fold higher antibody accumulation while increasing the ratio of the fully assembled antibody compared to that from the parental strain. Supplementation of a rhodanine-3-acetic acid derivative (R3AD_1c), an inhibitor of O-mannosylation, further increased the secretion of the correctly assembled antibody. These results indicated that the co-overexpression of chaperones is an effective way to produce the correctly assembled antibody in O. minuta.

  18. Cloning and characterization of three hypothetical secretion chaperone proteins from Xanthomonas axonopodis pv. citri.

    PubMed

    Tasic, Ljubica; Borin, Paula F L; Khater, Leti Cia; Ramos, Carlos H I

    2007-06-01

    Xanthomonas axonopodis pv. citri (Xac) causes citrus canker in plantations around the world and is of particular significance in Brazil where its incidence has risen exponentially over the past decade. Approximately one third of the predicted Xac open reading frames show no homology, or homology with very low score with that of known sequences. It is believed that Xac utilizes secretion systems to transfer virulence proteins into susceptible eukaryotic cells. This process is assisted by secretion chaperones that maintain virulence proteins partly or completely unfolded during translocation. We have cloned three of these hypothetical secretion chaperones: XAC0419 and XAC1346 from type III secretion system (TTSS) and XACb0033 from type IV secretion system (TFSS). All proteins were cloned in a pET23a vector (Novagen), expressed at 37 degrees C using a BL21(DE3)pLysS Escherichia coli strain and purified by ion exchange and gel-filtration chromatographic methods. Pure proteins were characterized using spectroscopic measurements: circular dichroism, and both static and lifetime emission fluorescence in the case of XACb0033. The analyzed proteins are stable at elevated temperatures (up to 65 degrees C) and exhibit alpha-helix content from approximately 30% (XACb003) to approximately 87% (XAC1346). XACb0033 exhibits lifetimes in the fluorescence experiments that indicate different neighborhoods for its tryptophan residues. These chaperones have the characteristics of TTSS and TFSS: all are small, with a high alpha-helix content, and without ATP-binding or ATP-hydrolyzing activity.

  19. Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics

    PubMed Central

    Conn, P. Michael

    2014-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases. PMID:24661201

  20. Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

    PubMed

    Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

    2015-11-01

    Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

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

    SciTech Connect

    Trent, J.D.

    1996-05-01

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

  2. Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders

    PubMed Central

    Parenti, Giancarlo; Andria, Generoso; Valenzano, Kenneth J

    2015-01-01

    Lysosomal storage disorders (LSDs) are a group of inborn metabolic diseases caused by mutations in genes that encode proteins involved in different lysosomal functions, in most instances acidic hydrolases. Different therapeutic approaches have been developed to treat these disorders. Pharmacological chaperone therapy (PCT) is an emerging approach based on small-molecule ligands that selectively bind and stabilize mutant enzymes, increase their cellular levels, and improve lysosomal trafficking and activity. Compared to other approaches, PCT shows advantages, particularly in terms of oral administration, broad biodistribution, and positive impact on patients' quality of life. After preclinical in vitro and in vivo studies, PCT is now being translated in the first clinical trials, either as monotherapy or in combination with enzyme replacement therapy, for some of the most prevalent LSDs. For some LSDs, the results of the first clinical trials are encouraging and warrant further development. Future research in the field of PCT will be directed toward the identification of novel chaperones, including new allosteric drugs, and the exploitation of synergies between chaperone treatment and other therapeutic approaches. PMID:25881001

  3. Elucidating the mechanism of substrate recognition by the bacterial Hsp90 molecular chaperone

    PubMed Central

    Street, Timothy O.; Zeng, Xiaohui; Pellarin, Riccardo; Bonomi, Massimiliano; Sali, Andrej; Kelly, Mark J.S.; Chu, Feixia; Agard, David A.

    2017-01-01

    Hsp90 is a conformationally dynamic molecular chaperone known to promote the folding and activation of a broad array of protein substrates (“clients”). Hsp90 is believed to preferentially interact with partially folded substrates, and it has been hypothesized that the chaperone can significantly alter substrate structure as a mechanism to alter the substrate functional state. However, critically testing the mechanism of substrate recognition and remodeling by Hsp90 has been challenging. Using a partially folded protein as a model system, we find that the bacterial Hsp90 adapts its conformation to the substrate, forming a binding site that spans the middle and C-terminal domains of the chaperone. Crosslinking and NMR measurements indicate that Hsp90 binds to a large partially-folded region of the substrate and significantly alters both its local and long-range structure. These findings implicate Hsp90’s conformational dynamics in its ability to bind and remodel partially folded proteins. Moreover, native-state hydrogen exchange indicates that Hsp90 can also interact with partially folded states only transiently populated from within a thermodynamically stable native state ensemble. These results suggest a general mechanism by which Hsp90 can recognize and remodel native proteins by binding and remodeling partially folded states that are transiently sampled from within the native ensemble. PMID:24726919

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

    SciTech Connect

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

    2015-02-09

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

  5. Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event

    PubMed Central

    Campos, Eric I.; Smits, Arne H.; Kang, Young-Hoon; Landry, Sébastien; Escobar, Thelma M.; Nayak, Shruti; Ueberheide, Beatrix M.; Durocher, Daniel; Vermeulen, Michiel; Hurwitz, Jerard; Reinberg, Danny

    2015-01-01

    SUMMARY Despite minimal disparity at the sequence level, mammalian H3 variants bind to distinct sets of polypeptides. Though histone H3.1 predominates in cycling cells, our knowledge of the soluble complexes that it forms en route to deposition or following eviction from chromatin remains limited. Here, we provide a comprehensive analysis of the H3.1-binding proteome, with emphasis on its interactions with histone chaperones and components of the replication fork. Quantitative mass spectrometry revealed 170 protein interactions, whereas a large-scale biochemical fractionation of H3.1 and associated enzymatic activities uncovered over twenty stable protein complexes in dividing human cells. The sNASP and ASF1 chaperones play pivotal roles in the processing of soluble histones, but do not associate with the active CDC45/MCM2-7/GINS (CMG) replicative helicase. We also find TONSL-MMS22L to function as a H3-H4 histone chaperone. It associates with the regulatory MCM5 subunit of the replicative helicase. PMID:26527279

  6. The crystal structure of the Hsp90 co-chaperone Cpr7 from Saccharomyces cerevisiae.

    PubMed

    Qiu, Yu; Ge, Qiangqiang; Wang, Mingxing; Lv, Hui; Ebrahimi, Mohammad; Niu, Liwen; Teng, Maikun; Li, Xu

    2017-02-09

    The versatility of Hsp90 can be attributed to the variety of co-chaperone proteins that modulate the role of Hsp90 in many cellular processes. As a co-chaperone of Hsp90, Cpr7 is essential for accelerating the cell growth in an Hsp90-containing trimeric complex. Here, we report the crystal structure of Cpr7 at a resolution of 1.8Å. It consists of an N-terminal PPI domain and a C-terminal TPR domain, and exhibits a U-shape conformation. Our studies revealed the aggregation state of Cpr7 in solution and the interaction properties between Cpr7 and the MEEVD sequence from the C-terminus of Hsp90. In addition, the structure and sequence analysis between Cpr7 and homologues revealed the structure basis both for the function differences between Cpr6 and Cpr7 and the functional complements between Cns1 and Cpr7. Our studies facilitate the understanding of Cpr7 and provide decent insights into the molecular mechanisms of the Hsp90 co-chaperone pathway.

  7. Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders.

    PubMed

    Parenti, Giancarlo; Andria, Generoso; Valenzano, Kenneth J

    2015-07-01

    Lysosomal storage disorders (LSDs) are a group of inborn metabolic diseases caused by mutations in genes that encode proteins involved in different lysosomal functions, in most instances acidic hydrolases. Different therapeutic approaches have been developed to treat these disorders. Pharmacological chaperone therapy (PCT) is an emerging approach based on small-molecule ligands that selectively bind and stabilize mutant enzymes, increase their cellular levels, and improve lysosomal trafficking and activity. Compared to other approaches, PCT shows advantages, particularly in terms of oral administration, broad biodistribution, and positive impact on patients' quality of life. After preclinical in vitro and in vivo studies, PCT is now being translated in the first clinical trials, either as monotherapy or in combination with enzyme replacement therapy, for some of the most prevalent LSDs. For some LSDs, the results of the first clinical trials are encouraging and warrant further development. Future research in the field of PCT will be directed toward the identification of novel chaperones, including new allosteric drugs, and the exploitation of synergies between chaperone treatment and other therapeutic approaches.

  8. Structure of the Spt16 Middle Domain Reveals Functional Features of the Histone Chaperone FACT*

    PubMed Central

    Kemble, David J.; Whitby, Frank G.; Robinson, Howard; McCullough, Laura L.; Formosa, Tim; Hill, Christopher P.

    2013-01-01

    The histone chaperone FACT is an essential and abundant heterodimer found in all eukaryotes. Here we report a crystal structure of the middle domain of the large subunit of FACT (Spt16-M) to reveal a double pleckstrin homology architecture. This structure was found previously in the Pob3-M domain of the small subunit of FACT and in the related histone chaperone Rtt106, although Spt16-M is distinguished from these structures by the presence of an extended α-helix and a C-terminal addition. Consistent with our finding that the double pleckstrin homology structure is common to these three histone chaperones and reports that Pob3 and Rtt106 double pleckstrin homology domains bind histones H3-H4, we also find that Spt16-M binds H3-H4 with low micromolar affinity. Our structure provides a framework for interpreting a large body of genetic data regarding the physiological functions of FACT, including the identification of potential interaction surfaces for binding histones or other proteins. PMID:23417676

  9. Pharmacological Enhancement of α-Glucosidase by the Allosteric Chaperone N-acetylcysteine

    PubMed Central

    Porto, Caterina; Ferrara, Maria C; Meli, Massimiliano; Acampora, Emma; Avolio, Valeria; Rosa, Margherita; Cobucci-Ponzano, Beatrice; Colombo, Giorgio; Moracci, Marco; Andria, Generoso; Parenti, Giancarlo

    2012-01-01

    Pompe disease (PD) is a metabolic myopathy due to the deficiency of the lysosomal enzyme α-glucosidase (GAA). The only approved treatment for this disorder, enzyme replacement with recombinant human GAA (rhGAA), has shown limited therapeutic efficacy in some PD patients. Pharmacological chaperone therapy (PCT), either alone or in combination with enzyme replacement, has been proposed as an alternative therapeutic strategy. However, the chaperones identified so far also are active site-directed molecules and potential inhibitors of target enzymes. We demonstrated that N-acetylcysteine (NAC) is a novel allosteric chaperone for GAA. NAC improved the stability of rhGAA as a function of pH and temperature without disrupting its catalytic activity. A computational analysis of NAC–GAA interactions confirmed that NAC does not interact with GAA catalytic domain. NAC enhanced the residual activity of mutated GAA in cultured PD fibroblasts and in COS7 cells overexpressing mutated GAA. NAC also enhanced rhGAA efficacy in PD fibroblasts. In cells incubated with NAC and rhGAA, GAA activities were 3.7–8.7-fold higher than those obtained in cells treated with rhGAA alone. In a PD mouse model the combination of NAC and rhGAA resulted in better correction of enzyme activity in liver, heart, diaphragm and gastrocnemia, compared to rhGAA alone. PMID:22990675

  10. Distinct Prion Domain Sequences Ensure Efficient Amyloid Propagation by Promoting Chaperone Binding or Processing In Vivo

    PubMed Central

    Langlois, Christine R.; Serio, Tricia R.

    2016-01-01

    Prions are a group of proteins that can adopt a spectrum of metastable conformations in vivo. These alternative states change protein function and are self-replicating and transmissible, creating protein-based elements of inheritance and infectivity. Prion conformational flexibility is encoded in the amino acid composition and sequence of the protein, which dictate its ability not only to form an ordered aggregate known as amyloid but also to maintain and transmit this structure in vivo. But, while we can effectively predict amyloid propensity in vitro, the mechanism by which sequence elements promote prion propagation in vivo remains unclear. In yeast, propagation of the [PSI+] prion, the amyloid form of the Sup35 protein, has been linked to an oligopeptide repeat region of the protein. Here, we demonstrate that this region is composed of separable functional elements, the repeats themselves and a repeat proximal region, which are both required for efficient prion propagation. Changes in the numbers of these elements do not alter the physical properties of Sup35 amyloid, but their presence promotes amyloid fragmentation, and therefore maintenance, by molecular chaperones. Rather than acting redundantly, our observations suggest that these sequence elements make complementary contributions to prion propagation, with the repeat proximal region promoting chaperone binding to and the repeats promoting chaperone processing of Sup35 amyloid. PMID:27814358

  11. Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its potential implications for Parkinson disease

    PubMed Central

    Jung, Olive; Patnaik, Samarjit; Marugan, Juan; Sidransky, Ellen; Westbroek, Wendy

    2017-01-01

    Gaucher disease, caused by pathological mutations GBA1, encodes the lysosome-resident enzyme glucocerebrosidase, which cleaves glucosylceramide into glucose and ceramide. In Gaucher disease, glucocerebrosidase deficiency leads to lysosomal accumulation of substrate, primarily in cells of the reticulo-endothelial system. Gaucher disease has broad clinical heterogeneity, and mutations in GBA1 are a risk factor for the development of different synucleinopathies. Insights into the cell biology and biochemistry of glucocerebrosidase have led to new therapeutic approaches for Gaucher disease including small chemical chaperones. Such chaperones facilitate proper enzyme folding and translocation to lysosomes, thereby preventing premature breakdown of the enzyme in the proteasome. This review discusses recent work developing chemical chaperones as a therapy for Gaucher disease, with implications for the treatment of synucleinopathies. It focuses on the development of non-inhibitory glucocerebrosidase chaperones and their therapeutic advantages over inhibitory chaperones, as well as the challenges involved in identifying and validating chemical chaperones. PMID:27098312

  12. Lead induces the expression of endoplasmic reticulum chaperones GRP78 and GRP94 in vascular endothelial cells via the JNK-AP-1 pathway.

    PubMed

    Shinkai, Yasuhiro; Yamamoto, Chika; Kaji, Toshiyuki

    2010-04-01

    Lead, a ubiquitous heavy metal, is an important industrial and environmental pollutant that can target the vascular endothelium. To clarify the effects of lead on the unfolded protein response (UPR) and their significance in cytotoxicity, we examined the expression and function of endoplasmic reticulum (ER) chaperones glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94) in vascular endothelial cells. We used bovine aortic endothelial cells as an in vitro model of the vascular endothelium. Exposure of vascular endothelial cells to lead nitrate resulted in a marked induction of GRP78 and GRP94 messenger RNA levels. In response to lead, the expression of GRP78 and GRP94 proteins also significantly increased in a dose- and time-dependent manner. In addition, small interfering RNA (siRNA)-mediated knockdown of GRP78 significantly enhanced lead-induced cytotoxicity. Compared with other metal(loid)s, including cadmium chloride, zinc sulfate, copper sulfate, and sodium arsenite, lead nitrate was found to be the most potent metal to induce these chaperones in endothelial cells. In the examined UPR pathways, lead increased the phosphorylation of inositol-requiring enzyme 1 (IRE1) and c-jun N-terminal kinase (JNK). Interestingly, the lead-induced upregulation of GRP78 and GRP94 was almost completely blocked by the JNK inhibitor SP600125 or activator protein-1 (AP-1) inhibitor curcumin. Taken together, these results suggest that lead induces ER stress, but the induction of GRP78 and GRP94 expression via the JNK-AP-1 pathway functions as a defense mechanism against lead-induced cytotoxicity in vascular endothelial cells.

  13. Functional characterization of human nucleosome assembly protein-2 (NAP1L4) suggests a role as a histone chaperone

    SciTech Connect

    Rodriguez, P.; Chu, Lee Lee; Kim, Jungho; Pelletier, J.

    1997-09-15

    Histones are thought to play a key role in regulating gene expression at the level of DNA packaging. Recent evidence suggests that transcriptional activation requires competition of transcription factors with histones for binding to regulatory regions and that there may be several mechanisms by which this is achieved. We have characterized a human nucleosome assembly protein, NAP-2, previously identified by positional cloning at 11p15.5, a region implicated in several disease processes including Wilms tumor (WT) etiology. The deduced amino acid sequence of NAP-2 indicates that it encodes a protein with a potential nuclear localization motif and two clusters of highly acidic residues. Functional analysis of recombinant NAP-2 protein purified from Escherichia coli demonstrates that this protein can interact with both core and linker histones. We demonstrate that recombinant NAP-2 can transfer histones onto naked DNA templates. Deletion mutagenesis of NAP-2 demonstrates that both NH3- and COOH-terminal domains are required for histone transfer activity. Subcellular localization studies of NAP-2 indicate that it can shuttle between the cytoplasm and the nucleus, suggesting a role as a histone chaperone. Given the potential role of the human NAP-2 gene (HGMW-approved symbol NAP1L4) in WT etiology, we have elucidated the exon/intron structure of this gene and have analyzed the mutational status of NAP-2 in sporadic WTs. Our results, coupled with tumor suppression assays in G401 WT cells, do not support a role for NAP-2 in the etiology of WT. A putative role for NAP-2 in regulating cellular differentiation is discussed. 59 refs., 7 figs., 1 tab.

  14. LAMP-2C Inhibits MHC Class II Presentation of Cytoplasmic Antigens by Disrupting Chaperone-Mediated Autophagy.

    PubMed

    Pérez, Liliana; McLetchie, Shawna; Gardiner, Gail J; Deffit, Sarah N; Zhou, Delu; Blum, Janice S

    2016-03-15

    Cells use multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein-2 (LAMP-2) regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA), which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy and phagocytosis. Yet, far less is known about LAMP-2C function. Whereas LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, we used ectopic gene expression. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact macroautophagy. The gene expression of other LAMP2 isoforms and proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins that are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA that can selectively skew MHCII presentation of cytoplasmic Ags.

  15. Structural and functional significance of the FGL sequence of the periplasmic chaperone Caf1M of Yersinia pestis.

    PubMed

    Chapman, D A; Zavialov, A V; Chernovskaya, T V; Karlyshev, A V; Zav'yalova, G A; Vasiliev, A M; Dudich, I V; Abramov, V M; Zav'yalov, V P; MacIntyre, S

    1999-04-01

    The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 beta-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 beta-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.

  16. Structural and Functional Significance of the FGL Sequence of the Periplasmic Chaperone Caf1M of Yersinia pestis

    PubMed Central

    Chapman, David A. G.; Zavialov, Anton V.; Chernovskaya, Tatiana V.; Karlyshev, Andrey V.; Zav’yalova, Galina A.; Vasiliev, Anatoly M.; Dudich, Igor V.; Abramov, Vyacheslav M.; Zav’yalov, Vladimir P.; MacIntyre, Sheila

    1999-01-01

    The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 β-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 β-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones. PMID:10198004

  17. Interaction of new antidepressants with sigma-1 receptor chaperones and their potentiation of neurite outgrowth in PC12 cells.

    PubMed

    Ishima, Tamaki; Fujita, Yuko; Hashimoto, Kenji

    2014-03-15

    The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [(3)H](+)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine>sertraline>fluoxetine>escitalopram>citalopram>paroxetine>duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5-hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect.

  18. The role of the cytosolic HSP70 chaperone system in diseases caused by misfolding and aberrant trafficking of ion channels.

    PubMed

    Young, Jason C

    2014-03-01

    Protein-folding diseases are an ongoing medical challenge. Many diseases within this group are genetically determined, and have no known cure. Among the examples in which the underlying cellular and molecular mechanisms are well understood are diseases driven by misfolding of transmembrane proteins that normally function as cell-surface ion channels. Wild-type forms are synthesized and integrated into the endoplasmic reticulum (ER) membrane system and, upon correct folding, are trafficked by the secretory pathway to the cell surface. Misfolded mutant forms traffic poorly, if at all, and are instead degraded by the ER-associated proteasomal degradation (ERAD) system. Molecular chaperones can assist the folding of the cytosolic domains of these transmembrane proteins; however, these chaperones are also involved in selecting misfolded forms for ERAD. Given this dual role of chaperones, diseases caused by the misfolding and aberrant trafficking of ion channels (referred to here as ion-channel-misfolding diseases) can be regarded as a consequence of insufficiency of the pro-folding chaperone activity and/or overefficiency of the chaperone ERAD role. An attractive idea is that manipulation of the chaperones might allow increased folding and trafficking of the mutant proteins, and thereby partial restoration of function. This Review outlines the roles of the cytosolic HSP70 chaperone system in the best-studied paradigms of ion-channel-misfolding disease--the CFTR chloride channel in cystic fibrosis and the hERG potassium channel in cardiac long QT syndrome type 2. In addition, other ion channels implicated in ion-channel-misfolding diseases are discussed.

  19. The ATPase cycle of the endoplasmic chaperone Grp94.

    PubMed

    Frey, Stephan; Leskovar, Adriane; Reinstein, Jochen; Buchner, Johannes

    2007-12-07

    Grp94, the Hsp90 paralog of the endoplasmic reticulum, plays a crucial role in protein secretion. Like cytoplasmic Hsp90, Grp94 is regulated by nucleotide binding to its N-terminal domain. However, the question of whether Grp94 hydrolyzes ATP was controversial. This sets Grp94 apart from other members of the Hsp90 family where a slow but specific turnover of ATP has been unambiguously established. In this study we aimed at analyzing the nucleotide binding properties and the potential ATPase activity of Grp94. We show here that Grp94 has an ATPase activity comparable with that of yeast Hsp90 with a k(cat) of 0.36 min(-1) at 25 degrees C. Kinetic and equilibrium constants of the partial reactions of the ATPase cycle were determined using transient kinetic methods. Nucleotide binding appears to be tighter compared with other Hsp90s investigated, with dissociation constants (K(D)) of approximately 4 microm for ADP, ATP, and AMP-PCP. Interestingly, all nucleotides and inhibitors (radicicol, 5'-N-ethylcarboxamidoadenosine) studied here bind with similar rate constants for association (0.2-0.3 x 10(6) M(-1) s(-1)). Furthermore, there is a marked difference from cytosolic Hsp90s in that after binding, the ATP molecule does not seem to become trapped by conformational changes in Grp94. Grp94 stays predominantly in the open state concerning the nucleotide-binding pocket as evidenced by kinetic analyses. Thus, Grp94 shows mechanistically important differences in the interaction with adenosine nucleotides, but the basic hydrolysis reaction seems to be conserved between cytosolic and endoplasmic members of the Hsp90 family.

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

    PubMed

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

    2016-03-22

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

  1. Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex

    PubMed Central

    Daniel Ricketts, M; Frederick, Brian; Hoff, Henry; Tang, Yong; Schultz, David C.; Singh Rai, Taranjit; Grazia Vizioli, Maria; Adams, Peter D.; Marmorstein, Ronen

    2015-01-01

    Histone chaperones bind specific histones to mediate their storage, eviction or deposition from/or into chromatin. The HIRA histone chaperone complex, composed of HIRA, ubinuclein-1 (UBN1) and CABIN1, cooperates with the histone chaperone ASF1a to mediate H3.3-specific binding and chromatin deposition. Here we demonstrate that the conserved UBN1 Hpc2-related domain (HRD) is a novel H3.3-specific-binding domain. Biochemical and biophysical studies show the UBN1-HRD preferentially binds H3.3/H4 over H3.1/H4. X-ray crystallographic and mutational studies reveal that conserved residues within the UBN1-HRD and H3.3 G90 as key determinants of UBN1–H3.3-binding specificity. Comparison of the structure with the unrelated H3.3-specific chaperone DAXX reveals nearly identical points of contact between the chaperone and histone in the proximity of H3.3 G90, although the mechanism for H3.3 G90 recognition appears to be distinct. This study points to UBN1 as the determinant of H3.3-specific binding and deposition by the HIRA complex. PMID:26159857

  2. Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity.

    PubMed

    Minagawa, Shun; Kondoh, Yasumitsu; Sueoka, Keigo; Osada, Hiroyuki; Nakamoto, Hitoshi

    2011-04-01

    Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.

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

    PubMed

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

    2016-08-19

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

  4. Cloning Expression Purification Crystallization and Preliminary X-ray Diffractino Studies of a 12R-LOX-chaperone Complex

    SciTech Connect

    G Deb; K Boeshanes; W Idler; B Ahvazi

    2011-12-31

    Lipoxygenases are a family of nonheme iron-containing dioxygenases. An Escherichia coli expression system producing the bacterial chaperones GroES and GroEL was engineered and successfully used to produce large quantities of recombinant human 12R-LOX (LOXR; MW 80.34 kDa; 701 amino-acid residues). The co-overproduction of the two chaperones with 12R-LOX resulted in increased solubility of 12R-LOX and allowed the purification of milligram amounts of active enzyme for structural studies by X-ray diffraction. The lipoxygenase protein was purified on an affinity column and a gel-filtration column with chaperone protein (MW 57.16 kDa). The LOXR-chaperone complex was crystallized with ligand by the hanging-drop vapor-diffusion method using 1.5 M ammonium hydrogen phosphate as precipitant. The crystals belonged to the monoclinic system, space group P2{sub 1}, with unit-cell parameters a = 138.97, b = 266.11, c = 152.26 {angstrom}, {beta} = 101.07{sup o}. Based on the calculated Matthews coefficient (3.1 {angstrom}3 Da{sup -1}), it is estimated that one molecule of LOXR complexed with two molecules of chaperone is present in the asymmetric unit of the crystal lattice. X-ray diffraction data were collected to 4 {angstrom} resolution using synchrotron radiation.

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

    PubMed Central

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

    2016-01-01

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

  6. The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution

    PubMed Central

    Sarowar, Samema; Hu, Olivia J.; Werneburg, Glenn T.; Thanassi, David G.

    2016-01-01

    ABSTRACT The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains of Escherichia coli use this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in the periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 β-strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function. IMPORTANCE Pili are critical virulence factors for many bacterial pathogens. Uropathogenic E. coli relies on P and type 1 pili assembled by the chaperone/usher pathway to adhere to the urinary tract and establish infection. Studying pilus assembly is important for understanding mechanisms of protein secretion, as well as for identifying points for

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

    PubMed Central

    Spratley, Samantha J.

    2016-01-01

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

  8. The Ydj1 molecular chaperone facilitates formation of active p60v-src in yeast.

    PubMed Central

    Dey, B; Caplan, A J; Boschelli, F

    1996-01-01

    Molecular chaperones have been implicated in the formation of active p60v-src tyrosine kinase. In Saccharomyces cerevisiae, expression of p60v-src causes cell death, a phenomenon that requires functional Hsp90. We show here that mutations in a member of a second class of chaperones, the yeast dnaJ homologue YDJ1, suppress the lethality caused by p60v-src. One p60v-src-resistant ydj1 mutant, ydj1-39, which has two point mutations in the highly conserved "J" domain, has reduced levels of v-src mRNA and protein. However, a ydj1 null mutant produces normal quantities of active p60v-src, indicating that Ydj1p facilitates, but is not essential for, the formation of active p60v-src. We also report p60v-src-resistance in a previously identified temperature-sensitive ydj1 mutant, ydj1-151. In this mutant, the level of p60v-src remains unaltered, but the protein is much less active in vivo. In addition, p60v-src immunoprecipitates from the ydj1-151 strain contained Hsp90 and Hsp70 in greater amounts than in wild-type strains. Ydj1 protein was also detected in p60v-src immunoprecipitates from both wild-type and ydj1-151 strains. These results indicate that Ydj1p participates in the formation of active p60v-src via molecular chaperone complexes. Images PMID:8741842

  9. Tubulin-specific Chaperones: Components of a Molecular Machine that Assembles the α/β Heterodimer

    PubMed Central

    Tian, Guoling; Cowan, Nicholas J.

    2016-01-01

    The tubulin heterodimer consists of one α- and one β-tubulin polypeptide. Neither protein can partition to the native state or assemble into polymerization competent heterodimers without the concerted action of a series of chaperone proteins including five tubulin-specific chaperones termed TBCA-TBCE. TBCA and TBCB bind to and stabilize newly synthesized quasi-native β- and α-tubulin polypeptides following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin, CCT. There is free exchange β-tubulin between TBCA and TBCD, and of α-tubulin between TBCB and TBCE, resulting in the formation of TBCD/β and TBCE/α, respectively. The latter two complexes interact, forming a supercomplex (TBCD/α/TBCD/β). Discharge of the native α/β heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound β-tubulin-bound (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the supercomplex and the release of GDP-bound heterodimer, which becomes polymerization competent following spontaneous E-site exchange with GTP. The tubulin-specific chaperones thus function together as a tubulin assembly machine, marrying the α- and β-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate α- or β-tubulin as stable independent entities in the absence of their cognate partners, and implies that each exists and is maintained in the heterodimer in a non-minimal energy state. Here we describe methods for the purification of recombinant TBC’s as biologically active proteins following their expression in a variety of host/vector systems. PMID:23973072

  10. Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type

    PubMed Central

    Jorge-Finnigan, Ana; Brasil, Sandra; Underhaug, Jarl; Ruíz-Sala, Pedro; Merinero, Begoña; Banerjee, Ruma; Desviat, Lourdes R.; Ugarte, Magdalena; Martinez, Aurora; Pérez, Belén

    2013-01-01

    Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed; six were found to enhance the thermal stability of purified recombinant ATR. Further studies using a well-established bacterial system in which the recombinant ATR protein was expressed in the presence of these six compounds, showed them all to increase the stability of the wild-type ATR and the p.Ile96Thr mutant proteins. Compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) significantly increased this stability and did not act as an inhibitor of the purified protein. Importantly, compound V increased the activity of ATR in patient-derived fibroblasts harboring the destabilizing p.Ile96Thr mutation in a hemizygous state to within control range. When cobalamin was coadministrated with compound V, mutant ATR activity further improved. Oral administration of low doses of compound V to C57BL/6J mice for 12 days, led to increase in steady-state levels of ATR protein in liver and brain (disease-relevant organs). These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperone-responsive mutations. PMID:23674520

  11. Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope.

    PubMed

    Flores-Pérez, Úrsula; Bédard, Jocelyn; Tanabe, Noriaki; Lymperopoulos, Panagiotis; Clarke, Adrian K; Jarvis, Paul

    2016-01-01

    The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone's interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import.

  12. HSP33 in eukaryotes - an evolutionary tale of a chaperone adapted to photosynthetic organisms.

    PubMed

    Segal, Na'ama; Shapira, Michal

    2015-06-01

    HSP33 was originally identified in bacteria as a redox-sensitive chaperone that protects unfolded proteins from aggregation. Here, we describe a eukaryote ortholog of HSP33 from the green algae Chlamydomonas reinhardtii, which appears to play a protective role under light-induced oxidizing conditions. The algal HSP33 exhibits chaperone activity, as shown by citrate synthase aggregation assays. Studies from the Jakob laboratory established that activation of the bacterial HSP33 upon its oxidation initiates by the release of pre-bound Zn from the well conserved Zn-binding motif Cys-X-Cys-Xn -Cys-X-X-Cys, and is followed by significant structural changes (Reichmann et al., ). Unlike the bacterial protein, the HSP33 from C. reinhardtii had lost the first cysteine residue of its center, diminishing Zn-binding activity under all conditions. As a result, the algal protein can be easily activated by minor structural changes in response to oxidation and/or excess heat. An attempt to restore the missing first cysteine did not have a major effect on Zn-binding and on the mode of activation. Replacement of all remaining cysteines abolished completely any residual Zn binding, although the chaperone activation was maintained. A phylogenetic analysis of the algal HSP33 showed that it clusters with the cyanobacterial protein, in line with its biochemical localization to the chloroplast. Indeed, expression of the algal HSP33 increases in response to light-induced oxidative stress, which is experienced routinely by photosynthetic organisms. Despite the fact that no ortholog could be found in higher eukaryotes, its abundance in all algal species examined could have a biotechnological relevance.

  13. Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone.

    PubMed

    Chlenski, Alexandre; Guerrero, Lisa J; Salwen, Helen R; Yang, Qiwei; Tian, Yufeng; Morales La Madrid, Andres; Mirzoeva, Salida; Bouyer, Patrice G; Xu, David; Walker, Matthew; Cohn, Susan L

    2011-01-01

    Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+) concentrations are low, high extracellular concentrations of Ca(2+) activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+) concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the

  14. Liposomes as chaperone mimics with controllable affinity toward heat-denatured formate dehydrogenase from Candida boidinii.

    PubMed

    Yoshimoto, Makoto; Kozono, Ryohei; Tsubomura, Naoki

    2015-01-20

    Chaperone machinery in living systems can catch denatured enzymes and induce their reactivation. Chaperone mimics are beneficial for applying enzymatic reactions in vitro. In this work, the affinity between liposomes and thermally denatured enzymes was controlled to stabilize the enzyme activity. The model enzyme is formate dehydrogenase from Candida boidinii (CbFDH) which is a homodimer and negatively charged in the phosphate buffer solution (pH 7.2) used. The activity of free CbFDH readily decreased at 58 °C following the first-order kinetics with the half-life t1/2 of 27 min. The turbidity measurements showed that the denatured enzyme molecules formed aggregates. The liposomes composed of zwitterionic phosphatidylcholines (PCs) stabilized the CbFDH activity at 58 °C, as revealed with six different PCs. The PC liposomes were indicated to bind to the aggregate-prone enzyme molecules, allowing reactivation at 25 °C. The cofactor β-reduced nicotinamide adenine dinucleotide (NADH) also stabilized the enzyme activity. The affinity between liposomes and denatured CbFDH could be modulated by incorporating cationic 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP) in PC membranes. The t1/2 values significantly increased in the presence of liposomes ([lipid] = 1.5 mM) composed of PC and DOTAP at the mole fraction f(D) of 0.1. On the other hand, the DOTAP-rich liposomes (f(D) ≥ 0.7) showed strong affinity toward denatured CbFDH, accelerating its deactivation. The liposomes with low charge density function as chaperone mimics that can efficiently catch the denatured enzymes without interfering with their intramolecular interaction for reactivation.

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.

    2009-06-05

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

  17. The Mitochondrial Chaperone TRAP1 Promotes Neoplastic Growth by Inhibiting Succinate Dehydrogenase

    PubMed Central

    Sciacovelli, Marco; Guzzo, Giulia; Morello, Virginia; Frezza, Christian; Zheng, Liang; Nannini, Nazarena; Calabrese, Fiorella; Laudiero, Gabriella; Esposito, Franca; Landriscina, Matteo; Defilippi, Paola; Bernardi, Paolo; Rasola, Andrea

    2013-01-01

    Summary We report that the mitochondrial chaperone TRAP1, which is induced in most tumor types, is required for neoplastic growth and confers transforming potential to noncancerous cells. TRAP1 binds to and inhibits succinate dehydrogenase (SDH), the complex II of the respiratory chain. The respiratory downregulation elicited by TRAP1 interaction with SDH promotes tumorigenesis by priming the succinate-dependent stabilization of the proneoplastic transcription factor HIF1α independently of hypoxic conditions. These findings provide a mechanistic clue to explain the switch to aerobic glycolysis of tumors and identify TRAP1 as a promising antineoplastic target. PMID:23747254

  18. Molecular Chaperones and the Assembly of the Prion Ure2p in Vitro*

    PubMed Central

    Savistchenko, Jimmy; Krzewska, Joanna; Fay, Nicolas; Melki, Ronald

    2008-01-01

    The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94–354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3]. PMID:18400756

  19. Membrane chaperoning by members of the PspA/IM30 protein family

    PubMed Central

    Thurotte, Adrien; Brüser, Thomas; Mascher, Thorsten; Schneider, Dirk

    2017-01-01

    ABSTRACT PspA, IM30 (Vipp1) and LiaH, which all belong to the PspA/IM30 protein family, form high molecular weight oligomeric structures. For all proteins membrane binding and protection of the membrane structure and integrity has been shown or postulated. Here we discuss the possible membrane chaperoning activity of PspA, IM30 and LiaH and propose that larger oligomeric structures bind to stressed membrane regions, followed by oligomer disassembly and membrane stabilization by protein monomers or smaller/different oligomeric scaffolds.

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

    PubMed

    Tyagi, Nutan; Tyagi, Rishi

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies. PMID:25365782

  2. Endoplasmic reticulum chaperone gp96 in macrophages is essential for protective immunity during Gram-negative pneumonia.

    PubMed

    Anas, Adam A; de Vos, Alex F; Hoogendijk, Arie J; van Lieshout, Miriam H P; van Heijst, Jeroen W J; Florquin, Sandrine; Li, Zihai; van 't Veer, Cornelis; van der Poll, Tom

    2016-01-01

    Klebsiella pneumoniae is among the most common Gram-negative bacteria that cause pneumonia. Gp96 is an endoplasmic reticulum chaperone that is essential for the trafficking and function of Toll-like receptors (TLRs) and integrins. To determine the role of gp96 in myeloid cells in host defence during Klebsiella pneumonia, mice homozygous for the conditional Hsp90b1 allele encoding gp96 were crossed with mice expressing Cre-recombinase under control of the LysM promoter to generate LysMcre-Hsp90b1-flox mice. LysMcre-Hsp90b1-flox mice showed absence of gp96 protein in macrophages and partial depletion in monocytes and granulocytes. This was accompanied by almost complete absence of TLR2 and TLR4 on macrophages. Likewise, integrin subunits CD11b and CD18 were not detectable on macrophages, while being only slightly reduced on monocytes and granulocytes. Gp96-deficient macrophages did not release pro-inflammatory cytokines in response to Klebsiella and displayed reduced phagocytic capacity independent of CD18. LysMcre-Hsp90b1-flox mice were highly vulnerable to lower airway infection induced by K. pneumoniae, as reflected by enhanced bacterial growth and a higher mortality rate. The early inflammatory response in Hsp90b1-flox mice was characterized by strongly impaired recruitment of granulocytes into the lungs, accompanied by attenuated production of pro-inflammatory cytokines, while the inflammatory response during late-stage pneumonia was not dependent on the presence of gp96. Blocking CD18 did not reproduce the impaired host defence of LysMcre-Hsp90b1-flox mice during Klebsiella pneumonia. These data indicate that macrophage gp96 is essential for protective immunity during Gram-negative pneumonia by regulating TLR expression.

  3. Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106

    SciTech Connect

    Su, Dan; Hu, Qi; Li, Qing; Thompson, James R; Cui, Gaofeng; Fazly, Ahmed; Davies, Brian A; Botuyan, Maria Victoria; Zhang, Zhiguo; Mer, Georges

    2013-04-08

    Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)2 tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)2. We show that the Rtt106-(H3-H4)2 interaction is important for gene silencing and the DNA damage response.

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakd