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Sample records for protein chaperone thio-oxidoreductase

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. RNA helicase proteins as chaperones and remodelers

    PubMed Central

    Jarmoskaite, Inga; Russell, Rick

    2014-01-01

    Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are to promote rearrangements of structured RNAs and to remodel RNA-protein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. While all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of their biological substrates and mechanisms. PMID:24635478

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  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. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Acid-denatured Green Fluorescent Protein (GFP) as model substrate to study the chaperone activity of protein disulfide isomerase.

    PubMed

    Mares, Rosa E; Meléndez-López, Samuel G; Ramos, Marco A

    2011-01-01

    Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  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. Engineering of chaperone systems and of the unfolded protein response

    PubMed Central

    Khan, Saeed U.

    2008-01-01

    Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR. PMID:19003179

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Ric-8 Proteins Are Molecular Chaperones That Direct Nascent G Protein α Subunit Membrane Association

    PubMed Central

    Gabay, Meital; Pinter, Mary E.; Wright, Forrest A.; Chan, PuiYee; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Tall, Gregory G.

    2013-01-01

    Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide–binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A– or Ric-8B–deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A−/− ES cells, which resulted from reduced steady-state amounts of Gαi, Gαq, and Gα13 proteins to <5% of those of wild-type cells. The amounts of Gαs and total Gβ protein were partially reduced in Ric-8A−/− cells compared to those in wild-type cells, and only the amount of Gαs was reduced substantially in Ric-8B−/− cells. The abundances of mRNAs encoding the G protein α subunits were largely unchanged by loss of Ric-8A or Ric-8B. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous Gαi and Gαq were efficiently translated in Ric-8A−/− cells but integrated into endomembranes poorly; however, the reduced amounts of G protein α subunits that reached the membrane still bound to nascent Gβγ. Finally, Gαi, Gαq, and Gβ1 proteins exhibited accelerated rates of degradation in Ric-8A−/− cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent Gα subunits with cellular membranes. PMID:22114146

  17. 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

  18. 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

  19. Proteomic analysis of exported chaperone/co-chaperone complexes of P. falciparum reveals an array of complex protein-protein interactions

    PubMed Central

    Zhang, Qi; Ma, Cheng; Oberli, Alexander; Zinz, Astrid; Engels, Sonja; Przyborski, Jude M.

    2017-01-01

    Malaria parasites modify their human host cell, the mature erythrocyte. This modification is mediated by a large number of parasite proteins that are exported to the host cell, and is also the underlying cause for the pathology caused by malaria infection. Amongst these proteins are many Hsp40 co-chaperones, and a single Hsp70. These proteins have been implicated in several processes in the host cell, including a potential role in protein transport, however the further molecular players in this process remain obscure. To address this, we have utilized chemical cross-linking followed by mass spectrometry and immunoblotting to isolate and characterize proteins complexes containing an exported Hsp40 (PFE55), and the only known exported Hsp70 (PfHsp70x). Our data reveal that both of these proteins are contained in high molecular weight protein complexes. These complexes are found both in the infected erythrocyte, and within the parasite-derived compartment referred to as the parasitophorous vacuole. Surprisingly, our data also reveal an association of PfHsp70x with components of PTEX, a putative protein translocon within the membrane of the parasitophorous vacuole. Our results suggest that the P. falciparum- infected human erythrocyte contains numerous high molecular weight protein complexes, which may potentially be involved in host cell modification. PMID:28218284

  20. 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.

  1. 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

  2. Visualization of a radical B12 enzyme with its G-protein chaperone

    SciTech Connect

    Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; Banerjee, Ruma; Drennan, Catherine L.

    2015-02-09

    G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

  3. Visualization of a radical B12 enzyme with its G-protein chaperone

    DOE PAGES

    Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; ...

    2015-02-09

    G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms ofmore » IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.« less

  4. 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

  5. Peptide-chaperone-directed transdermal protein delivery requires energy.

    PubMed

    Ruan, Renquan; Jin, Peipei; Zhang, Li; Wang, Changli; Chen, Chuanjun; Ding, Weiping; Wen, Longping

    2014-11-03

    The biologically inspired transdermal enhanced peptide TD1 has been discovered to specifically facilitate transdermal delivery of biological macromolecules. However, the biological behavior of TD1 has not been fully defined. In this study, we find that energy is required for the TD1-mediated transdermal protein delivery through rat and human skins. Our results show that the permeation activity of TD1-hEGF, a fusion protein composed of human epidermal growth factor (hEGF) and the TD1 sequence connected with a glycine-serine linker (GGGGS), can be inhibited by the energy inhibitor, rotenone or oligomycin. In addition, adenosine triphosphate (ATP), the essential energetic molecule in organic systems, can effectively facilitate the TD1 directed permeation of the protein-based drug into the skin in a dose-dependent fashion. Our results here demonstrate a novel energy-dependent permeation process during the TD1-mediated transdermal protein delivery that could be valuable for the future development of promising new transdermal drugs.

  6. 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

  7. Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins

    NASA Astrophysics Data System (ADS)

    Rouzina, Ioulia; Vo, My-Nuong; Stewart, Kristen; Musier-Forsyth, Karin; Cruceanu, Margareta; Williams, Mark

    2006-03-01

    Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes.

  8. 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.

  9. 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

  10. 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.

  11. Structural and functional homology between periplasmic bacterial molecular chaperones and small heat shock proteins.

    PubMed

    Zav'yalov, V P; Zav'yalova, G A; Denesyuk, A I; Gaestel, M; Korpela, T

    1995-07-01

    The periplasmic Yersinia pestis molecular chaperone Caf1M belongs to a superfamily of bacterial proteins for one of which (PapD protein of Escherichia coli) the immunoglobulin-like fold was solved by X-ray analysis. The N-terminal domain of Caf1M was found to share a 20% amino acid sequence identity with an inclusion body-associated protein IbpB of Escherichia coli. One of the regions that was compared, was 32 amino acids long, and displayed more than 40% identity, probability of random coincidence was 1.2 x 10(-4). IbpB is involved in a superfamily of small heat shock proteins which fulfil the function of molecular chaperone. On the basis of the revealed homology, an immunoglobulin-like one-domain model of IbpB three-dimensional structure was designed which could be a prototype conformation of sHsp's. The structure suggested is in good agreement with the known experimental data obtained for different members of sHsp's superfamily.

  12. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    PubMed

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

  13. 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

  14. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

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

    PubMed Central

    2016-01-01

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

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

    PubMed

    Lavado-Roldán, Angela; Fernández-Chacón, Rafael

    2016-07-01

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

  18. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

  1. 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

  2. Functional similarity between the chloroplast translocon component, Tic40, and the human co-chaperone, Hsp70-interacting protein (Hip).

    PubMed

    Bédard, Jocelyn; Kubis, Sybille; Bimanadham, Sarat; Jarvis, Paul

    2007-07-20

    Tic40 is a component of the protein import apparatus of the inner envelope of chloroplasts, but its role in the import mechanism has not been clearly defined. The C terminus of Tic40 shares weak similarity with the C-terminal Sti1 domains of the mammalian Hsp70-interacting protein (Hip) and Hsp70/Hsp90-organizing protein (Hop) co-chaperones. Additionally, Tic40 may possess a tetratricopeptide repeat (TPR) protein-protein interaction domain, another characteristic feature of Hip/Hop co-chaperones. To investigate the functional importance of different parts of the Tic40 protein and to determine whether the homology between Tic40 and co-chaperones is functionally significant, different Tic40 deletion and Tic40:Hip fusion constructs were generated and assessed for complementation activity in the Arabidopsis Tic40 knock-out mutant, tic40. Interestingly, all Tic40 deletion constructs failed to complement tic40, indicating that each part removed is essential for Tic40 function; these included a construct lacking the Sti1-like domain (DeltaSti1), a second lacking a central region, including the putative TPR domain (DeltaTPR), and a third lacking the predicted transmembrane anchor region. Moreover, the DeltaSti1 and DeltaTPR constructs caused strong dominant-negative, albino phenotypes in tic40 transformants, indicating that the truncated Tic40 proteins interfere with the residual chloroplast protein import that occurs in tic40 plants. Remarkably, the Tic40:Hip fusion constructs showed that the Sti1 domain of human Hip is functionally equivalent to the Sti1-like region of Tic40, strongly suggesting a co-chaperone role for the Tic40 protein. Supporting this notion, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated the in vivo interaction of Tic40 with Tic110, a protein believed to recruit stromal chaperones to protein import sites.

  3. The early-onset torsion dystonia-associated protein, torsinA, displays molecular chaperone activity in vitro

    PubMed Central

    Burdette, Alexander J.; Churchill, Perry F.; Caldwell, Guy A.

    2010-01-01

    TorsinA is a member of the AAA+ ATPase family of proteins and, notably, is the only known ATPase localized to the ER lumen. It has been suggested to act as a molecular chaperone, while a mutant form associated with early-onset torsion dystonia, a dominantly inherited movement disorder, appears to result in a net loss of function in vivo. Thus far, no studies have examined the chaperone activity of torsinA in vitro. Here we expressed and purified both wild-type (WT) and mutant torsinA fusion proteins in bacteria and examined their ability to function as molecular chaperones by monitoring suppression of luciferase and citrate synthase (CS) aggregation. We also assessed their ability to hold proteins in an intermediate state for refolding. As measured by light scattering and SDS-PAGE, both WT and mutant torsinA effectively, and similarly, suppressed protein aggregation compared to controls. This function was not further enhanced by the presence of ATP. Further, we found that while neither form of torsinA could protect CS from heat-induced inactivation, they were both able to reactivate luciferase when ATP and rabbit reticulocyte lysate were added. This suggests that torsinA holds luciferase in an intermediate state, which can then be refolded in the presence of other chaperones. These data provide conclusive evidence that torsinA acts as a molecular chaperone in vitro and suggests that early-onset torsion dystonia is likely not a consequence of a loss in torsinA chaperone activity but might be an outcome of insufficient torsinA localization at the ER to manage protein folding or trafficking. PMID:20169475

  4. Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK

    PubMed Central

    Eaton, Daniel S.; Crosson, Sean

    2016-01-01

    ABSTRACT Growth in a surface-attached bacterial community, or biofilm, confers a number of advantages. However, as a biofilm matures, high-density growth imposes stresses on individual cells, and it can become less advantageous for progeny to remain in the community. Thus, bacteria employ a variety of mechanisms to control attachment to and dispersal from surfaces in response to the state of the environment. The freshwater oligotroph Caulobacter crescentus can elaborate a polysaccharide-rich polar organelle, known as the holdfast, which enables permanent surface attachment. Holdfast development is strongly inhibited by the small protein HfiA; mechanisms that control HfiA levels in the cell are not well understood. We have discovered a connection between the essential general protein chaperone, DnaK, and control of C. crescentus holdfast development. C. crescentus mutants partially or completely lacking the C-terminal substrate binding “lid” domain of DnaK exhibit enhanced bulk surface attachment. Partial or complete truncation of the DnaK lid domain increases the probability that any single cell will develop a holdfast by 3- to 10-fold. These results are consistent with the observation that steady-state levels of an HfiA fusion protein are significantly diminished in strains that lack the entire lid domain of DnaK. While dispensable for growth, the lid domain of C. crescentus DnaK is required for proper chaperone function, as evidenced by observed dysregulation of HfiA and holdfast development in strains expressing lidless DnaK mutants. We conclude that DnaK is an important molecular determinant of HfiA stability and surface adhesion control. IMPORTANCE Regulatory control of cell adhesion ensures that bacterial cells can transition between free-living and surface-attached states. We define a role for the essential protein chaperone, DnaK, in the control of Caulobacter crescentus cell adhesion. C. crescentus surface adhesion is mediated by an envelope

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. In Vitro Thermodynamic Dissection of Human Copper Transfer from Chaperone to Target Protein

    PubMed Central

    Niemiec, Moritz S.; Weise, Christoph F.; Wittung-Stafshede, Pernilla

    2012-01-01

    Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer. PMID:22574136

  11. Nucleic acid chaperons: a theory of an RNA-assisted protein folding

    PubMed Central

    Biro, Jan C

    2005-01-01

    Background Proteins are assumed to contain all the information necessary for unambiguous folding (Anfinsen's principle). However, ab initio structure prediction is often not successful because the amino acid sequence itself is not sufficient to guide between endless folding possibilities. It seems to be a logical to try to find the "missing" information in nucleic acids, in the redundant codon base. Results mRNA energy dot plots and protein residue contact maps were found to be rather similar. The structure of mRNA is also conserved if the protein structure is conserved, even if the sequence similarity is low. These observations led me to suppose that some similarity might exist between nucleic acid and protein folding. I found that amino acid pairs, which are co-located in the protein structure, are preferentially coded by complementary codons. This codon complementarity is not perfect; it is suboptimal where the 1st and 3rd codon residues are complementary to each other in reverse orientation, while the 2nd codon letters may be, but are not necessarily, complementary. Conclusion Partial complementary coding of co-locating amino acids in protein structures suggests that mRNA assists in protein folding and functions not only as a template but even as a chaperon during translation. This function explains the role of wobble bases and answers the mystery of why we have a redundant codon base. PMID:16137324

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

    PubMed Central

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

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

  13. Mechanistic basis for the recognition of a misfolded protein by the molecular chaperone Hsp90.

    PubMed

    Oroz, Javier; Kim, Jin Hae; Chang, Bliss J; Zweckstetter, Markus

    2017-02-20

    The critical toxic species in over 40 human diseases are misfolded proteins. Their interaction with molecular chaperones such as Hsp90, which preferentially interacts with metastable proteins, is essential for the blocking of disease progression. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the three-dimensional structure of the misfolded cytotoxic monomer of the amyloidogenic human protein transthyretin, which is characterized by the release of the C-terminal β-strand and perturbations of the A-B loop. The misfolded transthyretin monomer, but not the wild-type protein, binds to human Hsp90. In the bound state, the Hsp90 dimer predominantly populates an open conformation, and transthyretin retains its globular structure. The interaction surface for the transthyretin monomer comprises the N-terminal and middle domains of Hsp90 and overlaps with that of the Alzheimer's-disease-related protein tau. Taken together, the data suggest that Hsp90 uses a mechanism for the recognition of aggregation-prone proteins that is largely distinct from those of other Hsp90 clients.

  14. Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness to stress

    PubMed Central

    Escusa-Toret, Stéphanie; Vonk, Willianne I. M.; Frydman, Judith

    2014-01-01

    The extensive links between proteotoxic stress, protein aggregation and pathologies ranging from aging to neurodegeneration underscore the importance of understanding how cells manage protein misfolding. Using live-cell imaging, we here determine the fate of stress-induced misfolded proteins from their initial appearance until their elimination. Upon denaturation, misfolded proteins are sequestered from the bulk cytoplasm into dynamic ER-associated puncta that move and coalesce into larger structures in an energy-dependent but cytoskeleton-independent manner. These puncta, which we name Q-bodies, concentrate different misfolded and stress-denatured proteins en-route to degradation, but do not contain amyloid aggregates, which localize instead to the IPOD. Q-body formation and clearance depends on an intact cortical ER and a complex chaperone network that is affected by rapamycin and impaired during chronological aging. Importantly, Q-body formation enhances cellular fitness during stress. We conclude that spatial sequestration of misfolded proteins in Q-bodies is an early quality control strategy occurring synchronously with degradation to clear the cytoplasm from potentially toxic species. PMID:24036477

  15. Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

    PubMed Central

    Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

    2012-01-01

    Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

  16. Structural insights into yeast histone chaperone Hif1: a scaffold protein recruiting protein complexes to core histones.

    PubMed

    Liu, Hejun; Zhang, Mengying; He, Wei; Zhu, Zhongliang; Teng, Maikun; Gao, Yongxiang; Niu, Liwen

    2014-09-15

    Yeast Hif1 [Hat1 (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hif1 consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.

  17. Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1

    PubMed Central

    Mitterer, Valentin; Gantenbein, Nadine; Birner-Gruenberger, Ruth; Murat, Guillaume; Bergler, Helmut; Kressler, Dieter; Pertschy, Brigitte

    2016-01-01

    After their cytoplasmic synthesis, ribosomal proteins need to be transported into the nucleus, where they assemble with ribosomal RNA into pre-ribosomal particles. Due to their physicochemical properties, they need protection from aggregation on this path. Newly synthesized ribosomal protein Rps3 forms a dimer that is associated with one molecule of its specific chaperone Yar1. Here we report that redundant pathways contribute to the nuclear import of Rps3, with the classical importin α/β pathway (Kap60/Kap95 in yeast) constituting a main import route. The Kap60/Kap95 heterodimer mediates efficient nuclear import of Rps3 by recognition of an N-terminal monopartite nuclear localization signal (NLS). This Rps3-NLS is located directly adjacent to the Yar1-binding site and, upon binding of Kap60 to Rps3, Yar1 is displaced from the ribosomal protein in vitro. While Yar1 does not directly interact with Kap60 in vitro, affinity purifications of Yar1 and Rps3, however, revealed that Kap60 is present in the Rps3/Yar1 complex in vivo. Indeed we could reconstitute such a protein complex containing Rps3 and both Yar1 and Kap60 in vitro. Our data suggest that binding of Yar1 to one N-domain and binding of Kap60 to the second N-domain of dimerized Rps3 orchestrates import and protection of the ribosomal protein. PMID:27819319

  18. Sti1 and Cdc37 can stabilize Hsp90 in chaperone complexes with a protein kinase.

    PubMed

    Lee, Paul; Shabbir, Arsalan; Cardozo, Christopher; Caplan, Avrom J

    2004-04-01

    Hsp90 functions in association with several cochaperones for folding of protein kinases and transcription factors, although the relative contribution of each to the overall reaction is unknown. We assayed the role of nine different cochaperones in the activation of Ste11, a Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase. Studies on signaling via this protein kinase pathway was measured by alpha-factor-stimulated induction of FIG1 or lacZ, and repression of HHF1. Several cochaperone mutants tested had reduced FIG1 induction or HHF1 repression, although to differing extents. The greatest defects were in cpr7Delta, sse1Delta, and ydj1Delta mutants. Assays of Ste11 kinase activity revealed a pattern of defects in the cochaperone mutant strains that were similar to the gene expression studies. Overexpression of CDC37, a chaperone required for protein kinase folding, suppressed defects the sti1Delta mutant back to wild-type levels. CDC37 overexpression also restored stable Hsp90 binding to the Ste11 protein kinase domain in the sti1Delta mutant strain. These data suggest that Cdc37 and Sti1 have functional overlap in stabilizing Hsp90:client complexes. Finally, we show that Cns1 functions in MAP kinase signaling in association with Cpr7.

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

    PubMed

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

    2016-07-20

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

  20. Protein Disulfide Isomerase Chaperone ERP-57 Decreases Plasma Membrane Expression of the Human GnRH Receptor

    PubMed Central

    Yánez, Rodrigo Ayala; Conn, P. Michael

    2012-01-01

    Retention of misfolded proteins by the endoplasmic reticulum (ER) is a quality control mechanism involving the participation of endogenous chaperones such as calnexin (CANX) which interact and restrict plasma membrane expression of gonadotropin releasing hormone receptor (GnRHR), a G protein coupled receptor. CANX also interacts with ERP-57, a thiol oxidoreductase chaperone present in the ER. CANX along with ERP-57, promotes the formation of disulfide bond bridges in nascent proteins. The human GnRH receptor (hGnRHR) is stabilized by two disulfide bond bridges (Cys14-Cys200 and Cys114-Cys196), that, when broken, its expression at plasma membrane decreases. To determine if the presence of chaperones CANX and ERP-57 exert an influence over membrane routing and second messenger activation, we assessed the effect of various mutants including those with broken bridges (Cys→Ala) along with the wild type hGnRHR. The effect of chaperones on mutants was insignificant, whereas the overexpression of ERP-57 led to a wild type hGnRHR retention which was further enhanced by cotransfection with CANX cDNA disclosing receptor retention by ERP-57 augmented by CANX, suggesting a quality control mechanism. PMID:20029959

  1. 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.

  2. 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.

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

    PubMed Central

    2016-01-01

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

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

    SciTech Connect

    Qualley, Dominic F. Sokolove, Victoria L.; Ross, James L.

    2015-03-13

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

  5. Design of heat shock-resistant surfaces to prevent protein aggregation: Enhanced chaperone activity of immobilized α-Crystallin.

    PubMed

    Ray, Namrata; Roy, Sarita; Singha, Santiswarup; Chandra, Bappaditya; Dasgupta, Anjan Kr; Sarkar, Amitabha

    2014-05-21

    α-Crystallin is a multimeric protein belonging to the family of small heat shock proteins, which function as molecular chaperones by resisting heat and oxidative stress induced aggregation of other proteins. We immobilized α-Crystallin on a self-assembled monolayer on glass surface and studied its activity in terms of the prevention of aggregation of aldolase. We discovered that playing with grafted protein density led to interesting variations in the chaperone activity of immobilized α-Crystallin. This result is in accordance with the hypothesis that dynamicity of subunits plays a vital role in the functioning of α-Crystallin and might be able to throw light on the structure-activity relationship. We showed that the chaperone activity of a certain number of immobilized α-Crystallins was superior compared to a solution containing an equivalent number of the protein and 10 times the number of the protein at temperatures >60 °C. The α-Crystallin grafted surfaces retained activity on reuse. This could also lead to the design of potent heat-shock resistant surfaces that can find wide applications in storage and shipping of protein based biopharmaceuticals.

  6. Fidelity drive: a mechanism for chaperone proteins to maintain stable mutation rates in prokaryotes over evolutionary time.

    PubMed

    Xue, Julian Z; Kaznatcheev, Artem; Costopoulos, Andre; Guichard, Frederic

    2015-01-07

    We show a mechanism by which chaperone proteins can play a key role in maintaining the long-term evolutionary stability of mutation rates in prokaryotes with perfect genetic linkage. Since chaperones can reduce the phenotypic effects of mutations, higher mutation rate, by affecting chaperones, can increase the phenotypic effects of mutations. This in turn leads to greater mutation effect among the proteins that control mutation repair and DNA replication, resulting in large changes in mutation rate. The converse of this is that when mutation rate is low and chaperones are functioning well, then the rate of change in mutation rate will also be low, leading to low mutation rates being evolutionarily frozen. We show that the strength of this recursion is critical to determining the long-term evolutionary patterns of mutation rate among prokaryotes. If this recursion is weak, then mutation rates can grow without bound, leading to the extinction of the lineage. However, if this recursion is strong, then we can reproduce empirical patterns of prokaryotic mutation rates, where mutation rates remain stable over evolutionary time, and where most mutation rates are low, but with a significant fraction of high mutators.

  7. Antioxidants Complement the Requirement for Protein Chaperone Function to Maintain β-Cell Function and Glucose Homeostasis.

    PubMed

    Han, Jaeseok; Song, Benbo; Kim, Jiun; Kodali, Vamsi K; Pottekat, Anita; Wang, Miao; Hassler, Justin; Wang, Shiyu; Pennathur, Subramaniam; Back, Sung Hoon; Katze, Michael G; Kaufman, Randal J

    2015-08-01

    Proinsulin misfolding in the endoplasmic reticulum (ER) initiates a cell death response, although the mechanism(s) remains unknown. To provide insight into how protein misfolding may cause β-cell failure, we analyzed mice with the deletion of P58(IPK)/DnajC3, an ER luminal co-chaperone. P58(IPK-/-) mice become diabetic as a result of decreased β-cell function and mass accompanied by induction of oxidative stress and cell death. Treatment with a chemical chaperone, as well as deletion of Chop, improved β-cell function and ameliorated the diabetic phenotype in P58(IPK-/-) mice, suggesting P58(IPK) deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58(IPK-/-) mice and corrected abnormal localization of MafA, a critical transcription factor for β-cell function. Antioxidant feeding also preserved β-cell function in Akita mice that express mutant misfolded proinsulin. Therefore defective protein folding in the β-cell causes oxidative stress as an essential proximal signal required for apoptosis in response to ER stress. Remarkably, these findings demonstrate that antioxidant feeding restores cell function upon deletion of an ER molecular chaperone. Therefore antioxidant or chemical chaperone treatment may be a promising therapeutic approach for type 2 diabetes.

  8. Direct Metal Transfer Between Periplasmic Proteins Identifies a Bacterial Copper Chaperone

    SciTech Connect

    Bagai, I.; Rensing, C.; Blackburn, N.; McEvoy, M.M.

    2009-05-11

    Transition metals require exquisite handling within cells to ensure that cells are not harmed by an excess of free metal species. In Gram-negative bacteria, copper is required in only small amounts in the periplasm, not in the cytoplasm, so a key aspect of protection under excess metal conditions is to export copper from the periplasm. Additional protection could be conferred by a periplasmic chaperone to limit the free metal species prior to export. Using isothermal titration calorimetry, we have demonstrated that two periplasmic proteins, CusF and CusB, of the Escherichia coli Cu(I)/Ag(I) efflux system undergo a metal-dependent interaction. Through the development of a novel X-ray absorption spectroscopy approach using selenomethionine labeling to distinguish the metal sites of the two proteins, we have demonstrated transfer of Cu(I) occurs between CusF and CusB. The interaction between these proteins is highly specific, as a homologue of CusF with a 51% identical sequence and a similar affinity for metal, did not function in metal transfer. These experiments establish a metallochaperone activity for CusF in the periplasm of Gram-negative bacteria, serving to protect the periplasm from metal-mediated damage.

  9. Direct metal transfer between periplasmic proteins identifies a bacterial copper chaperone.

    PubMed

    Bagai, Ireena; Rensing, Christopher; Blackburn, Ninian J; McEvoy, Megan M

    2008-11-04

    Transition metals require exquisite handling within cells to ensure that cells are not harmed by an excess of free metal species. In gram-negative bacteria, copper is required in only small amounts in the periplasm, not in the cytoplasm, so a key aspect of protection under excess metal conditions is to export copper from the periplasm. Additional protection could be conferred by a periplasmic chaperone to limit the free metal species prior to export. Using isothermal titration calorimetry, we have demonstrated that two periplasmic proteins, CusF and CusB, of the Escherichia coli Cu(I)/Ag(I) efflux system undergo a metal-dependent interaction. Through the development of a novel X-ray absorption spectroscopy approach using selenomethionine labeling to distinguish the metal sites of the two proteins, we have demonstrated transfer of Cu(I) occurs between CusF and CusB. The interaction between these proteins is highly specific, as a homologue of CusF with a 51% identical sequence and a similar affinity for metal, did not function in metal transfer. These experiments establish a metallochaperone activity for CusF in the periplasm of gram-negative bacteria, serving to protect the periplasm from metal-mediated damage.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  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. Gene expression and molecular characterization of a chaperone protein HtpG from Bacillus licheniformis.

    PubMed

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

    2016-04-01

    Heat shock protein 90 (Hsp90/HtpG) is a highly abundant and ubiquitous ATP-dependent molecular chaperone consisting of three flexibly linked regions, an N-terminal nucleotide-binding domain, middle domain, and a C-terminal domain. Here the putative htpG gene of Bacillus licheniformis was cloned and heterologously expressed in Escherichia coli M15 cells. Native-gel electrophoresis, size exclusion chromatography, and cross-linking analysis revealed that the recombinant protein probably exists as a mixture of monomer, dimer and other oligomers in solution. The optimal conditions for the ATPase activity of B. licheniformis HtpG (BlHtpG) were 45°C and pH 7.0 in the presence of 0.5mM Mg(2+) ions. The molecular architecture of this protein was stable at higher temperatures with a transition point (Tm) of 45°C at neutral pH, whereas the Tm value was reduced to 40.8°C at pH 10.5. Acrylamide quenching experiment further indicated that the dynamic quenching constant (Ksv) of BlHtpG became larger at higher pH values. BlHtpG also experienced a significant change in the protein conformation upon the addition of ATP and organic solvents. Collectively, our experiment data may provide insights into the molecular properties of BlHtpG and identify the alteration of protein structure to forfeit the ATPase activity at alkaline conditions.

  17. At the Start of the Sarcomere: A Previously Unrecognized Role for Myosin Chaperones and Associated Proteins during Early Myofibrillogenesis

    PubMed Central

    Myhre, J. Layne; Pilgrim, David B.

    2012-01-01

    The development of striated muscle in vertebrates requires the assembly of contractile myofibrils, consisting of highly ordered bundles of protein filaments. Myofibril formation occurs by the stepwise addition of complex proteins, a process that is mediated by a variety of molecular chaperones and quality control factors. Most notably, myosin of the thick filament requires specialized chaperone activity during late myofibrillogenesis, including that of Hsp90 and its cofactor, Unc45b. Unc45b has been proposed to act exclusively as an adaptor molecule, stabilizing interactions between Hsp90 and myosin; however, recent discoveries in zebrafish and C. elegans suggest the possibility of an earlier role for Unc45b during myofibrillogenesis. This role may involve functional control of nonmuscle myosins during the earliest stages of myogenesis, when premyofibril scaffolds are first formed from dynamic cytoskeletal actin. This paper will outline several lines of evidence that converge to build a model for Unc45b activity during early myofibrillogenesis. PMID:22400118

  18. Levels of metacaspase1 and chaperones related to protein quality control in alcoholic and nonalcoholic steatohepatitis.

    PubMed

    Mendoza, Alejandro S; Dorce, Jacques; Peng, Yue; French, Barbara A; Tillman, Brittany; Li, Jun; French, Samuel W

    2015-02-01

    Efficient management of misfolded or aggregated proteins in ASH and NASH is crucial for continued hepatic viability. Cellular protein quality control systems play an important role in the pathogenesis and progression of ASH and NASH. In a recent study, elevated Mca1 expression counteracted aggregation and accumulation of misfolded proteins and extended the life span of the yeast Saccharomyces cerevisiae (Hill et al, 2014). Mca1 may also associate with Ssa1 and Hsp104 in disaggregation and fragmentation of aggregated proteins and their subsequent degradation through the ER-associated degradation (ERAD) pathway. If degradation is not available, protection of the cellular environment from a misfolded protein is accomplished by its sequestration into two distinct inclusion bodies (Kaganovich et al., 2008) called the JUNQ (JUxta Nuclear Quality control compartment) and the IPOD (Insoluble Protein Deposit). Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 all play important roles in protein quality control systems. This study aims to measure the expression of Mca1 and related chaperones involved in protein quality control in alcoholic steatohepatitis (ASH), and nonalcoholic steatohepatitis (NASH) compared with normal control liver biopsies. Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 expressions were measured in three to six formalin-fixed paraffin embedded ASH and NASH liver biopsies and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. Mca1, Hsp104, Ydj1 and p62 were significantly upregulated compared to control (p<0.05) in ASH specimens. Hsp40 and VCP/p97 were also uptrending in ASH. In NASH, the only significant difference was the increased expression of Hsp104 compared to control (p<0.05). Ssa1 levels were uptrending in both ASH and NASH specimens. The upregulation of Mca1, Hsp104, Ydj1 and p62 in ASH may be elicited as a response to the chronic exposure of the hepatocytes to the toxicity of alcohol

  19. The ClpE protein involved in biogenesis of the CS31A capsule-like antigen is a member of a periplasmic chaperone family in gram-negative bacteria.

    PubMed

    Bertin, Y; Girardeau, J P; Der Vartanian, M; Martin, C

    1993-03-15

    The putative chaperone-like protein ClpE, required for biogenesis of the Escherichia coli capsule-like antigen CS31A, was compared with ten known periplasmic chaperones from E. coli, Klebsiella pneumoniae, Bordetella pertussis, Haemophilus influenzae and Yersinia pestis. The amino acid sequence alignment was superimposed onto the three-dimensional structure of the PapD chaperone of uropathogenic E. coli, and amino acid residues involved in maintaining the structure integrity of the suggested binding site were found identical in most of the 11 chaperones. Construction of a phylogenetic tree to investigate the relationship within the chaperone family has revealed interesting degrees of relatedness between the different proteins.

  20. Human pancreas-specific protein disulfide-isomerase (PDIp) can function as a chaperone independently of its enzymatic activity by forming stable complexes with denatured substrate proteins.

    PubMed

    Fu, Xin-Miao; Zhu, Bao Ting

    2010-07-01

    Members of the PDI (protein disulfide-isomerase) family are critical for the correct folding of secretory proteins by catalysing disulfide bond formation as well as by serving as molecular chaperones to prevent protein aggregation. In the present paper, we report that the chaperone activity of the human pancreas-specific PDI homologue (PDIp) is independent of its enzymatic activity on the basis of the following lines of evidence. First, alkylation of PDIp by iodoacetamide fully abolishes its enzymatic activity, whereas it still retains most of its chaperone activity in preventing the aggregation of reduced insulin B chain and denatured GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Secondly, mutation of the cysteine residues in PDIp's active sites completely abolishes its enzymatic activity, but does not affect its chaperone activity. Thirdly, the b-b' fragment of PDIp, which does not contain the active sites and is devoid of enzymatic activity, still has chaperone activity. Mechanistically, we found that both the recombinant PDIp expressed in Escherichia coli and the natural PDIp present in human or monkey pancreas can form stable complexes with thermal-denatured substrate proteins independently of their enzymatic activity. The high-molecular-mass soluble complexes between PDIp and GAPDH are formed in a stoichiometric manner (subunit ratio of 1:3.5-4.5), and can dissociate after storage for a certain time. As a proof-of-concept for the biological significance of PDIp in intact cells, we demonstrated that its selective expression in E. coli confers strong protection of these cells against heat shock and oxidative-stress-induced death independently of its enzymatic activity.

  1. DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.

    PubMed

    Lira, C B B; Gui, K E; Perez, A M; da Silveira, R C V; Gava, L M; Ramos, C H I; Cano, M I N

    2009-02-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.

  2. Thermotolerance and molecular chaperone function of an SGT1-like protein from the psychrophilic yeast, Glaciozyma antarctica.

    PubMed

    Yusof, Nur Athirah; Hashim, Noor Haza Fazlin; Beddoe, Travis; Mahadi, Nor Muhammad; Illias, Rosli Md; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul

    2016-07-01

    The ability of eukaryotes to adapt to an extreme range of temperatures is critically important for survival. Although adaptation to extreme high temperatures is well understood, reflecting the action of molecular chaperones, it is unclear whether these molecules play a role in survival at extremely low temperatures. The recent genome sequencing of the yeast Glaciozyma antarctica, isolated from Antarctic sea ice near Casey Station, provides an opportunity to investigate the role of molecular chaperones in adaptation to cold temperatures. We isolated a G. antarctica homologue of small heat shock protein 20 (HSP20), GaSGT1, and observed that the GaSGT1 mRNA expression in G. antarctica was markedly increased following culture exposure at low temperatures. Additionally, we demonstrated that GaSGT1 overexpression in Escherichia coli protected these bacteria from exposure to both high and low temperatures, which are lethal for growth. The recombinant GaSGT1 retained up to 60 % of its native luciferase activity after exposure to luciferase-denaturing temperatures. These results suggest that GaSGT1 promotes cell thermotolerance and employs molecular chaperone-like activity toward temperature assaults.

  3. 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

  4. Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

    PubMed Central

    Wu, Hao; Wang, Wei; Naiyer, Nada; Fichtenbaum, Eric; Qualley, Dominic F.; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic

  5. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site

    PubMed Central

    Pillet, Benjamin; García-Gómez, Juan J.; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-01-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S

  6. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site.

    PubMed

    Pillet, Benjamin; García-Gómez, Juan J; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-10-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S

  7. The Chaperone-Mediated Autophagy Receptor Organizes in Dynamic Protein Complexes at the Lysosomal Membrane ▿ †

    PubMed Central

    Bandyopadhyay, Urmi; Kaushik, Susmita; Varticovski, Lyuba; Cuervo, Ana Maria

    2008-01-01

    Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which specific cytosolic proteins are sent to lysosomes for degradation. Substrate proteins bind to the lysosomal membrane through the lysosome-associated membrane protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene, and this binding is limiting for their degradation via CMA. However, the mechanisms of substrate binding and uptake remain unknown. We report here that LAMP-2A organizes at the lysosomal membrane into protein complexes of different sizes. The assembly and disassembly of these complexes are a very dynamic process directly related to CMA activity. Substrate proteins only bind to monomeric LAMP-2A, while the efficient translocation of substrates requires the formation of a particular high-molecular-weight LAMP-2A complex. The two major chaperones related to CMA, hsc70 and hsp90, play critical roles in the functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we have identified a novel function for hsc70 in the disassembly of LAMP-2A from these complexes, whereas the presence of lysosome-associated hsp90 is essential to preserve the stability of LAMP-2A at the lysosomal membrane. PMID:18644871

  8. 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

  9. Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.

    PubMed

    Belviso, Benny D; Galliani, Angela; Lasorsa, Alessia; Mirabelli, Valentina; Caliandro, Rocco; Arnesano, Fabio; Natile, Giovanni

    2016-07-05

    Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the

  10. 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

  11. 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.

  12. Virus-Induced Chaperone-Enriched (VICE) domains function as nuclear protein quality control centers during HSV-1 infection.

    PubMed

    Livingston, Christine M; Ifrim, Marius F; Cowan, Ann E; Weller, Sandra K

    2009-10-01

    Virus-Induced Chaperone-Enriched (VICE) domains form adjacent to nuclear viral replication compartments (RC) during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70), the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42 degrees C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.

  13. Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

    PubMed

    Nakamoto, Hitoshi; Fujita, Kensaku; Ohtaki, Aguru; Watanabe, Satoru; Narumi, Shoichi; Maruyama, Takahiro; Suenaga, Emi; Misono, Tomoko S; Kumar, Penmetcha K R; Goloubinoff, Pierre; Yoshikawa, Hirofumi

    2014-02-28

    In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

  14. On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases†

    PubMed Central

    Naik, Subhashchandra; Zhang, Na; Gao, Phillip; Fisher, Mark T.

    2013-01-01

    Correcting aberrant folds that develop during protein folding disease states is now an active research endeavor that is attracting increasing attention from both academic and industrial circles. One particular approach focuses on developing or identifying small molecule correctors or pharmacological chaperones that specifically stabilize the native fold. Unfortunately, the limited screening platforms available to rapidly identify or validate potential drug candidates are usually inadequate or slow because the folding disease proteins in question are often transiently folded and/or aggregation-prone, complicating and/or interfering with the assay outcomes. In this review, we outline and discuss the numerous platform options currently being employed to identify small molecule therapeutics for folding diseases. Finally, we describe a new stability screening approach that is broad based and is easily applicable toward a very large number of both common and rare protein folding diseases. The label free screening method described herein couples the promiscuity of the GroEL binding to transient aggregation-prone hydrophobic folds with surface plasmon resonance enabling one to rapidly identify potential small molecule pharmacological chaperones. PMID:23339304

  15. A Structural Model of the Sgt2 Protein and Its Interactions with Chaperones and the Get4/Get5 Complex*

    PubMed Central

    Chartron, Justin W.; Gonzalez, Grecia M.; Clemons, William M.

    2011-01-01

    The insertion of tail-anchored transmembrane (TA) proteins into the appropriate membrane is a post-translational event that requires stabilization of the transmembrane domain and targeting to the proper destination. Sgt2 is a heat-shock protein cognate (HSC) co-chaperone that preferentially binds endoplasmic reticulum-destined TA proteins and directs them to the GET pathway via Get4 and Get5. Here, we present the crystal structure from a fungal Sgt2 homolog of the tetratrico-repeat (TPR) domain and part of the linker that connects to the C-terminal domain. The linker extends into the two-carboxylate clamp of the TPR domain from a symmetry-related molecule mimicking the binding to HSCs. Based on this structure, we provide biochemical evidence that the Sgt2 TPR domain has the ability to directly bind multiple HSC family members. The structure allows us to propose features involved in this lower specificity relative to other TPR containing co-chaperones. We further show that a dimer of Sgt2 binds a single Get5 and use small angle x-ray scattering to characterize the domain arrangement of Sgt2 in solution. These results allow us to present a structural model of the Sgt2-Get4/Get5-HSC complex. PMID:21832041

  16. 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

  17. Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins.

    PubMed

    Lu, Xinyao; He, Shuying; Zong, Hong; Song, Jian; Chen, Wen; Zhuge, Bin

    2016-09-01

    1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK-dnaJ-grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool.

  18. Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production.

    PubMed

    Crowley, P J; Brady, L J

    2016-02-01

    The respective contributions of components of the protein translocation/maturation machinery to cell surface biogenesis in Streptococcus mutans are not fully understood. Here we used a genetic approach to characterize the effects of deletion of genes encoding the ribosome-associated chaperone RopA (Trigger Factor), the surface-localized foldase PrsA, and the membrane-localized chaperone insertases YidC1 and YidC2, both singly and in combination, on bacterial growth, chain length, self-aggregation, cell surface hydrophobicity, autolysis, and antigenicity of surface proteins P1 (AgI/II, PAc), WapA, GbpC, and GtfD. The single and double deletion mutants, as well as additional mutant strains lacking components of the signal recognition particle pathway, were also evaluated for their effects on mutacin production and genetic competence.

  19. Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production

    PubMed Central

    Crowley, Paula J.; Brady, L. Jeannine

    2015-01-01

    Summary The respective contributions of components of the protein translocation/maturation machinery on cell surface biogenesis in Streptococcus mutans are not fully understood. Here we used a genetic approach to characterize the effects of deletion of genes encoding the ribosome-associated chaperone RopA (Trigger Factor), the surface-localized foldase PrsA, and the membrane-localized chaperone insertases YidC1 and YidC2, both singly and in combination, on bacterial growth, chain length, self-aggregation, cell surface hydrophobicity, autolysis, and antigenicity of surface proteins P1 (AgI/II, PAc), WapA, GbpC and GtfD. The single and double deletion mutants, as well as additional mutant strains lacking components of the signal recognition particle (SRP) pathway, were also evaluated for effects on mutacin production and genetic competence. PMID:26386361

  20. 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

  1. 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

  2. Heat shock protein 70 chaperone overexpression ameliorates phenotypes of the spinal and bulbar muscular atrophy transgenic mouse model by reducing nuclear-localized mutant androgen receptor protein.

    PubMed

    Adachi, Hiroaki; Katsuno, Masahisa; Minamiyama, Makoto; Sang, Chen; Pagoulatos, Gerassimos; Angelidis, Charalampos; Kusakabe, Moriaki; Yoshiki, Atsushi; Kobayashi, Yasushi; Doyu, Manabu; Sobue, Gen

    2003-03-15

    Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR). The nuclear inclusions consisting of the mutant AR protein are characteristic and combine with many components of ubiquitin-proteasome and molecular chaperone pathways, raising the possibility that misfolding and altered degradation of mutant AR may be involved in the pathogenesis. We have reported that the overexpression of heat shock protein (HSP) chaperones reduces mutant AR aggregation and cell death in a neuronal cell model (Kobayashi et al., 2000). To determine whether increasing the expression level of chaperone improves the phenotype in a mouse model, we cross-bred SBMA transgenic mice with mice overexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motor function of the SBMA model mice. In double-transgenic mice, the nuclear-localized mutant AR protein, particularly that of the large complex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting the enhanced degradation of mutant AR. These findings suggest that HSP70 overexpression ameliorates SBMA phenotypes in mice by reducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study may provide the basis for the development of an HSP70-related therapy for SBMA and other polyQ diseases.

  3. glsA, a Volvox gene required for asymmetric division and germ cell specification, encodes a chaperone-like protein.

    PubMed

    Miller, S M; Kirk, D L

    1999-02-01

    The gls genes of Volvox are required for the asymmetric divisions that set apart cells of the germ and somatic lineages during embryogenesis. Here we used transposon tagging to clone glsA, and then showed that it is expressed maximally in asymmetrically dividing embryos, and that it encodes a 748-amino acid protein with two potential protein-binding domains. Site-directed mutagenesis of one of these, the J domain (by which Hsp40-class chaperones bind to and activate specific Hsp70 partners) abolishes the capacity of glsA to rescue mutants. Based on this and other considerations, including the fact that the GlsA protein is associated with the mitotic spindle, we discuss how it might function, in conjunction with an Hsp70-type partner, to shift the division plane in asymmetrically dividing cells.

  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. 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.

  6. 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

    ABSTRACT DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCE Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single

  7. Convergent Synthesis of Homogeneous Glc1Man9GlcNAc2-Protein and Derivatives as Ligands of Molecular Chaperones in Protein Quality Control

    PubMed Central

    Amin, Mohammed N.; Huang, Wei; Mizanur, Rahman M.

    2011-01-01

    A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal1Glc1Man9GlcNAc2-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc1Man9GlcNAc2-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc1Man9GlcNAc2-RNase had high affinity to lectin CRT, while the synthetic Man9GlcNAc2-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal1Glc1Man9GlcNAc2-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between mis-folded and folded glycoproteins in the protein quality control cycle. PMID:21819116

  8. Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase

    PubMed Central

    Amm, Ingo; Norell, Derrick; Wolf, Dieter H.

    2015-01-01

    The Saccharomyces cerevisiae heat shock proteins Hsp31, Hsp32, Hsp33 and Hsp34 belong to the DJ-1/ThiJ/PfpI superfamily which includes the human protein DJ-1 (PARK7) as the most prominent member. Mutations in the DJ-1 gene are directly linked to autosomal recessive, early-onset Parkinson’s disease. DJ-1 acts as an oxidative stress-induced chaperone preventing aggregation and fibrillation of α-synuclein, a critical factor in the development of the disease. In vivo assays in Saccharomyces cerevisiae using the model substrate ΔssCPY*Leu2myc (ΔssCL*myc) as an aggregation-prone misfolded cytoplasmic protein revealed an influence of the Hsp31 chaperone family on the steady state level of this substrate. In contrast to the ubiquitin ligase of the N-end rule pathway Ubr1, which is known to be prominently involved in the degradation process of misfolded cytoplasmic proteins, the absence of the Hsp31 chaperone family does not impair the degradation of newly synthesized misfolded substrate. Also degradation of substrates with strong affinity to Ubr1 like those containing the type 1 N-degron arginine is not affected by the absence of the Hsp31 chaperone family. Epistasis analysis indicates that one function of the Hsp31 chaperone family resides in a pathway overlapping with the Ubr1-dependent degradation of misfolded cytoplasmic proteins. This pathway gains relevance in late growth phase under conditions of nutrient limitation. Additionally, the Hsp31 chaperones seem to be important for maintaining the cellular Ssa Hsp70 activity which is important for Ubr1-dependent degradation. PMID:26466368

  9. A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells.

    PubMed

    Castorena, Kathryn M; Weeks, Spencer A; Stapleford, Kenneth A; Cadwallader, Amy M; Miller, David J

    2007-08-01

    The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.

  10. The ER stress sensor PERK luminal domain functions as a molecular chaperone to interact with misfolded proteins

    SciTech Connect

    Wang, Peng; Li, Jingzhi; Sha, Bingdong

    2016-11-29

    PERK is one of the major sensor proteins which can detect the protein-folding imbalance generated by endoplasmic reticulum (ER) stress. It remains unclear how the sensor protein PERK is activated by ER stress. It has been demonstrated that the PERK luminal domain can recognize and selectively interact with misfolded proteins but not native proteins. Moreover, the PERK luminal domain may function as a molecular chaperone to directly bind to and suppress the aggregation of a number of misfolded model proteins. The data strongly support the hypothesis that the PERK luminal domain can interact directly with misfolded proteins to induce ER stress signaling. To illustrate the mechanism by which the PERK luminal domain interacts with misfolded proteins, the crystal structure of the human PERK luminal domain was determined to 3.2 Å resolution. Two dimers of the PERK luminal domain constitute a tetramer in the asymmetric unit. Superimposition of the PERK luminal domain molecules indicated that the β-sandwich domain could adopt multiple conformations. It is hypothesized that the PERK luminal domain may utilize its flexible β-sandwich domain to recognize and interact with a broad range of misfolded proteins.

  11. The ER stress sensor PERK luminal domain functions as a molecular chaperone to interact with misfolded proteins.

    PubMed

    Wang, Peng; Li, Jingzhi; Sha, Bingdong

    2016-12-01

    PERK is one of the major sensor proteins which can detect the protein-folding imbalance generated by endoplasmic reticulum (ER) stress. It remains unclear how the sensor protein PERK is activated by ER stress. It has been demonstrated that the PERK luminal domain can recognize and selectively interact with misfolded proteins but not native proteins. Moreover, the PERK luminal domain may function as a molecular chaperone to directly bind to and suppress the aggregation of a number of misfolded model proteins. The data strongly support the hypothesis that the PERK luminal domain can interact directly with misfolded proteins to induce ER stress signaling. To illustrate the mechanism by which the PERK luminal domain interacts with misfolded proteins, the crystal structure of the human PERK luminal domain was determined to 3.2 Å resolution. Two dimers of the PERK luminal domain constitute a tetramer in the asymmetric unit. Superimposition of the PERK luminal domain molecules indicated that the β-sandwich domain could adopt multiple conformations. It is hypothesized that the PERK luminal domain may utilize its flexible β-sandwich domain to recognize and interact with a broad range of misfolded proteins.

  12. 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.

  13. The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

    PubMed Central

    Mehnert, Martin; Sommermeyer, Franziska; Berger, Maren; Kumar Lakshmipathy, Sathish; Gauss, Robert; Aebi, Markus; Jarosch, Ernst; Sommer, Thomas

    2015-01-01

    Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1. PMID:25428985

  14. A Hypothetical Protein of Alteromonas macleodii AltDE1 (amad1_06475) Predicted to be a Cold-Shock Protein with RNA Chaperone Activity

    PubMed Central

    Oany, Arafat Rahman; Ahmad, Shah Adil Ishtiyaq; Kibria, KM Kaderi; Hossain, Mohammad Uzzal; Jyoti, Tahmina Pervin

    2014-01-01

    Alteromonas macleodii AltDE1 is a deep sea protobacteria that is distinct from the surface isolates of the same species. This study was designed to elucidate the biological function of amad1_06475, a hypothetical protein of A. macleodii AltDE1. The 70 residues protein sequence showed considerable homology with cold-shock proteins (CSPs) and RNA chaperones from different organisms. Multiple sequence alignment further supported the presence of conserved csp domain on the protein sequence. The three-dimensional structure of the protein was also determined, and verified by PROCHECK, Verify3D, and QMEAN programs. The predicted structure contained five anti-parallel β-strands and RNA-binding motifs, which are characteristic features of prokaryotic CSPs. Finally, the binding of a thymidine-rich oligonucleotide and a single uracil molecule in the active site of the protein further strengthens our prediction about the function of amad1_06475 as a CSP and thereby acting as a RNA chaperone. The binding was performed by molecular docking tools and was compared with similar binding of 3PF5 (PDB) and 2HAX (PDB), major CSPs of Bacillus subtilis and Bacillus caldolyticus, respectively. PMID:25574135

  15. Chaperoning to the metabolic party: The emerging therapeutic role of heat-shock proteins in obesity and type 2 diabetes

    PubMed Central

    Henstridge, Darren C.; Whitham, Martin; Febbraio, Mark A.

    2014-01-01

    Background From their initial, accidental discovery 50 years ago, the highly conserved Heat Shock Proteins (HSPs) continue to exhibit fundamental roles in the protection of cell integrity. Meanwhile, in the midst of an obesity epidemic, research demonstrates a key involvement of low grade inflammation, and mitochondrial dysfunction amongst other mechanisms, in the pathology of insulin resistance and type 2 diabetes mellitus (T2DM). In particular, tumor necrosis factor alpha (TNFα), endoplasmic reticulum (ER) and oxidative stress all appear to be associated with obesity and stimulate inflammatory kinases such as c jun amino terminal kinase (JNK), inhibitor of NF-κβ kinase (IKK) and protein kinase C (PKC) which in turn, inhibit insulin signaling. Mitochondrial dysfunction in skeletal muscle has also been proposed to be prominent in the pathogenesis of T2DM either by reducing the ability to oxidize fatty acids, leading to the accumulation of deleterious lipid species in peripheral tissues such as skeletal muscle and liver, or by altering the cellular redox state. Since HSPs act as molecular chaperones and demonstrate crucial protective functions in stressed cells, we and others have postulated that the manipulation of HSP expression in metabolically relevant tissues represents a therapeutic avenue for obesity-induced insulin resistance. Scope of Review This review summarizes the literature from both animal and human studies, that has examined how HSPs, particularly the inducible HSP, Heat Shock Protein 72 (Hsp72) alters glucose homeostasis and the possible approaches to modulating Hsp72 expression. A summation of the role of chemical chaperones in metabolic disorders is also included. Major Conclusions Targeted manipulation of Hsp72 or use of chemical chaperiones may have clinical utility in treating metabolic disorders such as insulin resistance and T2DM. PMID:25379403

  16. Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones

    PubMed Central

    Campo, Mark Del; Mohr, Sabine; Jiang, Yue; Jia, Huijue; Jankowsky, Eckhard

    2009-01-01

    The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements the mitochondrial translation and group I and II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I as well as group II introns, and functions indistinguishably from CYT-19 to resolve different non-native secondary and/or tertiary structures in the Tetrahymena thermophila LSU-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and vaccinia virus DExH-box protein NPH-II gave little if any group I or II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. By contrast, DbpA binds group I and II intron RNAs non-specifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and II intron splicing correlates primarily with their RNA-unwinding activity, which for the protein preparations used here was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Further, this correlation holds for all group I and II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their

  17. Alpha casein micelles show not only molecular chaperone-like aggregation inhibition properties but also protein refolding activity from the denatured state.

    PubMed

    Sakono, Masafumi; Motomura, Konomi; Maruyama, Tatsuo; Kamiya, Noriho; Goto, Masahiro

    2011-01-07

    Casein micelles are a major component of milk proteins. It is well known that casein micelles show chaperone-like activity such as inhibition of protein aggregation and stabilization of proteins. In this study, it was revealed that casein micelles also possess a high refolding activity for denatured proteins. A buffer containing caseins exhibited higher refolding activity for denatured bovine carbonic anhydrase than buffers including other proteins. In particular, a buffer containing α-casein showed about a twofold higher refolding activity compared with absence of α-casein. Casein properties of surface hydrophobicity, a flexible structure and assembly formation are thought to contribute to this high refolding activity. Our results indicate that casein micelles stabilize milk proteins by both chaperone-like activity and refolding properties.

  18. Isolation of a Latimeria menadoensis heat shock protein 70 (Lmhsp70) that has all the features of an inducible gene and encodes a functional molecular chaperone.

    PubMed

    Modisakeng, Keoagile W; Jiwaji, Meesbah; Pesce, Eva-Rachele; Robert, Jacques; Amemiya, Chris T; Dorrington, Rosemary A; Blatch, Gregory L

    2009-08-01

    Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system.

  19. 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

  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. 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

  3. Heterochromatin protein 1 (HP1) connects the FACT histone chaperone complex to the phosphorylated CTD of RNA polymerase II

    PubMed Central

    Kwon, So Hee; Florens, Laurence; Swanson, Selene K.; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2010-01-01

    Heterochromatin protein 1 (HP1) is well known as a silencing protein found at pericentric heterochromatin. Most eukaryotes have at least three isoforms of HP1 that play differential roles in heterochromatin and euchromatin. In addition to its role in heterochromatin, HP1 proteins have been shown to function in transcription elongation. To gain insights into the transcription functions of HP1, we sought to identify novel HP1-interacting proteins. Biochemical and proteomic approaches revealed that HP1 interacts with the histone chaperone complex FACT (facilitates chromatin transcription). HP1c interacts with the SSRP1 (structure-specific recognition protein 1) subunit and the intact FACT complex. Moreover, HP1c guides the recruitment of FACT to active genes and links FACT to active forms of RNA polymerase II. The absence of HP1c partially impairs the recruitment of FACT into heat-shock loci and causes a defect in heat-shock gene expression. Thus, HP1c functions to recruit the FACT complex to RNA polymerase II. PMID:20889714

  4. Purification of the outer membrane usher protein and periplasmic chaperone-subunit complexes from the P and type 1 pilus systems.

    PubMed

    Henderson, Nadine S; Thanassi, David G

    2013-01-01

    Understanding molecular mechanisms of protein secretion by bacteria requires the purification of secretion machinery components and the isolation of complexes between the secretion machinery and substrate proteins. Here, we describe methods for the purification of proteins from the chaperone/usher pathway, which is a conserved secretion pathway dedicated to the assembly of polymeric surface fibers termed pili or fimbriae in gram-negative bacteria. Specifically, we describe the isolation of the PapC and FimD usher proteins from the bacterial outer membrane, and the purification of PapD-PapG and FimC-FimH chaperone--subunit complexes from the periplasm. These Pap and Fim proteins belong to the P and type 1 pilus systems of uropathogenic Escherichia coli, respectively.

  5. 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

  6. Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA

    PubMed Central

    Cosgriff, Sarah; Chintakayala, Kiran; Chim, Ya Tsz A; Chen, Xinyong; Allen, Stephanie; Lovering, Andrew L; Grainger, David C

    2010-01-01

    The Escherichia coli curved DNA-binding protein A (CbpA) is a nucleoid-associated DNA-binding factor and chaperone that is expressed at high levels as cells enter stationary phase. Using a combination of genetics, biochemistry, structural modelling and single-molecule atomic force microscopy we have examined dimerization of, and DNA binding by, CbpA. Our data show that CbpA dimerization is driven by a hydrophobic surface comprising amino acid side chains W287 and L290 located on the same side of an α helix close to the C-terminus of CbpA. Derivatives of CbpA that are unable to dimerize are also unable to bind DNA. Free in solution, CbpA can exist as either a monomer or dimer. However, when bound to DNA, CbpA forms large aggregates that can protect DNA from degradation by nucleases. These CbpA–DNA aggregates are similar in morphology to protein–DNA complexes formed by the DNA-binding protein from starved cells (Dps), the only other stationary phase-specific nucleoid protein. Conversely, protein–DNA complexes formed by Fis, the major growth phase nucleoid protein, have a markedly different appearance. PMID:20633229

  7. Liposome chaperon in cell-free membrane protein synthesis: one-step preparation of KcsA-integrated liposomes and electrophysiological analysis by the planar bilayer method.

    PubMed

    Ando, M; Akiyama, M; Okuno, D; Hirano, M; Ide, T; Sawada, S; Sasaki, Y; Akiyoshi, K

    2016-02-01

    Chaperoning functions of liposomes were investigated using cell-free membrane protein synthesis. KcsA potassium channel-reconstituted liposomes were prepared directly using cell-free protein synthesis. In the absence of liposomes, all synthesized KcsA protein aggregated. In the presence of liposomes, however, synthesized KcsA spontaneously integrated into the liposome membrane. The KscA-reconstituted liposomes were transferred to the planar bilayer across a small hole in a thin plastic sheet and the channel function of KcsA was examined. The original electrophysiological activities, such as voltage- and pH-dependence, were observed. These results suggested that in cell-free membrane protein synthesis, liposomes act as chaperones, preventing aggregation and assisting in folding and tetrameric formation, thereby allowing full channel activity.

  8. Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance.

    PubMed Central

    Knauf, U; Jakob, U; Engel, K; Buchner, J; Gaestel, M

    1994-01-01

    Small heat shock proteins (sHsps) show a very rapid stress- and mitogen-dependent phosphorylation by MAPKAP kinase 2. Based on this observation, phosphorylation of sHsps was thought to play a key role in mediating thermoresistance immediately after heat shock, before the increased synthesis of heat shock proteins becomes relevant. We have analysed the phosphorylation dependence of the chaperone and thermoresistance-mediating properties of the small heat shock protein Hsp25. Surprisingly, overexpression of Hsp25 mutants, which are not phosphorylated in the transfected cells, confers the same thermoresistant phenotype as overexpression of wild type Hsp25, which is either mono- or bis-phosphorylated at serine residues 15 and 86 within the cells. Furthermore, in vitro phosphorylated Hsp25 shows the same oligomerization properties and the same chaperone activity as the nonphosphorylated protein. No differences between phosphorylated and nonphosphorylated Hsp25 are detected in preventing thermal aggregation of unfolding proteins and assisting refolding of denatured proteins. The results suggest that chaperone properties of the small heat shock proteins contribute to the increased cellular thermoresistance in a phosphorylation-independent manner. Images PMID:7905823

  9. 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.

  10. 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

  11. 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.

  12. Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα

    PubMed Central

    Kant, Ravi; Zeng, Baisen; Thomas, Celestine J; Bothner, Brian; Sprang, Stephen R

    2016-01-01

    Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding. DOI: http://dx.doi.org/10.7554/eLife.19238.001 PMID:28008853

  13. Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα.

    PubMed

    Kant, Ravi; Zeng, Baisen; Thomas, Celestine J; Bothner, Brian; Sprang, Stephen R

    2016-12-23

    Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding.

  14. The Molecular Chaperone Hsp70 Activates Protein Phosphatase 5 (PP5) by Binding the Tetratricopeptide Repeat (TPR) Domain*

    PubMed Central

    Connarn, Jamie N.; Assimon, Victoria A.; Reed, Rebecca A.; Tse, Eric; Southworth, Daniel R.; Zuiderweg, Erik R. P.; Gestwicki, Jason E.; Sun, Duxin

    2014-01-01

    Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development. PMID:24327656

  15. 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.

  16. 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.

  17. 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.

  18. The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome

    PubMed Central

    Reeg, Sandra; Jung, Tobias; Castro, José P.; Davies, Kelvin J.A.; Henze, Andrea; Grune, Tilman

    2016-01-01

    One hallmark of aging is the accumulation of protein aggregates, promoted by the unfolding of oxidized proteins. Unraveling the mechanism by which oxidized proteins are degraded may provide a basis to delay the early onset of features, such as protein aggregate formation, that contribute to the aging phenotype. In order to prevent aggregation of oxidized proteins, cells recur to the 20S proteasome, an efficient turnover proteolysis complex. It has previously been shown that upon oxidative stress the 26S proteasome, another form, dissociates into the 20S form. A critical player implicated in its dissociation is the Heat Shock Protein 70 (Hsp70), which promotes an increase in free 20S proteasome and, therefore, an increased capability to degrade oxidized proteins. The aim of this study was to test whether or not Hsp70 is involved in cooperating with the 20S proteasome for a selective degradation of oxidatively damaged proteins. Our results demonstrate that Hsp70 expression is induced in HT22 cells as a result of mild oxidative stress conditions. Furthermore, Hsp70 prevents the accumulation of oxidized proteins and directly promotes their degradation by the 20S proteasome. In contrast the expression of the Heat shock cognate protein 70 (Hsc70) was not changed in recovery after oxidative stress and Hsc70 has no influence on the removal of oxidatively damaged proteins. We were able to demonstrate in HT22 cells, in brain homogenates from 129/SV mice and in vitro, that there is an increased interaction of Hsp70 with oxidized proteins, but also with the 20S proteasome, indicating a role of Hsp70 in mediating the interaction of oxidized proteins with the 20S proteasome. Thus, our data clearly implicate an involvement of Hsp70 oxidatively damaged protein degradation by the 20S proteasome. PMID:27498116

  19. Chaperoned amyloid proteins for immune manipulation: α-Synuclein/Hsp70 shifts immunity toward a modulatory phenotype

    PubMed Central

    Labrador-Garrido, Adahir; Cejudo-Guillén, Marta; Klippstein, Rebecca; De Genst, Erwin J; Tomas-Gallardo, Laura; Leal, María M; Villadiego, Javier; Toledo-Aral, Juan J; Dobson, Christopher M; Pozo, David; Roodveldt, Cintia

    2014-01-01

    α-Synuclein (αSyn) is a 140-residue amyloid-forming protein whose aggregation is linked to Parkinson's disease (PD). It has also been found to play a critical role in the immune imbalance that accompanies disease progression, a characteristic that has prompted the search for an effective αSyn-based immunotherapy. In this study, we have simultaneously exploited two important features of certain heat-shock proteins (HSPs): their classical “chaperone” activities and their recently discovered and diverse “immunoactive” properties. In particular, we have explored the immune response elicited by immunization of C57BL/6 mice with an αSyn/Hsp70 protein combination in the absence of added adjuvant. Our results show differential effects for mice immunized with the αSyn/Hsp70 complex, including a restrained αSyn-specific (IgM and IgG) humoral response as well as minimized alterations in the Treg (CD4+CD25+Foxp3+) and Teff (CD4+Foxp3−) cell populations, as opposed to significant changes in mice immunized with αSyn and Hsp70 alone. Furthermore, in vitro-stimulated splenocytes from immunized mice showed the lowest relative response against αSyn challenge for the “αSyn/Hsp70” experimental group as measured by IFN-γ and IL-17 secretion, and higher IL-10 levels when stimulated with LPS. Finally, serum levels of Th1-cytokine IFN-γ and immunomodulatory IL-10 indicated a unique shift toward an immunomodulatory/immunoprotective phenotype in mice immunized with the αSyn/Hsp70 complex. Overall, we propose the use of functional “HSP-chaperoned amyloid/aggregating proteins” generated with appropriate HSP-substrate protein combinations, such as the αSyn/Hsp70 complex, as a novel strategy for immune-based intervention against synucleinopathies and other amyloid or “misfolding” neurodegenerative disorders. PMID:25866630

  20. Iron–Sulfur Cluster Biogenesis Chaperones: Evidence for Emergence of Mutational Robustness of a Highly Specific Protein–Protein Interaction

    PubMed Central

    Delewski, Wojciech; Paterkiewicz, Bogumiła; Manicki, Mateusz; Schilke, Brenda; Tomiczek, Bartłomiej; Ciesielski, Szymon J.; Nierzwicki, Lukasz; Czub, Jacek; Dutkiewicz, Rafal; Craig, Elizabeth A.; Marszalek, Jaroslaw

    2016-01-01

    Biogenesis of iron–sulfur clusters (FeS) is a highly conserved process involving Hsp70 and J-protein chaperones. However, Hsp70 specialization differs among species. In most eukaryotes, including Schizosaccharomyces pombe, FeS biogenesis involves interaction between the J-protein Jac1 and the multifunctional Hsp70 Ssc1. But, in Saccharomyces cerevisiae and closely related species, Jac1 interacts with the specialized Hsp70 Ssq1, which emerged through duplication of SSC1. As little is known about how gene duplicates affect the robustness of their protein interaction partners, we analyzed the functional and evolutionary consequences of Ssq1 specialization on the ubiquitous J-protein cochaperone Jac1, by comparing S. cerevisiae and S. pombe. Although deletion of JAC1 is lethal in both species, alanine substitutions within the conserved His–Pro–Asp (HPD) motif, which is critical for Jac1:Hsp70 interaction, have species-specific effects. They are lethal in S. pombe, but not in S. cerevisiae. These in vivo differences correlated with in vitro biochemical measurements. Charged residues present in the J-domain of S. cerevisiae Jac1, but absent in S. pombe Jac1, are important for tolerance of S. cerevisiae Jac1 to HPD alterations. Moreover, Jac1 orthologs from species that encode Ssq1 have a higher sequence divergence. The simplest interpretation of our results is that Ssq1’s coevolution with Jac1 resulted in expansion of their binding interface, thus increasing the efficiency of their interaction. Such an expansion could in turn compensate for negative effects of HPD substitutions. Thus, our results support the idea that the robustness of Jac1 emerged as consequence of its highly efficient and specific interaction with Ssq1. PMID:26545917

  1. 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.

  2. Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli▿†

    PubMed Central

    Prasad, Shivcharan; Khadatare, Prashant B.; Roy, Ipsita

    2011-01-01

    The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium. PMID:21551288

  3. 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...

  4. InvB is a type III secretion-associated chaperone for the Salmonella enterica effector protein SopE.

    PubMed

    Lee, Sang Ho; Galán, Jorge E

    2003-12-01

    SopE is a bacteriophage-encoded effector protein of Salmonella enterica serovar Typhimurium that is translocated into the cytosol of eukaryotic cells by a type III secretion system (TTSS) (W.-D. Hardt, H. Urlaub, and J. E. Galán, Proc. Natl. Acad. Sci. USA 95:2574-2579, 1998; M. W. Wood, R. Rosqvist, P. B. Mullan, M. H. Edwards, and E. E. Galyov, Mol. Microbiol. 22:327-338, 1996). In this study, we provide evidence that an unlinked gene carried within the Salmonella pathogenicity island 1 (SPI-1), invB (K. Eichelberg, C. Ginocchio, and J. E. Galán, J. Bacteriol. 176:4501-4510, 1994), is required for the secretion of SopE through the SPI-1 TTSS. Furthermore, far-Western blotting analysis shows that SopE directly interacts with InvB through a domain located at its amino terminus. We conclude that InvB is the TTSS-associated chaperone for SopE.

  5. 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.

  6. A Cytosolic Chaperone Complexes with Dynamic Membrane J-Proteins and Mobilizes a Nonenveloped Virus out of the Endoplasmic Reticulum

    PubMed Central

    Walczak, Christopher Paul; Ravindran, Madhu Sudhan; Inoue, Takamasa; Tsai, Billy

    2014-01-01

    Nonenveloped viruses undergo conformational changes that enable them to bind to, disrupt, and penetrate a biological membrane leading to successful infection. We assessed whether cytosolic factors play any role in the endoplasmic reticulum (ER) membrane penetration of the nonenveloped SV40. We find the cytosolic SGTA-Hsc70 complex interacts with the ER transmembrane J-proteins DnaJB14 (B14) and DnaJB12 (B12), two cellular factors previously implicated in SV40 infection. SGTA binds directly to SV40 and completes ER membrane penetration. During ER-to-cytosol transport of SV40, SGTA disengages from B14 and B12. Concomitant with this, SV40 triggers B14 and B12 to reorganize into discrete foci within the ER membrane. B14 must retain its ability to form foci and interact with SGTA-Hsc70 to promote SV40 infection. Our results identify a novel role for a cytosolic chaperone in the membrane penetration of a nonenveloped virus and raise the possibility that the SV40-induced foci represent cytosol entry sites. PMID:24675744

  7. The J-domain proteins of Arabidopsis thaliana: an unexpectedly large and diverse family of chaperones.

    PubMed

    Miernyk, J A

    2001-07-01

    A total of 89 J-domain proteins were identified in the genome of the model flowering plant Arabidopsis thaliana. The deduced amino acid sequences of the J-domain proteins were analyzed for an assortment of structural features and motifs. Based on the results of sequence comparisons and structure and function predictions, 51 distinct families were identified. The families ranged in size from 1 to 6 members. Subcellular localizations of the A thaliana J-domain proteins were predicted; species were found in both the soluble and membrane compartments of all cellular organelles. Based on digital Northern analysis, the J-domain proteins could be separated into groups of low, medium, and moderate expression levels. This genomics-based analysis of the A thaliana J-domain proteins establishes a framework for detailed studies of biological function and specificity. It additionally provides a comprehensive basis for evolutionary comparisons.

  8. Structural Features and Chaperone Activity of the NudC Protein Family

    SciTech Connect

    Zheng, Meiying; Cierpicki, Tomasz; Burdette, Alexander J.; Utepbergenov, Darkhan; Janczyk, Pawe; #322; #321; .; Derewenda, Urszula; Stukenberg, P. Todd; Caldwell, Kim A.; Derewenda, Zygmunt S.

    2012-05-25

    The NudC family consists of four conserved proteins with representatives in all eukaryotes. The archetypal nudC gene from Aspergillus nidulans is a member of the nud gene family that is involved in the maintenance of nuclear migration. This family also includes nudF, whose human orthologue, Lis1, codes for a protein essential for brain cortex development. Three paralogues of NudC are known in vertebrates: NudC, NudC-like (NudCL), and NudC-like 2 (NudCL2). The fourth distantly related member of the family, CML66, contains a NudC-like domain. The three principal NudC proteins have no catalytic activity but appear to play as yet poorly defined roles in proliferating and dividing cells. We present crystallographic and NMR studies of the human NudC protein and discuss the results in the context of structures recently deposited by structural genomics centers (i.e., NudCL and mouse NudCL2). All proteins share the same core CS domain characteristic of proteins acting either as cochaperones of Hsp90 or as independent small heat shock proteins. However, while NudC and NudCL dimerize via an N-terminally located coiled coil, the smaller NudCL2 lacks this motif and instead dimerizes as a result of unique domain swapping. We show that NudC and NudCL, but not NudCL2, inhibit the aggregation of several target proteins, consistent with an Hsp90-independent heat shock protein function. Importantly, and in contrast to several previous reports, none of the three proteins is able to form binary complexes with Lis1. The availability of structural information will be of help in further studies on the cellular functions of the NudC family.

  9. 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.

  10. 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.

  11. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    SciTech Connect

    FLANAGAN,J.M.; BEWLEY,M.C.

    2001-12-03

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of -5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol {approx}1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in folding are only completed post-translationally since {approx}40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to aggregation and

  12. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    SciTech Connect

    FLANAGAN,J.M.BEWLEY,M.C.

    2002-10-01

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of {approx}5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol {approx}1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in ffolding are only completed post-translationally since {approx}40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to

  13. Determinants of rodent longevity in the chaperone-protein degradation network.

    PubMed

    Rodriguez, Karl A; Valentine, Joseph M; Kramer, David A; Gelfond, Jonathan A; Kristan, Deborah M; Nevo, Eviatar; Buffenstein, Rochelle

    2016-05-01

    Proteostasis is an integral component of healthy aging, ensuring maintenance of protein structural and functional integrity with concomitant impact upon health span and longevity. In most metazoans, increasing age is accompanied by a decline in protein quality control resulting in the accrual of damaged, self-aggregating cytotoxic proteins. A notable exception to this trend is observed in the longest-lived rodent, the naked mole-rat (NMR, Heterocephalus glaber) which maintains proteostasis and proteasome-mediated degradation and autophagy during aging. We hypothesized that high levels of the proteolytic degradation may enable better maintenance of proteostasis during aging contributing to enhanced species maximum lifespan potential (MLSP). We test this by examining proteasome activity, proteasome-related HSPs, the heat-shock factor 1 (HSF1) transcription factor, and several markers of autophagy in the liver and quadriceps muscles of eight rodent species with divergent MLSP. All subterranean-dwelling species had higher levels of proteasome activity and autophagy, possibly linked to having to dig in soils rich in heavy metals and where underground atmospheres have reduced oxygen availability. Even after correcting for phylogenetic relatedness, a significant (p < 0.02) positive correlation between MLSP, HSP25, HSF1, proteasome activity, and autophagy-related protein 12 (ATG12) was observed, suggesting that the proteolytic degradation machinery and maintenance of protein quality play a pivotal role in species longevity among rodents.

  14. The yeast Hsp70 homolog Ssb: a chaperone for general de novo protein folding and a nanny for specific intrinsically disordered protein domains.

    PubMed

    Hübscher, Volker; Mudholkar, Kaivalya; Rospert, Sabine

    2017-02-01

    Activation of the heterotrimeric kinase SNF1 via phosphorylation of a specific residue within the α subunit is essential for the release from glucose repression in the yeast Saccharomyces cerevisiae. When glucose is available, SNF1 is maintained in the dephosphorylated, inactive state by the phosphatase Glc7-Reg1. Recent findings suggest that Bmh and Ssb combine their unique client-binding properties to interact with the regulatory region of the SNF1 α subunit and by that stabilize a conformation of SNF1, which is accessible for Glc7-Reg1-dependent dephosphorylation. Together, the 14-3-3 protein Bmh and the Hsp70 homolog Ssb comprise a novel chaperone module, which is required to maintain proper glucose repression in the yeast S. cerevisiae.

  15. 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

  16. The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein*

    PubMed Central

    Prior, Kim-Kristin; Wittig, Ilka; Leisegang, Matthias S.; Groenendyk, Jody; Weissmann, Norbert; Michalak, Marek; Jansen-Dürr, Pidder; Shah, Ajay M.; Brandes, Ralf P.

    2016-01-01

    Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum. PMID:26861875

  17. The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein.

    PubMed

    Prior, Kim-Kristin; Wittig, Ilka; Leisegang, Matthias S; Groenendyk, Jody; Weissmann, Norbert; Michalak, Marek; Jansen-Dürr, Pidder; Shah, Ajay M; Brandes, Ralf P

    2016-03-25

    Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.

  18. ClpB chaperone passively threads soluble denatured proteins through its central pore.

    PubMed

    Nakazaki, Yosuke; Watanabe, Yo-Hei

    2014-12-01

    ClpB disaggregase forms a ring-shaped hexamer that threads substrate proteins through the central pore using energy from ATP. The ClpB protomer consists of an N-terminal domain, a middle domain, and two AAA+ modules. These two AAA+ modules bind and hydrolyze ATP and construct the core of the hexameric ring. Here, we investigated the roles of the two AAA+ modules in substrate threading. BAP is an engineered ClpB that can bind ClpP proteolytic chamber; substrates threaded by BAP are degraded by ClpP. We combined BAP with conserved motif mutations in two AAA+ modules and measured the steady-state rates of threading of soluble denatured proteins by these mutants over a range of substrate concentrations. By fitting the data to the Michaelis-Menten equation, k(cat) and K(m) values were determined. We found that the kinetic parameters of the substrate threading correlate with the type of mutation introduced rather than the ATPase activity of the mutant. Moreover, some mutants having no or marginal ATPase activity could thread denatured proteins significantly. These results indicate that ClpB can passively thread soluble denatured proteins.

  19. Compartmentalization of ER-Bound Chaperone Confines Protein Deposit Formation to the Aging Yeast Cell.

    PubMed

    Saarikangas, Juha; Caudron, Fabrice; Prasad, Rupali; Moreno, David F; Bolognesi, Alessio; Aldea, Martí; Barral, Yves

    2017-03-20

    In order to produce rejuvenated daughters, dividing budding yeast cells confine aging factors, including protein aggregates, to the aging mother cell. The asymmetric inheritance of these protein deposits is mediated by organelle and cytoskeletal attachment and by cell geometry. Yet it remains unclear how deposit formation is restricted to the aging lineage. Here, we show that selective membrane anchoring and the compartmentalization of the endoplasmic reticulum (ER) membrane confine protein deposit formation to aging cells during division. Supporting the idea that the age-dependent deposit forms through coalescence of smaller aggregates, two deposits rapidly merged when placed in the same cell by cell-cell fusion. The deposits localized to the ER membrane, primarily to the nuclear envelope (NE). Strikingly, weakening the diffusion barriers that separate the ER membrane into mother and bud compartments caused premature formation of deposits in the daughter cells. Detachment of the Hsp40 protein Ydj1 from the ER membrane elicited a similar phenotype, suggesting that the diffusion barriers and farnesylated Ydj1 functioned together to confine protein deposit formation to mother cells during division. Accordingly, fluorescence correlation spectroscopy measurements in dividing cells indicated that a slow-diffusing, possibly client-bound Ydj1 fraction was asymmetrically enriched in the mother compartment. This asymmetric distribution depended on Ydj1 farnesylation and intact diffusion barriers. Taking these findings together, we propose that ER-anchored Ydj1 binds deposit precursors and prevents them from spreading into daughter cells during division by subjecting them to the ER diffusion barriers. This ensures that the coalescence of precursors into a single deposit is restricted to the aging lineage.

  20. 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.

  1. 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.

  2. Aging, Protein Aggregation, Chaperones, and Neurodegenerative Disorders: Mechanisms of Coupling and Therapeutic Opportunities

    PubMed Central

    Cohen, Ehud

    2012-01-01

    Late onset is a key unifying feature of human neurodegenerative maladies such as Alzheimer’s and Parkinson’s diseases and prion disorders. While sporadic cases typically emerge during the patient’s seventh decade of life or later, mutation-linked, familial cases manifest during the fifth or sixth decade. This common temporal emergence pattern raises the prospect that slowing aging may prevent the accumulation of toxic protein aggregates that lead to the development of these disorders, postpone the onset of these maladies, and alleviate their symptoms once emerged. Invertebrate-based studies indicated that reducing the activity of insulin/IGF signaling (IIS), a prominent aging regulatory pathway, protects from neurodegeneration-linked toxic protein aggregation. The validity of this approach has been tested and confirmed in mammals as reducing the activity of the IGF-1 signaling pathway-protected Alzheimer’s model mice from the behavioral and biochemical impairments associated with the disease. Here I review the recent advances in the field, describe the known mechanistic links between toxic protein aggregation and the aging process, and delineate the future therapeutic potential of IIS reduction as a treatment for various neurodegenerative disorders. PMID:23908845

  3. Aging, protein aggregation, chaperones, and neurodegenerative disorders: mechanisms of coupling and therapeutic opportunities.

    PubMed

    Cohen, Ehud

    2012-10-01

    Late onset is a key unifying feature of human neurodegenerative maladies such as Alzheimer's and Parkinson's diseases and prion disorders. While sporadic cases typically emerge during the patient's seventh decade of life or later, mutation-linked, familial cases manifest during the fifth or sixth decade. This common temporal emergence pattern raises the prospect that slowing aging may prevent the accumulation of toxic protein aggregates that lead to the development of these disorders, postpone the onset of these maladies, and alleviate their symptoms once emerged. Invertebrate-based studies indicated that reducing the activity of insulin/IGF signaling (IIS), a prominent aging regulatory pathway, protects from neurodegeneration-linked toxic protein aggregation. The validity of this approach has been tested and confirmed in mammals as reducing the activity of the IGF-1 signaling pathway-protected Alzheimer's model mice from the behavioral and biochemical impairments associated with the disease. Here I review the recent advances in the field, describe the known mechanistic links between toxic protein aggregation and the aging process, and delineate the future therapeutic potential of IIS reduction as a treatment for various neurodegenerative disorders.

  4. Stress response in the ascidian Ciona intestinalis: transcriptional profiling of genes for the heat shock protein 70 chaperone system under heat stress and endoplasmic reticulum stress.

    PubMed

    Fujikawa, Tetsuya; Munakata, Takeo; Kondo, Shin-ichi; Satoh, Nori; Wada, Shuichi

    2010-03-01

    The genome of Ciona intestinalis contains eight genes for HSP70 superfamily proteins, 36 genes for J-proteins, a gene for a J-like protein, and three genes for BAG family proteins. To understand the stress responses of genes in the HSP70 chaperone system comprehensively, the transcriptional profiles of these 48 genes under heat stress and endoplasmic reticulum (ER) stress were studied using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Heat stress treatment increased the messenger RNA (mRNA) levels of six HSP70 superfamily genes, eight J-protein family genes, and two BAG family genes. In the cytoplasmic group of the DnaK subfamily of the HSP70 family, Ci-HSPA1/6/7-like was the only heat-inducible gene and Ci-HSPA2/8 was the only constitutively active gene which showed striking simplicity in comparison with other animals that have been examined genome-wide so far. Analyses of the time course and temperature dependency of the heat stress responses showed that the induction of Ci-HSPA1/6/7-like expression rises to a peak after heat stress treatment at 28 degrees C (10 degrees C upshift from control temperature) for 1 h. ER stress treatment with Brefeldin A, a drug that is known to act as ER stress inducer, increased the mRNA levels of four HSP70 superfamily genes and four J-protein family genes. Most stress-inducible genes are conserved between Ciona and vertebrates, as expected from a close evolutionary relationship between them. The present study characterized the stress responses of HSP70 chaperone system genes in Ciona for the first time and provides essential data for comprehensive understanding of the functions of the HSP70 chaperone system.

  5. Simultaneous Platinum and Copper Ion Attachment to a Human Copper Chaperone Protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Cvitkovic, John; Yu, Corey; Dmitriev, Oleg; Kaminski, George; Bernholc, Jerry

    2015-03-01

    Cisplatin is a potent anti-cancer drug based on a platinum ion. However, its effectiveness is decreased by cellular resistance, which involves cisplatin attaching to copper transport proteins. One of such proteins is Atox1, where cisplatin attaches to the copper binding site. Surprisingly, it was shown that both cisplatin and copper can attach to Atox1 at the same time. To study this double metal ion attachment, we use the KS/FD DFT method, which combines Kohn-Sham DFT with frozen-density DFT to achieve efficient quantum-mechanical description of explicit solvent. Calculations have so far investigated copper ion attachment to CXXC motifs present in Atox1. The addition of the platinum ion and the competition between the two metals is currently being studied. These calculations start from a molecular mechanics (MM) structural model, in which glutathione groups provide additional ligands to the Pt ion. Our goals are to identify possible Cu-Pt structures and to determine whether copper/platinum attachment is competitive, independent, or cooperative. Results will be compared to the 1H, N1 5 -HSQC NMR experiments, in which binding of copper and cisplatin to Atox1 produces distinct secondary chemical shift signatures, allowing for kinetic studies of simultaneous metal binding.

  6. Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

    PubMed

    Harris, Julia M; Nguyen, Phil P; Patel, Milan J; Sporn, Zachary A; Hines, Justin K

    2014-07-01

    Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have

  7. 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.

  8. The molecular chaperone, Atp12p, from Homo sapiens. In vitro studies with purified wild type and mutant (E240K) proteins.

    PubMed

    Hinton, Ayana; Gatti, Domenico L; Ackerman, Sharon H

    2004-03-05

    Work in Saccharomyces cerevisiae has shown that Atp12p binds to unassembled alpha subunits of F(1) and in so doing prevents the alpha subunit from associating with itself in non-productive complexes during assembly of the F(1) moiety of the mitochondrial ATP synthase. We have developed a method to prepare recombinant Atp12p after expression of its human cDNA in bacterial cells. The molecular chaperone activity of HuAtp12p was studied using citrate synthase as a model substrate. Wild type HuAtp12p suppresses the aggregation of thermally inactivated citrate synthase. In contrast, the mutant protein HuAtp12p(E240K), which harbors a lysine at the position of the highly conserved Glu-240, fails to prevent citrate synthase aggregation at 43 degrees C. No significant differences were observed between the wild type and the mutant proteins as judged by sedimentation analysis, cysteine titration, tryptophan emission spectra, or limited proteolysis, which suggests that the E240K mutation alters the activity of HuAtp12p with minimal effects on the physical integrity of the protein. An additional important finding of this work is that the equilibrium chemical denaturation curve of HuAtp12p shows two components, the first of which is associated with protein aggregation. This result is consistent with a model for Atp12p structure in which there is a hydrophobic chaperone domain that is buried within the protein interior.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins.

    PubMed

    Koide, Takaki; Nishikawa, Yoshimi; Asada, Shinichi; Yamazaki, Chisato M; Takahara, Yoshifumi; Homma, Daisuke L; Otaka, Akira; Ohtani, Katsuki; Wakamiya, Nobutaka; Nagata, Kazuhiro; Kitagawa, Kouki

    2006-04-21

    The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa(-3)) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg(0)) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa(-3) and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa(-3) determined the binding affinity for HSP47. Maximal binding was observed when Yaa(-3) was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr(-3) and one Arg(0). The results revealed that HSP47 recognizes the Yaa(-3) and Arg(0) residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.

  14. Small heat shock protein IbpB acts as a robust chaperone in living cells by hierarchically activating its multi-type substrate-binding residues.

    PubMed

    Fu, Xinmiao; Shi, Xiaodong; Yin, Linxiang; Liu, Jiafeng; Joo, Keehyoung; Lee, Jooyoung; Chang, Zengyi

    2013-04-26

    As ubiquitous molecular chaperones, small heat shock proteins (sHSPs) are crucial for protein homeostasis. It is not clear why sHSPs are able to bind a wide spectrum of non-native substrate proteins and how such binding is enhanced by heat shock. Here, by utilizing a genetically incorporated photo-cross-linker (p-benzoyl-l-phenylalanine), we systematically characterized the substrate-binding residues in IbpB (a sHSP from Escherichia coli) in living cells over a wide spectrum of temperatures (from 20 to 50 °C). A total of 20 and 48 residues were identified at normal and heat shock temperatures, respectively. They are not necessarily hydrophobic and can be classified into three types: types I and II were activated at low and normal temperatures, respectively, and type III mediated oligomerization at low temperature but switched to substrate binding at heat shock temperature. In addition, substrate binding of IbpB in living cells began at temperatures as low as 25 °C and was further enhanced upon temperature elevation. Together, these in vivo data provide novel structural insights into the wide substrate spectrum of sHSPs and suggest that sHSP is able to hierarchically activate its multi-type substrate-binding residues and thus act as a robust chaperone in cells under fluctuating growth conditions.

  15. Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain.

    PubMed

    Bocian-Ostrzycka, Katarzyna M; Łasica, Anna M; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J; Drabik, Karolina; Dobosz, Aneta M; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K

    2015-01-01

    Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

  16. Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

    PubMed Central

    Bocian-Ostrzycka, Katarzyna M.; Łasica, Anna M.; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J.; Drabik, Karolina; Dobosz, Aneta M.; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K.

    2015-01-01

    Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity. PMID:26500620

  17. ANKYRIN REPEAT-CONTAINING PROTEIN 2A is an essential molecular chaperone for peroxisomal membrane-bound ASCORBATE PEROXIDASE3 in Arabidopsis.

    PubMed

    Shen, Guoxin; Kuppu, Sundaram; Venkataramani, Sujatha; Wang, Jing; Yan, Juqiang; Qiu, Xiaoyun; Zhang, Hong

    2010-03-01

    Arabidopsis thaliana ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) interacts with peroxisomal membrane-bound ASCORBATE PEROXIDASE3 (APX3). This interaction involves the C-terminal sequence of APX3 (i.e., a transmembrane domain plus a few basic amino acid residues). The specificity of the AKR2A-APX3 interaction suggests that AKR2A may function as a molecular chaperone for APX3 because binding of AKR2A to the transmembrane domain can prevent APX3 from forming aggregates after translation. Analysis of three akr2a mutants indicates that these mutant plants have reduced steady state levels of APX3. Reduced expression of AKR2A using RNA interference also leads to reduced steady state levels of APX3 and reduced targeting of APX3 to peroxisomes in plant cells. Since AKR2A also binds specifically to the chloroplast OUTER ENVELOPE PROTEIN7 (OEP7) and is required for the biogenesis of OEP7, AKR2A may serve as a molecular chaperone for OEP7 as well. The pleiotropic phenotype of akr2a mutants indicates that AKR2A plays many important roles in plant cellular metabolism and is essential for plant growth and development.

  18. ER stress is associated with reduced ABCA-1 protein levels in macrophages treated with advanced glycated albumin - reversal by a chemical chaperone.

    PubMed

    Castilho, Gabriela; Okuda, Ligia S; Pinto, Raphael S; Iborra, Rodgiro T; Nakandakare, Edna R; Santos, Celio X; Laurindo, Francisco R; Passarelli, Marisa

    2012-07-01

    ATP-binding cassette transporter A1 mediates the export of excess cholesterol from macrophages, contributing to the prevention of atherosclerosis. Advanced glycated albumin (AGE-alb) is prevalent in diabetes mellitus and is associated with the development of atherosclerosis. Independently of changes in ABCA-1 mRNA levels, AGE-alb induces oxidative stress and reduces ABCA-1 protein levels, which leads to macrophage lipid accumulation. These metabolic conditions are known to elicit endoplasmic reticulum (ER) stress. We sought to determine if AGE-alb induces ER stress and unfolded protein response (UPR) in macrophages and how disturbances to the ER could affect ABCA-1 content and cholesterol efflux in macrophages. AGE-alb induced a time-dependent increase in ER stress and UPR markers. ABCA-1 content and cellular cholesterol efflux were reduced by 33% and 47%, respectively, in macrophages treated with AGE-alb, and both were restored by treatment with 4-phenyl butyric acid (a chemical chaperone that alleviates ER stress), but not MG132 (a proteasome inhibitor). Tunicamycin, a classical ER stress inductor, also impaired ABCA-1 expression and cholesterol efflux (showing a decrease of 61% and 82%, respectively), confirming the deleterious effect of ER stress in macrophage cholesterol accumulation. Glycoxidation induces macrophage ER stress, which relates to the reduction in ABCA-1 and in reverse cholesterol transport, endorsing the adverse effect of macrophage ER stress in atherosclerosis. Thus, chemical chaperones that alleviate ER stress may represent a useful tool for the prevention and treatment of atherosclerosis in diabetes.

  19. Differential effects of mutant SOD1 on protein structure of skeletal muscle and spinal cord of familial amyotrophic lateral sclerosis: role of chaperone network.

    PubMed

    Wei, Rochelle; Bhattacharya, Arunabh; Hamilton, Ryan T; Jernigan, Amanda L; Chaudhuri, Asish R

    2013-08-16

    Protein misfolding is considered to be a potential contributing factor for motor neuron and muscle loss in diseases like Amyotrophic lateral sclerosis (ALS). Several independent studies have demonstrated using over-expressed mutated Cu/Zn-superoxide dismutase (mSOD1) transgenic mouse models which mimic familial ALS (f-ALS), that both muscle and motor neurons undergo degeneration during disease progression. However, it is unknown whether protein conformation of skeletal muscle and spinal cord is equally or differentially affected by mSOD1-induced toxicity. It is also unclear whether heat shock proteins (Hsp's) differentially modulate skeletal muscle and spinal cord protein structure during ALS disease progression. We report three intriguing observations utilizing the f-ALS mouse model and cell-free in vitro system; (i) muscle proteins are equally sensitive to misfolding as spinal cord proteins despite the presence of low level of soluble and absence of insoluble G93A protein aggregate, unlike in spinal cord, (ii) Hsp's levels are lower in muscle compared to spinal cord at any stage of the disease, and (iii) G93ASOD1 enzyme-induced toxicity selectively affects muscle protein conformation over spinal cord proteins. Together, these findings strongly suggest that differential chaperone levels between skeletal muscle and spinal cord may be a critical determinant for G93A-induced protein misfolding in ALS.

  20. 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

  1. Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer

    PubMed Central

    Godet, Julien; Kenfack, Cyril; Przybilla, Frédéric; Richert, Ludovic; Duportail, Guy; Mély, Yves

    2013-01-01

    The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (−)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11–55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger–dependent binding of NC to the G10 and G50 residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G10 and G50 the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway. PMID:23511968

  2. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    PubMed

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics.

  3. Plantation forestry under global warming: hybrid poplars with improved thermotolerance provide new insights on the in vivo function of small heat shock protein chaperones.

    PubMed

    Merino, Irene; Contreras, Angela; Jing, Zhong-Ping; Gallardo, Fernando; Cánovas, Francisco M; Gómez, Luis

    2014-02-01

    Climate-driven heat stress is a key factor affecting forest plantation yields. While its effects are expected to worsen during this century, breeding more tolerant genotypes has proven elusive. We report here a substantial and durable increase in the thermotolerance of hybrid poplar (Populus tremula×Populus alba) through overexpression of a major small heat shock protein (sHSP) with convenient features. Experimental evidence was obtained linking protective effects in the transgenic events with the unique chaperone activity of sHSPs. In addition, significant positive correlations were observed between phenotype strength and heterologous sHSP accumulation. The remarkable baseline levels of transgene product (up to 1.8% of total leaf protein) have not been reported in analogous studies with herbaceous species. As judged by protein analyses, such an accumulation is not matched either by endogenous sHSPs in both heat-stressed poplar plants and field-grown adult trees. Quantitative real time-polymerase chain reaction analyses supported these observations and allowed us to identify the poplar members most responsive to heat stress. Interestingly, sHSP overaccumulation was not associated with pleiotropic effects that might decrease yields. The poplar lines developed here also outperformed controls under in vitro and ex vitro culture conditions (callus biomass, shoot production, and ex vitro survival), even in the absence of thermal stress. These results reinforce the feasibility of improving valuable genotypes for plantation forestry, a field where in vitro recalcitrance, long breeding cycles, and other practical factors constrain conventional genetic approaches. They also provide new insights into the biological functions of the least understood family of heat shock protein chaperones.

  4. Plantation Forestry under Global Warming: Hybrid Poplars with Improved Thermotolerance Provide New Insights on the in Vivo Function of Small Heat Shock Protein Chaperones1[C][W

    PubMed Central

    Merino, Irene; Contreras, Angela; Jing, Zhong-Ping; Gallardo, Fernando; Cánovas, Francisco M.; Gómez, Luis

    2014-01-01

    Climate-driven heat stress is a key factor affecting forest plantation yields. While its effects are expected to worsen during this century, breeding more tolerant genotypes has proven elusive. We report here a substantial and durable increase in the thermotolerance of hybrid poplar (Populus tremula × Populus alba) through overexpression of a major small heat shock protein (sHSP) with convenient features. Experimental evidence was obtained linking protective effects in the transgenic events with the unique chaperone activity of sHSPs. In addition, significant positive correlations were observed between phenotype strength and heterologous sHSP accumulation. The remarkable baseline levels of transgene product (up to 1.8% of total leaf protein) have not been reported in analogous studies with herbaceous species. As judged by protein analyses, such an accumulation is not matched either by endogenous sHSPs in both heat-stressed poplar plants and field-grown adult trees. Quantitative real time-polymerase chain reaction analyses supported these observations and allowed us to identify the poplar members most responsive to heat stress. Interestingly, sHSP overaccumulation was not associated with pleiotropic effects that might decrease yields. The poplar lines developed here also outperformed controls under in vitro and ex vitro culture conditions (callus biomass, shoot production, and ex vitro survival), even in the absence of thermal stress. These results reinforce the feasibility of improving valuable genotypes for plantation forestry, a field where in vitro recalcitrance, long breeding cycles, and other practical factors constrain conventional genetic approaches. They also provide new insights into the biological functions of the least understood family of heat shock protein chaperones. PMID:24306533

  5. Water-structuring technology with the molecular chaperone proteins: indicated application of the α-crystallin domains and imidazole-containing peptidomimetics in cosmetic skin care systems or dermatological therapeutic drug carrier formulations.

    PubMed

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

    2011-01-01

    Changes in structural proteins and hydration during aging are responsible for altered skin morphologic and mechanical properties manifested as wrinkling, sagging, loss of elasticity, and apparent dryness. Impairment in protein hydration may add to the ultrastructural, mechanical, and biochemical changes in structural proteins in the aged skin. At Innovative Vision Products, Inc., we have pioneered a molecular chaperone protein-activated therapeutic or cosmetic platform to enable simultaneous analysis of water-binding and structuring characteristics for biology-related or skin aging and skin disease-related pathways. This cutting-edge technology has changed the hydration of proteins in photoaged skin which so that they are more compact and interact with water to limited degree. The mechanisms of skin diseases, aging, and cellular and signaling pathways mediated by targeting with molecular chaperone protein(s) are considered. Skin lesions that are growing, spreading, or pigmented, and those that occur on exposed areas of skin are likely to be treated by these emerging pharmacological chaperones that could have cosmetic or dermatological benefits. Examples of such chaperones are anti-/trans-glycation-imidazole-containing peptidomimetic(s) (natural L-carnosine derivatives and mimetics) combined with the molecular chaperone protein α-crystallin derived from a natural source, brine shrimp (Artemia franciscana) cysts, or with recombinant human αA-crystallin. This patented biotechnology represents an efficient tool with which to mitigate the consequences of free radical-induced skin damage. The article is organized to provide in one place all of the relevant technical information, such as high-performance nuclear magnetic resonance and electron spin resonance application tools, and to describe the entire process from sample preparation to data analysis, which is moving from biological studies to biotechnology batches of the product. The proposed biotechnology results in

  6. Rice ASR1 protein with reactive oxygen species scavenging and chaperone-like activities enhances acquired tolerance to abiotic stresses in Saccharomyces cerevisiae.

    PubMed

    Kim, Il-Sup; Kim, Young-Saeng; Yoon, Ho-Sung

    2012-03-01

    Abscisic acid stress ripening (ASR1) protein is a small hydrophilic, low molecular weight, and stress-specific plant protein. The gene coding region of ASR1 protein, which is induced under high salinity in rice (Oryza sativa Ilmi), was cloned into a yeast expression vector pVTU260 and transformed into yeast cells. Heterologous expression of ASR1 protein in transgenic yeast cells improved tolerance to abiotic stresses including hydrogen peroxide (H(2)O(2)), high salinity (NaCl), heat shock, menadione, copper sulfate, sulfuric acid, lactic acid, salicylic acid, and also high concentration of ethanol. In particular, the expression of metabolic enzymes (Fba1p, Pgk1p, Eno2p, Tpi1p, and Adh1p), antioxidant enzyme (Ahp1p), molecular chaperone (Ssb1p), and pyrimidine biosynthesis-related enzyme (Ura1p) was up-regulated in the transgenic yeast cells under oxidative stress when compared with wild-type cells. All of these enzymes contribute to an alleviated redox state to H2O2-induced oxidative stress. In the in vitro assay, the purified ASR1 protein was able to scavenge ROS by converting H(2)O(2) to H(2)O. Taken together, these results suggest that the ASR1 protein could function as an effective ROS scavenger and its expression could enhance acquired tolerance of ROS-induced oxidative stress through induction of various cell rescue proteins in yeast cells.

  7. 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

  8. Crystallization and preliminary X-ray diffraction analysis of YidC, a membrane-protein chaperone and insertase from Bacillus halodurans

    SciTech Connect

    Kumazaki, Kaoru; Tsukazaki, Tomoya; Nishizawa, Tomohiro; Tanaka, Yoshiki; Kato, Hideaki E.; Nakada-Nakura, Yoshiko; Hirata, Kunio; Mori, Yoshihiro; Suga, Hiroaki; Dohmae, Naoshi; Ishitani, Ryuichiro; Nureki, Osamu

    2014-07-23

    YidC, a membrane-protein chaperone/insertase from B. halodurans, was expressed, purified and crystallized in the lipidic cubic phase. An X-ray diffraction data set was collected to 2.4 Å resolution. YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, Bacillus halodurans YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4 Å resolution and belonged to space group P2{sub 1}, with unit-cell parameters a = 43.9, b = 60.6, c = 58.9 Å, β = 100.3°. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.

  9. 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.

  10. 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

  11. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    PubMed

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress.

  12. 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

  13. CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma.

    PubMed

    Qian, Jie; Yi, Huanfa; Guo, Chunqing; Yu, Xiaofei; Zuo, Daming; Chen, Xing; Kane, John M; Repasky, Elizabeth A; Subjeck, John R; Wang, Xiang-Yang

    2011-09-15

    We previously reported that scavenger receptor A (SRA/CD204), a binding structure on dendritic cells (DCs) for large stress/heat shock proteins (HSPs; e.g., hsp110 and grp170), attenuated an antitumor response elicited by large HSP-based vaccines. In this study, we show that SRA/CD204 interacts directly with exogenous hsp110, and lack of SRA/CD204 results in a reduction in the hsp110 binding and internalization by DCs. However, SRA(-/-) DCs pulsed with hsp110 or grp170-reconstituted gp100 chaperone complexes exhibit a profoundly increased capability of stimulating melanoma Ag gp100-specific naive T cells compared with wild-type (WT) DCs. Similar results were obtained when SRA/CD204 was silenced in DCs using short hairpin RNA-encoding lentiviruses. In addition, hsp110-stimulated SRA(-/-) DCs produced more inflammatory cytokines associated with increased NF-κB activation, implicating an immunosuppressive role for SRA/CD204. Immunization with the hsp110-gp100 vaccine resulted in a more robust gp100-specific CD8(+) T cell response in SRA(-/-) mice than in WT mice. Lastly, SRA/CD204 absence markedly improved the therapeutic efficacy of the hsp110-gp100 vaccine in mice established with B16 melanoma, which was accompanied by enhanced activation and tumor infiltration of CD8(+) T cells. Given the presence of multiple HSP-binding scavenger receptors on APCs, we propose that selective scavenger receptor interactions with HSPs may lead to highly distinct immunological consequences. Our findings provide new insights into the immune regulatory functions of SRA/CD204 and have important implications in the rational design of protein Ag-targeted recombinant chaperone vaccines for the treatment of cancer.

  14. 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.

  15. 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

  16. 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

  17. PDE5 inhibitors enhance the lethality of pemetrexed through inhibition of multiple chaperone proteins and via the actions of cyclic GMP and nitric oxide

    PubMed Central

    Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Gordon, Sarah; Dent, Paul

    2017-01-01

    Phosphodiesterase 5 (PDE5) inhibitors prevent the breakdown of cGMP that results in prolonged protein kinase G activation and the generation of nitric oxide. PDE5 inhibitors enhanced the anti-NSCLC cell effects of the NSCLC therapeutic pemetrexed. [Pemetrexed + sildenafil] activated an eIF2α – ATF4 – CHOP – Beclin1 pathway causing formation of toxic autophagosomes; activated a protective IRE1 – XBP-1 – chaperone induction pathway; and activated a toxic eIF2α – CHOP – DR4 / DR5 / CD95 induction pathway. [Pemetrexed + sildenafil] reduced the expression of c-FLIP-s, MCL-1 and BCL-XL that was blocked in a cell-type -dependent fashion by either over-expression of HSP90 / GRP78 / HSP70 / HSP27 or by blockade of eIF2α-CHOP signaling. Knock down of PKGI/II abolished the ability of sildenafil to enhance pemetrexed toxicity whereas pan-inhibition of NOS using L-NAME or knock down of [iNOS + eNOS] only partially reduced the lethal drug interaction. Pemetrexed reduced the ATPase activities of HSP90 and HSP70 in an ATM-AMPK-dependent fashion that was enhanced by sildenafil signaling via PKGI/II. The drug combination activated an ATM-AMPK-TSC2 pathway that was associated with reduced mTOR S2448 and ULK-1 S757 phosphorylation and increased ULK-1 S317 and ATG13 S318 phosphorylation. These effects were prevented by chaperone over-expression or by expression of an activated form of mTOR that prevented autophagosome formation and reduced cell killing. In two models of NSCLC, sildenafil enhanced the ability of pemetrexed to suppress tumor growth. Collectively we argue that the combination of [pemetrexed + PDE5 inhibitor] should be explored in a new NSCLC phase I trial. PMID:27903966

  18. 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.

  19. Receptor-associated protein (RAP) has two high-affinity binding sites for the low-density lipoprotein receptor-related protein (LRP): consequences for the chaperone functions of RAP.

    PubMed

    Jensen, Jan K; Dolmer, Klavs; Schar, Christine; Gettins, Peter G W

    2009-06-26

    RAP (receptor-associated protein) is a three domain 38 kDa ER (endoplasmic reticulum)-resident protein that is a chaperone for the LRP (low-density lipoprotein receptor-related protein). Whereas RAP is known to compete for binding of all known LRP ligands, neither the location, the number of binding sites on LRP, nor the domains of RAP involved in binding is known with certainty. We have systematically examined the binding of each of the three RAP domains (D1, D2 and D3) to tandem and triple CRs (complement-like repeats) that span the principal ligand-binding region, cluster II, of LRP. We found that D3 binds with low nanomolar affinity to all (CR)2 species examined. Addition of a third CR domain increases the affinity for D3 slightly. A pH change from 7.4 to 5.5 gave only a 6-fold increase in Kd for D3 at 37 degrees C, whereas temperature change from 22 degrees C to 37 degrees C has a similar small effect on affinity, raising questions about the recently proposed D3-destabilization mechanism of RAP release from LRP. Surprisingly, and in contrast to literature suggestions, D1 and D2 also bind to most (CR)2 and (CR)3 constructs with nanomolar affinity. Although this suggested that there might be three high-affinity binding sites in RAP for LRP, studies with intact RAP showed that only two binding sites are available in the intact chaperone. These findings suggest a new model for RAP to function as a folding chaperone and also for the involvement of YWTD domains in RAP release from LRP in the Golgi.

  20. Action of multiple endoplasmic reticulum chaperon-like proteins is required for proper folding and polarized localization of Kre6 protein essential in yeast cell wall β-1,6-glucan synthesis.

    PubMed

    Kurita, Tomokazu; Noda, Yoichi; Yoda, Koji

    2012-05-18

    Saccharomyces cerevisiae Kre6 is a type II membrane protein essential for cell wall β-1,6-glucan synthesis. Recently we reported that the majority of Kre6 is in the endoplasmic reticulum (ER), but a significant portion of Kre6 is found in the plasma membrane of buds, and this polarized appearance of Kre6 is required for β-1,6-glucan synthesis. An essential membrane protein, Keg1, and ER chaperon Rot1 bind to Kre6. In this study we found that in mutant keg1-1 cells, accumulation of Kre6 at the buds is diminished, binding of Kre6 to Keg1 is decreased, and Kre6 becomes susceptible to ER-associated degradation (ERAD), which suggests Keg1 participates in folding and transport of Kre6. All mutants of the calnexin cycle member homologues (cwh41, rot2, kre5, and cne1) showed defects in β-1,6-glucan synthesis, although the calnexin chaperon system is considered not functional in yeast. We found synthetic defects between them and keg1-1, and Cne1 co-immunoprecipitated with Keg1 and Kre6. A stronger binding of Cne1 to Kre6 was detected when two glucosidases (Cwh41 and Rot2) that remove glucose on N-glycan were functional. Skn1, a Kre6 homologue, was not detected by immunofluorescence in the wild type yeast, but in kre6Δ cells it became detectable and behaved like Kre6. In conclusion, the action of multiple ER chaperon-like proteins is required for proper folding and localization of Kre6 and probably Skn1 to function in β-1,6-glucan synthesis.

  1. Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast.

    PubMed

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H

    2015-02-20

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2.

  2. The endoplasmic reticulum-resident chaperone heat shock protein 47 protects the Golgi apparatus from the effects of O-glycosylation inhibition.

    PubMed

    Miyata, Shingo; Mizuno, Tatsunori; Koyama, Yoshihisa; Katayama, Taiichi; Tohyama, Masaya

    2013-01-01

    The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.

  3. Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3*

    PubMed Central

    Pradeepa, Madapura M.; Nikhil, Gupta; Hari Kishore, Annavarapu; Bharath, Giriyapura N.; Kundu, Tapas K.; Rao, Manchanahalli R. Satyanarayana

    2009-01-01

    The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids. Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis. PMID:19710011

  4. 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.

  5. 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

  6. Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA.

    PubMed

    Karlsson, Oskar; Berg, Anna-Lena; Hanrieder, Jörg; Arnerup, Gunnel; Lindström, Anna-Karin; Brittebo, Eva B

    2015-03-01

    The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease, and recent studies indicate that BMAA can be misincorporated into proteins. BMAA is a developmental neurotoxicant that can induce long-term learning and memory deficits, as well as regionally restricted neuronal degeneration and mineralization in the hippocampal CA1. The aim of the study was to characterize long-term changes (2 weeks to 6 months) further in the brain of adult rats treated neonatally (postnatal days 9-10) with BMAA (460 mg/kg) using immunohistochemistry (IHC), transmission electron microscopy, and laser capture microdissection followed by LC-MS/MS for proteomic analysis. The histological examination demonstrated progressive neurodegenerative changes, astrogliosis, microglial activation, and calcification in the hippocampal CA1 3-6 months after exposure. The IHC showed an increased staining for α-synuclein and ubiquitin in the area. The ultrastructural examination revealed intracellular deposition of abundant bundles of closely packed parallel fibrils in neurons, axons, and astrocytes of the CA1. Proteomic analysis of the affected site demonstrated an enrichment of chaperones (e.g., clusterin, GRP-78), cytoskeletal and intermediate filament proteins, and proteins involved in the antioxidant defense system. Several of the most enriched proteins (plectin, glial fibrillar acidic protein, vimentin, Hsp 27, and ubiquitin) are known to form complex astrocytic inclusions, so-called Rosenthal fibers, in the neurodegenerative disorder Alexander disease. In addition, TDP-43 and the negative regulator of autophagy, GLIPR-2, were exclusively detected. The present study demonstrates that neonatal exposure to BMAA may offer a novel model for the study of hippocampal fibril formation in vivo.

  7. Structural Characterization of the Yersinia pestis Type III Secretion System Needle Protein YscF in Complex with Its Heterodimeric Chaperone YscE/YscG

    SciTech Connect

    Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

    2008-05-03

    The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.

  8. Mutational Analysis of Glycogen Synthase Kinase 3β Protein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90

    PubMed Central

    Pasculescu, Adrian; Dai, Anna Yue; Williton, Kelly; Taylor, Lorne; Savitski, Mikhail M.; Bantscheff, Marcus; Woodgett, James R.; Pawson, Tony; Colwill, Karen

    2016-01-01

    The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms. Using model cell lines stably expressing these two GSK3β forms, we observed no interaction between WT GSK3β and HSP90, in stark contrast to KD GSK3β forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome. PMID:26755559

  9. 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.

  10. Serological detection of ‘Candidatus Liberibacter asiaticus’ in citrus, and identification by GeLC-MS/MS of a chaperone protein responding to cellular pathogens

    PubMed Central

    Ding, Fang; Duan, Yongping; Yuan, Qing; Shao, Jonathan; Hartung, John S.

    2016-01-01

    We describe experiments with antibodies against ‘Candidatus Liberibacter asiaticus used to detect the pathogen in infected plants. We used scFv selected to bind epitopes exposed on the surface of the bacterium in tissue prints, with secondary monoclonal antibodies directed at a FLAG epitope included at the carboxyl end of the scFv. Unexpectedly, the anti-FLAG secondary antibody produced positive results with CaLas diseased samples when the primary scFv were not used. The anti-FLAG monoclonal antibody (Mab) also identified plants infected with other vascular pathogens. We then identified a paralogous group of secreted chaperone proteins in the HSP-90 family that contained the amino acid sequence DDDDK identical to the carboxy-terminal sequence of the FLAG epitope. A rabbit polyclonal antibody against one of the same epitopes combined with a goat anti-rabbit secondary antibody produced very strong purple color in individual phloem cells, as expected for this pathogen. These results were entirely specific for CaLas-infected citrus. The simplicity, cost and ability to scale the tissue print assay makes this an attractive assay to complement PCR-based assays currently in use. The partial FLAG epitope may itself be useful as a molecular marker for the rapid screening of citrus plants for the presence of vascular pathogens. PMID:27381064

  11. The aqueous extract of Glycyrrhiza inflata can upregulate unfolded protein response-mediated chaperones to reduce tau misfolding in cell models of Alzheimer’s disease

    PubMed Central

    Chang, Kuo-Hsuan; Chen, I-Cheng; Lin, Hsuan-Yuan; Chen, Hsuan-Chiang; Lin, Chih-Hsin; Lin, Te-Hsien; Weng, Yu-Ting; Chao, Chih-Ying; Wu, Yih-Ru; Lin, Jung-Yaw; Lee-Chen, Guey-Jen; Chen, Chiung-Mei

    2016-01-01

    Background Alzheimer’s disease (AD) and several neurodegenerative disorders known as tauopathies are characterized by misfolding and aggregation of tau protein. Although several studies have suggested the potential of traditional Chinese medicine (TCM) as treatment for neurodegenerative diseases, the role of TCM in treating AD and tauopathies have not been well explored. Materials and methods Tau protein was coupled to the DsRed fluorophore by fusing a pro-aggregation mutant of repeat domain of tau (ΔK280 tauRD) with DsRed. The ΔK280 tauRD-DsRed fusion gene was then used to generate Tet-On 293 and SH-SY5Y cell clones as platforms to test the efficacy of 39 aqueous extracts of TCM in reducing tau misfolding and in neuroprotection. Results Seven TCM extracts demonstrated a significant reduction in tau misfolding and reactive oxidative species with low cytotoxicity in the ΔK280 tauRD-DsRed 293 cell model. Glycyrrhiza inflata and Panax ginseng also demonstrated the potential to improve neurite outgrowth in the ΔK280 tauRD-DsRed SH-SY5Y neuronal cell model. G. inflata further rescued the upregulation of ERN2 (pro-apoptotic) and downregulation of unfolded-protein-response-mediated chaperones ERP44, DNAJC3, and SERP1 in ΔK280 tauRD-DsRed 293 cells. Conclusion This in vitro study provides evidence that G. inflata may be a novel therapeutic for AD and tauopathies. Future applications of G. inflata on animal models of AD and tauopathies are warranted to corroborate its effect of reducing misfolding and potential disease modification. PMID:27013866

  12. 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

  13. The molecular chaperone TRiC/CCT binds to the Trp-Asp 40 (WD40) repeat protein WDR68 and promotes its folding, protein kinase DYRK1A binding, and nuclear accumulation.

    PubMed

    Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

    2014-11-28

    Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68.

  14. The molecular chaperone HSP70 binds to and stabilizes NOD2, an important protein involved in Crohn disease.

    PubMed

    Mohanan, Vishnu; Grimes, Catherine Leimkuhler

    2014-07-04

    Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand.

  15. Structural Model of Dodecameric Heat-shock Protein Hsp21 - Flexible N-terminal Arms Interact with Client Proteins while C-terminal Tails Maintain the Dodecamer and Chaperone Activity.

    PubMed

    Rutsdottir, Gudrun; Härmark, Johan; Weide, Yoran; Hebert, Hans; Rasmussen, Morten Ib; Wernersson, Sven; Respondek, Michal; Akke, Mikael; Højrup, Peter; Koeck, Philip J B; Söderberg, Christopher A G; Emanuelsson, Cecilia

    2017-03-21

    Small heat shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently there is only one high-resolution structure of a plant sHsp published, of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminals arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, crosslinking mass spectrometry, NMR and small angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI-motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly-facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which as expected disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

  16. In Silico Studies on Fungal Metabolite against Skin Cancer Protein (4,5-Diarylisoxazole HSP90 Chaperone)

    PubMed Central

    Kandasamy, Saravanakumar; Sahu, Sunil Kumar; Kandasamy, Kathiresan

    2012-01-01

    This work was to find out the dominant secondary metabolites derived from the fungus Trichoderma and to test them against skin cancer protein. The metabolites were extracted in 80% methanol from the fungal biomass of Trichoderma isolated from mangrove sediment. The crude methanol extract was purified and analysed for the secondary metabolites by GC-MS. Three predominant compounds (heptadecanoic acid, 16 methyl-, methyl ester; 9,12-octadecadienoic acid; cis-9-octadecenoic acid) identified in the extracts were screened against the skin cancer protein (Hsp90) by in-silico docking method. Of the compounds, heptadecanoic acid, 16 methyl, methyl ester was the most potent having the docking score of −11.4592 Kcal/mol. This value was better than the standard drug “dyclonine”. This work recommends the heptadecanoic acid, 16 methyl, methyl ester for further in vitro and in vivo studies towards its development as anticancer drug. PMID:22991673

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Masculinisation of the Zebra Finch Song System: Roles of Oestradiol and the Z-chromosome Gene Tubulin-Specific Chaperone Protein A

    PubMed Central

    Beach, L. Q.; Wade, J.

    2015-01-01

    Robust sex differences in brain and behaviour exist in zebra finches. Only males sing, and forebrain song control regions are more developed in males. The factors driving these differences are not clear, although numerous experiments have shown that oestradiol (E2) administered to female hatchlings partially masculinises brain and behaviour. Recent studies suggest that an increased expression of Z-chromosome genes in males (ZZ; females: ZW) might also play a role. The Z-gene tubulin-specific chaperone A (TBCA) exhibits increased expression in the lateral magnocellular nucleus of the anterior nidopallium (LMAN) of juvenile males compared to females; TBCA+ cells project to the robust nucleus of the arcopallium (RA). In the present study, we investigated the role of TBCA and tested hypotheses with respect to the interactive or additive effects of E2 and TBCA. We first examined whether E2 in hatchling zebra finches modulates TBCA expression in the LMAN. It affected neither the mRNA, nor protein in either sex. We then unilaterally delivered TBCA small interfering (si)RNA to the LMAN of developing females treated with E2 or vehicle and males treated with the aromatase inhibitor, fadrozole, or its control. In both sexes, decreasing TBCA in LMAN reduced RA cell number, cell size and volume. It also decreased LMAN volume in females. Fadrozole in males increased LMAN volume and RA cell size. TBCA siRNA delivered to the LMAN also decreased the projection from this brain region to the RA, as indicated by anterograde tract tracing. The results suggest that TBCA is involved in masculinising the song system. However, because no interactions between the siRNA and hormone manipulations were detected, TBCA does not appear to modulate effects of E2 in the zebra finch song circuit. PMID:25702708

  2. Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair.

    PubMed

    Sato, Koichi; Ishiai, Masamichi; Toda, Kazue; Furukoshi, Satoshi; Osakabe, Akihisa; Tachiwana, Hiroaki; Takizawa, Yoshimasa; Kagawa, Wataru; Kitao, Hiroyuki; Dohmae, Naoshi; Obuse, Chikashi; Kimura, Hiroshi; Takata, Minoru; Kurumizaka, Hitoshi

    2012-08-29

    Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.

  3. 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

  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. 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

  6. 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.

  7. A bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor σ32 of Escherichia coli.

    PubMed

    Perrody, Elsa; Cirinesi, Anne-Marie; Desplats, Carine; Keppel, France; Schwager, Françoise; Tranier, Samuel; Georgopoulos, Costa; Genevaux, Pierre

    2012-01-01

    The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ(32), which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ(32) facilitates RB43 bacteriophage proliferation is discussed.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  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. A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed.

    PubMed Central

    Agueli, C; Geraci, F; Giudice, G; Chimenti, L; Cascino, D; Sconzo, G

    2001-01-01

    In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters. PMID:11716770

  16. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    SciTech Connect

    Choudhury, Kamalika Roy; Bhattacharyya, Nitai P.

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. Protein Chaperones Q8ZP25_SALTY from Salmonella Typhimurium and HYAE_ECOLI from Escherichia coli Exhibit Thioredoxin-like Structures Despite Lack of Canonical Thioredoxin Active Site Sequence Motif

    SciTech Connect

    Parish, D.; Benach, J; Liu, G; Singarapu, K; Xiao, R; Acton, T; Hunt, J; Montelione, G; Szyperski, T; et. al.

    2008-01-01

    The structure of the 142-residue protein Q8ZP25 SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE ECOLI was previously classified as a (NiFe) hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.

  3. 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”.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Molecular modeling of the binding modes of the iron-sulfur protein to the Jac1 co-chaperone from Saccharomyces cerevisiae by all-atom and coarse-grained approaches.

    PubMed

    Mozolewska, Magdalena A; Krupa, Paweł; Scheraga, Harold A; Liwo, Adam

    2015-08-01

    The iron-sulfur protein 1 (Isu1) and the J-type co-chaperone Jac1 from yeast are part of a huge ATP-dependent system, and both interact with Hsp70 chaperones. Interaction of Isu1 and Jac1 is a part of the iron-sulfur cluster biogenesis system in mitochondria. In this study, the structure and dynamics of the yeast Isu1-Jac1 complex has been modeled. First, the complete structure of Isu1 was obtained by homology modeling using the I-TASSER server and YASARA software and thereafter tested for stability in the all-atom force field AMBER. Then, the known experimental structure of Jac1 was adopted to obtain initial models of the Isu1-Jac1 complex by using the ZDOCK server for global and local docking and the AutoDock software for local docking. Three most probable models were subsequently subjected to the coarse-grained molecular dynamics simulations with the UNRES force field to obtain the final structures of the complex. In the most probable model, Isu1 binds to the left face of the Γ-shaped Jac1 molecule by the β-sheet section of Isu1. Residues L105 , L109 , and Y163 of Jac1 have been assessed by mutation studies to be essential for binding (Ciesielski et al., J Mol Biol 2012; 417:1-12). These residues were also found, by UNRES/molecular dynamics simulations, to be involved in strong interactions between Isu1 and Jac1 in the complex. Moreover, N(95), T(98), P(102), H(112), V(159), L(167), and A(170) of Jac1, not yet tested experimentally, were also found to be important in binding.

  13. Molecular modeling of the binding modes of the Iron-sulfur protein to the Jac1 co-chaperone from Saccharomyces cerevisiae by all-atom and coarse-grained approaches

    PubMed Central

    Mozolewska, Magdalena A.; Krupa, Paweł; Scheraga, Harold A.; Liwo, Adam

    2015-01-01

    The Iron sulfur protein 1 (Isu1) from yeast, and the J-type co-chaperone Jac1, are part of a huge ATP-dependent system, and both interact with Hsp70 chaperones. Interaction of Isu1 and Jac1 is a part of the iron-sulfur cluster biogenesis system in mitochondria. In this study, the structure and dynamics of the yeast Isu1-Jac1 complex has been modeled. First, the complete structure of Isu1 was obtained by homology modeling using the I-TASSER server and YASARA software and thereafter tested for stability in the all-atom force field AMBER. Then, the known experimental structure of Jac1 was adopted to obtain initial models of the Isu1-Jac1 complex by using the ZDOCK server for global and local docking and the AutoDock software for local docking. Three most probable models were subsequently subjected to the coarse-grained molecular dynamics simulations with the UNRES force field to obtain the final structures of the complex. In the most probable model, Isu1 binds to the left face of the “Γ” shaped Jac1 molecule by the β-sheet section of Isu1. Residues L105, L109, and Y163 of Jac1 have been assessed by mutation studies to be essential for binding (Ciesielski et al., J. Mol. Biol. 2012, 417, 1–12). These residues were also found, by UNRES/MD simulations, to be involved in strong interactions between Isu1 and Jac1 in the complex. Moreover, N95, T98, P102, H112, V159, L167 and A170 of Jac1, not yet tested experimentally, were also found important in binding. PMID:25973573

  14. 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.

  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. 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

  17. 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

  18. The CoxD protein of Oligotropha carboxidovorans is a predicted AAA+ ATPase chaperone involved in the biogenesis of the CO dehydrogenase [CuSMoO2] cluster.

    PubMed

    Pelzmann, Astrid; Ferner, Marion; Gnida, Manuel; Meyer-Klaucke, Wolfram; Maisel, Tobias; Meyer, Ortwin

    2009-04-03

    CO dehydrogenase from the Gram-negative chemolithoautotrophic eubacterium Oligotropha carboxidovorans OM5 is a structurally characterized molybdenum-containing iron-sulfur flavoenzyme, which catalyzes the oxidation of CO (CO + H(2)O --> CO(2) + 2e(-) + 2H(+)). It accommodates in its active site a unique bimetallic [CuSMoO(2)] cluster, which is subject to post-translational maturation. Insertional mutagenesis of coxD has established its requirement for the assembly of the [CuSMoO(2)] cluster. Disruption of coxD led to a phenotype of the corresponding mutant OM5 D::km with the following characteristics: (i) It was impaired in the utilization of CO, whereas the utilization of H(2) plus CO(2) was not affected; (ii) Under appropriate induction conditions bacteria synthesized a fully assembled apo-CO dehydrogenase, which could not oxidize CO; (iii) Apo-CO dehydrogenase contained a [MoO(3)] site in place of the [CuSMoO(2)] cluster; and (iv) Employing sodium sulfide first and then the Cu(I)-(thiourea)(3) complex, the non-catalytic [MoO(3)] site could be reconstituted in vitro to a [CuSMoO(2)] cluster capable of oxidizing CO. Sequence information suggests that CoxD is a MoxR-like AAA+ ATPase chaperone related to the hexameric, ring-shaped BchI component of Mg(2+)-chelatases. Recombinant CoxD, which appeared in Escherichia coli in inclusion bodies, occurs exclusively in cytoplasmic membranes of O. carboxidovorans grown in the presence of CO, and its occurrence coincided with GTPase activity upon sucrose density gradient centrifugation of cell extracts. The presumed function of CoxD is the partial unfolding of apo-CO dehydrogenase to assist in the stepwise introduction of sulfur and copper in the [MoO(3)] center of the enzyme.

  19. 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

  20. 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

  1. PqqD Is a Novel Peptide Chaperone That Forms a Ternary Complex with the Radical S-Adenosylmethionine Protein PqqE in the Pyrroloquinoline Quinone Biosynthetic Pathway*

    PubMed Central

    Latham, John A.; Iavarone, Anthony T.; Barr, Ian; Juthani, Prerak V.; Klinman, Judith P.

    2015-01-01

    Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and KD were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the KD, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (KD ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the KD for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein. PMID:25817994

  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. 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

  4. 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.

  5. 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.

  6. 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

  7. Neuronal cells but not muscle cells are resistant to oxidative stress mediated protein misfolding and cell death: role of molecular chaperones.

    PubMed

    Bhattacharya, Arunabh; Wei, Rochelle; Hamilton, Ryan T; Chaudhuri, Asish R

    2014-04-18

    Our recent study in a mouse model of familial-Amyotrophic Lateral Sclerosis (f-ALS) revealed that muscle proteins are equally sensitive to misfolding as spinal cord proteins despite the presence of low mutant CuZn-superoxide dismutase, which is considered to be the key toxic element for initiation and progression of f-ALS. More importantly, we observed differential level of heat shock proteins (Hsp's) between skeletal muscle and spinal cord tissues prior to the onset and during disease progression; spinal cord maintains significantly higher level of Hsp's compared to skeletal muscle. In this study, we report two important observations; (i) muscle cells (but not neuronal cells) are extremely vulnerable to protein misfolding and cell death during challenge with oxidative stress and (ii) muscle cells fail to mount Hsp's during challenge unlike neuronal cells. These two findings can possibly explain why muscle atrophy precedes the death of motor neurons in f-ALS mice.

  8. 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.

  9. 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

  10. Localization of the chaperone proteins GRP78 and HSP60 on the luminal surface of bovine oviduct epithelial cells and their association with spermatozoa.

    PubMed

    Boilard, M; Reyes-Moreno, C; Lachance, C; Massicotte, L; Bailey, J L; Sirard, M-A; Leclerc, P

    2004-12-01

    Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. To identify the oviduct factors involved in their survival, sperm cells were preincubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively, and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility, and acrosomal integrity were improved compared with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose-regulated protein 78 (GRP78). Their identities and association with spermatozoa were confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa, and the presence of

  11. Enhanced and sustained CD8+ T cell responses with an adenoviral vector-based hepatitis C virus vaccine encoding NS3 linked to the MHC class II chaperone protein invariant chain.

    PubMed

    Mikkelsen, Marianne; Holst, Peter Johannes; Bukh, Jens; Thomsen, Allan Randrup; Christensen, Jan Pravsgaard

    2011-02-15

    Potent and broad cellular immune responses against the nonstructural (NS) proteins of hepatitis C virus (HCV) are associated with spontaneous viral clearance. In this study, we have improved the immunogenicity of an adenovirus (Ad)-based HCV vaccine by fusing NS3 from HCV (Strain J4; Genotype 1b) to the MHC class II chaperone protein invariant chain (Ii). We found that, after a single vaccination of C57BL/6 or BALB/c mice with Ad-IiNS3, the HCV NS3-specific CD8(+) T cell responses were significantly enhanced, accelerated, and prolonged compared with the vaccine encoding NS3 alone. The AdIiNS3 vaccination induced polyfunctional CD8(+) T cells characterized by coproduction of IFN-γ, TNF-α and IL-2, and this cell phenotype is associated with good viral control. The memory CD8(+) T cells also expressed high levels of CD27 and CD127, which are markers of long-term survival and maintenance of T cell memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice demonstrated that this protection was mediated primarily through IFN-γ production. On the basis of these promising results, we suggest that this vaccination technology should be evaluated further in the chimpanzee HCV challenge model.

  12. The Box H/ACA snoRNP Assembly Factor Shq1p is a Chaperone Protein Homologous to Hsp90 Cochaperones that Binds to the Cbf5p Enzyme

    SciTech Connect

    Godin, Katherine S.; Walbott, Helene; Leulliot, Nicolas; van Tilbeurgh, Herman; Varani, Gabriele

    2009-05-06

    Box H/ACA small nucleolar (sno) ribonucleoproteins (RNPs) are responsible for the formation of pseudouridine in a variety of RNAs and are essential for ribosome biogenesis, modification of spliceosomal RNAs, and telomerase stability. A mature snoRNP has been reconstituted in vitro and is composed of a single RNA and four proteins. However, snoRNP biogenesis in vivo requires multiple factors to coordinate a complex and poorly understood assembly and maturation process. Among the factors required for snoRNP biogenesis in yeast is Shq1p, an essential protein necessary for stable expression of box H/ACA snoRNAs. We have found that Shq1p consists of two independent domains that contain casein kinase 1 phosphorylation sites. We also demonstrate that Shq1p binds the pseudourydilating enzyme Cbf5p through the C-terminal domain, in synergy with the N-terminal domain. The NMR solution structure of the N-terminal domain has striking homology to the ‘Chord and Sgt1’ domain of known Hsp90 cochaperones, yet Shq1p does not interact with the yeast Hsp90 homologue in vitro. Surprisingly, Shq1p has stand-alone chaperone activity in vitro. This activity is harbored by the C-terminal domain, but it is increased by the presence of the N-terminal domain. These results provide the first evidence of a specific biochemical activity for Shq1p and a direct link to the H/ACA snoRNP.

  13. 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

  14. 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.

  15. The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A.

    PubMed

    Jousselin, Ambre; Manzano, Caroline; Biette, Alexandra; Reed, Patricia; Pinho, Mariana G; Rosato, Adriana E; Kelley, William L; Renzoni, Adriana

    2015-12-28

    Expression of the methicillin-resistant S. aureus (MRSA) phenotype results from the expression of the extra penicillin-binding protein 2A (PBP2A), which is encoded by mecA and acquired horizontally on part of the SCCmec cassette. PBP2A can catalyze dd-transpeptidation of peptidoglycan (PG) because of its low affinity for β-lactam antibiotics and can functionally cooperate with the PBP2 transglycosylase in the biosynthesis of PG. Here, we focus upon the role of the membrane-bound PrsA foldase protein as a regulator of β-lactam resistance expression. Deletion of prsA altered oxacillin resistance in three different SCCmec backgrounds and, more importantly, caused a decrease in PBP2A membrane amounts without affecting mecA mRNA levels. The N- and C-terminal domains of PrsA were found to be critical features for PBP2A protein membrane levels and oxacillin resistance. We propose that PrsA has a role in posttranscriptional maturation of PBP2A, possibly in the export and/or folding of newly synthesized PBP2A. This additional level of control in the expression of the mecA-dependent MRSA phenotype constitutes an opportunity to expand the strategies to design anti-infective agents.

  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. 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.

  18. 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.

  19. 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.

  20. Reduction of brain barrier tight junctional proteins by lead exposure: role of activation of nonreceptor tyrosine kinase Src via chaperon GRP78.

    PubMed

    Song, Han; Zheng, Gang; Shen, Xue-Feng; Liu, Xin-Qin; Luo, Wen-Jing; Chen, Jing-Yuan

    2014-04-01

    Lead (Pb) has long been recognized as a neurodevelopmental toxin. Developing blood-brain barrier (BBB) is known to be a target of Pb neurotoxicity; however, the underlying mechanisms are still unclear. Recent evidence suggests that intracellular nonreceptor protein tyrosine kinase Src regulates tight junctional proteins (TJPs). This study was designed to investigate whether Pb acted on the Src-mediated cascade event leading to an altered TJP expression at BBB. Rats aged 20-22 days were exposed to Pb in drinking water (0, 100, 200, and 300 ppm Pb) for eight weeks. Electron microscopic and Western blot analyses revealed a severe leakage of BBB and significantly decreased expressions of TJP occludin and ZO-1. When cultured brain endothelial RBE4 cells were exposed to 10μM Pb for 24 h, expressions of phosphor-Src and an upstream regulator GRP78 were significantly increased by 6.42-fold and 8.29-fold (p < 0.01), respectively. Inactivation of Src pathway by a Src-specific inhibitor reversed Pb-induced downregulation of occludin, but not ZO-1; small interfering RNA knockdown of GRP78 attenuated Pb-induced Src phosphorylation and occludin reduction. Furthermore, Pb exposure caused redistribution of GRP78 from endoplasmic reticulum to cytosol and toward cell member. However, the data from immunoneutralization studies did not show the involvement of cell-surface GRP78 in regulating Src phosphorylation upon Pb exposure, suggesting that the cytosolic GRP78, rather than cell-surface GRP78, was responsible to Pb-induced Src activation and ensuing occludin reduction. Taken together, this study provides the evidence of a novel linkage of GRP78, Src activation to downregulation of occludin, and BBB disruption during Pb exposure.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. Inherent chaperone-like activity of aspartic proteases reveals a distant evolutionary relation to double-ψ barrel domains of AAA-ATPases

    PubMed Central

    Hulko, Michael; Lupas, Andrei N.; Martin, Jörg

    2007-01-01

    Chaperones and proteases share the ability to interact with unfolded proteins. Here we show that enzymatically inactive forms of the aspartic proteases HIV-1 protease and pepsin have inherent chaperone-like activity and can prevent the aggregation of denatured substrate proteins. In contrast to proteolysis, which requires dimeric enzymes, chaperone-like activity could be observed also with monomeric domains. The involvement of the active site cleft in the chaperone-like function was demonstrated by the inhibitory effect of peptide substrate inhibitors. The high structural similarity between aspartic proteases and the N-terminal double-ψ barrels of Cdc48-like proteins, which are involved in the unfolding and dissociation of proteins, suggests that they share a common ancestor. The latent chaperone-like activity in aspartic proteases can be seen as a relic that has further evolved to serve substrate binding in the context of proteolytic activity. PMID:17384229

  13. 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

  14. Transglutaminase type 2: A multifunctional protein chaperone?

    PubMed Central

    Rossin, Federica; D’Eletto, Manuela; Farrace, Maria Grazia; Piacentini, Mauro

    2014-01-01

    Macroautophagy selectively degrades dysfunctional mitochondria by a process known as mitophagy. The purpose of the study published in Cell Death and Differentiation was to investigate the involvement of transglutaminase 2 (TG2) in the turnover and degradation of damaged mitochondria and its effects on cell metabolism. PMID:27308365

  15. 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.

  16. 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.

  17. Identification of chaperones in a MPP+-induced and ATRA/TPA-differentiated SH-SY5Y cell PD model

    PubMed Central

    Xie, Hongrong; Hu, Hui; Chang, Ming; Huang, Dongya; Gu, Xiaobo; Xiong, Xinli; Xiong, Ran; Hu, Linsen; Li, Gang

    2016-01-01

    Parkinson’s disease (PD) is characterized by the pathological accumulation of misfolded proteins. Molecular chaperones assist in the proper folding of proteins and removal of irreversibly misfolded proteins. This study aims to identify potential chaperones associated with protein misfolding and accumulation in PD. ATRA/TPA-differentiated SH-SY5Y cells were treated with 1 mM of MPP+ for 48 hours. Proteins were analyzed by 2D-DIGE followed by MALDI-ToF MS. The treatment of differentiated SH-SY5Y cells by MPP+ led to the unambiguous identification of 10 protein spots, which corresponds to six proteins. Among these six proteins, four were chaperone proteins including nucleophosmin (NPM1), chaperonin-containing TCP-1 subunit 2 (CCT2 or CCTβ), heat shock 90 kDa protein 1 beta (HSP90AB1 or HSP90-β), and tyrosin3/tryptopha5-monoxygenase activation protein, zeta polypeptide (14-3-3ζ, gene symbol: Ywhaz). To our knowledge, this is the first report that linked the upregulation of chaperones after MPP+ treatment with SH-SY5Y cells. However, the NPM1 protein was identified for the first time in the PD model. The upregulation of four chaperone proteins provided evidence that these chaperones have a complementary effect on protein misfolding in the pathogenesis of PD, and hold promise as a good therapeutic target for PD treatment. PMID:28078037

  18. 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

  19. 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…

  20. 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.

  1. 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.

  2. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis

    PubMed Central

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  3. 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

  4. 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.

  5. 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

  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. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  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. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. Delay of diabetic cataract in rats by the antiglycating potential of cumin through modulation of alpha-crystallin chaperone activity.

    PubMed

    Kumar, Pasupulati Anil; Reddy, Paduru Yadagiri; Srinivas, P N B S; Reddy, Geereddy Bhanuprakash

    2009-07-01

    alpha-Crystallin, a molecular chaperone of the eye lens, plays an important role in maintaining the transparency of the lens by preventing the aggregation/inactivation of several proteins and enzymes in addition to its structural role. alpha-Crystallin is a long-lived protein and is susceptible to several posttranslational modifications during aging, more so in certain clinical conditions such as diabetes. Nonenzymatic glycation of lens proteins and decline in the chaperone-like function of alpha-crystallin have been reported in diabetic conditions. Therefore, inhibitors of nonenzymatic protein glycation appear to be a potential target to preserve the chaperone activity of alpha-crystallin and to combat cataract under hyperglycemic conditions. In this study, we investigated the antiglycating potential of cumin in vitro and its ability to modulate the chaperone-like activity of alpha-crystallin vis-à-vis the progression of diabetic cataract in vivo. Aqueous extract of cumin was tested for its antiglycating ability against fructose-induced glycation of goat lens total soluble protein (TSP), alpha-crystallin from goat lens and a nonlenticular protein bovine serum albumin (BSA). The antiglycating potential of cumin was also investigated by feeding streptozotocin (STZ)-induced diabetic rats with diet containing 0.5% cumin powder. The aqueous extract of cumin prevented in vitro glycation of TSP, alpha-crystallin and BSA. Slit lamp examination revealed that supplementation of cumin delayed progression and maturation of STZ-induced cataract in rats. Cumin was effective in preventing glycation of TSP and alpha-crystallin in diabetic lens. Interestingly, feeding of cumin to diabetic rats not only prevented loss of chaperone activity but also attenuated the structural changes of alpha-crystallin in lens. These results indicated that cumin has antiglycating properties that may be attributed to the modulation of chaperone activity of alpha-crystallin, thus delaying cataract in

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. Over-expression of inducible HSP70 chaperone suppresses neuropathology and improves motor function in SCA1 mice.

    PubMed

    Cummings, C J; Sun, Y; Opal, P; Antalffy, B; Mestril, R; Orr, H T; Dillmann, W H; Zoghbi, H Y

    2001-07-01

    Many neurodegenerative diseases are caused by gain-of-function mechanisms in which the disease-causing protein is altered, becomes toxic to the cell, and aggregates. Among these 'proteinopathies' are Alzheimer's and Parkinson's disease, prion disorders and polyglutamine diseases. Members of this latter group, also known as triplet repeat diseases, are caused by the expansion of unstable CAG repeats coding for glutamine within the respective proteins. Spinocerebellar ataxia type 1 (SCA1) is one such disease, characterized by loss of motor coordination due to the degeneration of cerebellar Purkinje cells and brain stem neurons. In SCA1 and several other polyglutamine diseases, the expanded protein aggregates into nuclear inclusions (NIs). Because these NIs accumulate molecular chaperones, ubiquitin and proteasomal subunits--all components of the cellular protein re-folding and degradation machinery--we hypothesized that protein misfolding and impaired protein clearance might underlie the pathogenesis of polyglutamine diseases. Over-expressing specific chaperones reduces protein aggregation in transfected cells and suppresses neurodegeneration in invertebrate animal models of polyglutamine disorders. To determine whether enhancing chaperone activity could mitigate the phenotype in a mammalian model, we crossbred SCA1 mice with mice over-expressing a molecular chaperone (inducible HSP70 or iHSP70). We found that high levels of HSP70 did indeed afford protection against neurodegeneration.

  6. 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

  7. J protein mutations and resulting proteostasis collapse

    PubMed Central

    Koutras, Carolina; Braun, Janice E. A.

    2014-01-01

    Despite a century of intensive investigation the effective treatment of protein aggregation diseases remains elusive. Ordinarily, molecular chaperones ensure that proteins maintain their functional conformation. The appearance of misfolded proteins that aggregate implies the collapse of the cellular chaperone quality control network. That said, the cellular chaperone network is extensive and functional information regarding the detailed action of specific chaperones is not yet available. J proteins (DnaJ/Hsp40) are a family of chaperone cofactors that harness Hsc70 (heat shock cognate protein of 70 kDa) for diverse conformational cellular tasks and, as such, represent novel clinically relevant targets for diseases resulting from the disruption of proteostasis. Here we review incisive reports identifying mutations in individual J protein chaperones and the proteostasis collapse that ensues. PMID:25071450

  8. 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.

  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. 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. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Extracellular and circulating redox- and metalloregulated eRNA and eRNP: copper ion-structured RNA cytokines (angiotropin ribokines) and bioaptamer targets imparting RNA chaperone and novel biofunctions to S100-EF-hand and disease-associated proteins.

    PubMed

    Wissler, Josef H

    2004-06-01

    were visualized by 3D-rapid prototyping of accurate molecular image models based on crystallographic or NMR data. For S100A12-homologous proteins, receptor- and metalloregulated RNA chaperone-shaped protein assemblies were investigated. They suggest insight into signaling cascades as to how eRNA transmits its cytokine (ribokine) bioinformation from the extracellular RNA biosphere into cells. Proteomics of the extracellular RNA biosphere demonstrate the presence of nucleic acid-binding domain homologies in defense-, aging-, and disease-associated neuronal and other proteins as targets for RNA orphans. By structural relationships found to transmissible processes, proteinaceous transfer ("infectivity") and feedback of bioinformation beyond the central dogma of molecular biology are considered in terms of metalloregulated RNA bioaptamer function, nucleic acid-binding domains, and protein conformation.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

    PubMed Central

    Weinstock, Matthew T.; Jacobsen, Michael T.; Kay, Michael S.

    2014-01-01

    Mirror-image proteins (composed of d-amino acids) are promising therapeutic agents and drug discovery tools, but as synthesis of larger d-proteins becomes feasible, a major anticipated challenge is the folding of these proteins into their active conformations. In vivo, many large and/or complex proteins require chaperones like GroEL/ES to prevent misfolding and produce functional protein. The ability of chaperones to fold d-proteins is unknown. Here we examine the ability of GroEL/ES to fold a synthetic d-protein. We report the total chemical synthesis of a 312-residue GroEL/ES-dependent protein, DapA, in both l- and d-chiralities, the longest fully synthetic proteins yet reported. Impressively, GroEL/ES folds both l- and d-DapA. This work extends the limits of chemical protein synthesis, reveals ambidextrous GroEL/ES folding activity, and provides a valuable tool to fold d-proteins for drug development and mirror-image synthetic biology applications. PMID:25071217

  5. The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein.

    PubMed

    Treweek, Teresa M; Thorn, David C; Price, William E; Carver, John A

    2011-06-01

    α(S)-Casein, the major milk protein, comprises α(S1)- and α(S2)-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α(S)-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α(S)-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α(S2)-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α(S1)-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α(S1)-, α- and α(S)-casein were all significantly less effective. We conclude that α(S1)- and α-casein stabilise each other to facilitate optimal chaperone activity of α(S)-casein. This work highlights the interdependency of casein proteins for their structural stability.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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…

  13. 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

  14. 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.

  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. 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

  17. 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

  18. 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.

  19. 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

  20. Cellular Pathology of Pelizaeus-Merzbacher Disease Involving Chaperones Associated with Endoplasmic Reticulum Stress

    PubMed Central

    Inoue, Ken

    2017-01-01

    Disease-causing mutations in genes encoding membrane proteins may lead to the production of aberrant polypeptides that accumulate in the endoplasmic reticulum (ER). These mutant proteins have detrimental conformational changes or misfolding events, which result in the triggering of the unfolded protein response (UPR). UPR is a cellular pathway that reduces ER stress by generally inhibiting translation, increasing ER chaperones levels, or inducing cell apoptosis in severe ER stress. This process has been implicated in the cellular pathology of many neurological disorders, including Pelizaeus-Merzbacher disease (PMD). PMD is a rare pediatric disorder characterized by the failure in the myelination process of the central nervous system (CNS). PMD is caused by mutations in the PLP1 gene, which encodes a major myelin membrane protein. Severe clinical PMD phenotypes appear to be the result of cell toxicity, due to the accumulation of PLP1 mutant proteins and not due to the lack of functional PLP1. Therefore, it is important to clarify the pathological mechanisms by which the PLP1 mutants negatively impact the myelin-generating cells, called oligodendrocytes, to overcome this devastating disease. This review discusses how PLP1 mutant proteins change protein homeostasis in the ER of oligodendrocytes, especially focusing on the reaction of ER chaperones against the accumulation of PLP1 mutant proteins that cause PMD. PMID:28286750

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

    PubMed

    Echeverría, Pablo C; Bernthaler, Andreas; Dupuis, Pierre; Mayer, Bernd; Picard, Didier

    2011-01-01

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

  2. Structural and Biochemical Characterization of SrcA, a Multi-cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

    SciTech Connect

    Cooper, C.; Zhang, K; Andres, S; Fnag, Y; Kaniuk, N; Hannemann, M; Brumell, J; Foster, L; Junop, M; Coombes, B

    2010-01-01

    Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 {angstrom} revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

  3. Molecular chaperones in ectothermic marine animals: biochemical function and gene expression.

    PubMed

    Hofmann, Gretchen E; Buckley, Bradley A; Place, Sean P; Zippay, Mackenzie L

    2002-08-01

    The intertidal zone has historically functioned as an important natural laboratory for testing ideas about how physical factors such as temperature influence organismal physiology and in turn influence the distribution patterns of organisms. Key to our understanding of how the physical environment helps structure organismal distribution is the identification of physiological processes that have ecological relevance. We have focused on biochemical- and molecular-level physiology that would contribute to thermal tolerance and maintenance of a functional intracellular protein pool in the face of extreme and fluctuating environmental temperatures. Past research has addressed processes central to protein homeostasis (e.g., protein ubiquitination) and the molecular ecology of molecular chaperones, a.k.a. heat shock proteins (Hsps), in ectothermic animals. In this presentation, we focus on two new developments regarding the biology of heat shock proteins as molecular chaperones in intertidal organisms. First, we present data on the functional characteristics of the transcriptional factor, HSF1 and discuss how these data relate to the plasticity of Hsp gene expression observed in intertidal organisms in nature. Second, we present data on the biochemical function of heat shock proteins purified from our non-model study organisms and discuss the temperature relationships of these molecules as they assist in protein folding in situ.

  4. Co-chaperone Specificity in Gating of the Polypeptide Conducting Channel in the Membrane of the Human Endoplasmic Reticulum*

    PubMed Central

    Schorr, Stefan; Klein, Marie-Christine; Gamayun, Igor; Melnyk, Armin; Jung, Martin; Schäuble, Nico; Wang, Qian; Hemmis, Birgit; Bochen, Florian; Greiner, Markus; Lampel, Pavel; Urban, Sabine Katharina; Hassdenteufel, Sarah; Dudek, Johanna; Chen, Xing-Zhen; Wagner, Richard; Cavalié, Adolfo; Zimmermann, Richard

    2015-01-01

    In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic polypeptide-conducting channel, the heterotrimeric Sec61 complex. Previous work has characterized the Sec61 complex as a potential ER Ca2+ leak channel in HeLa cells and identified ER lumenal molecular chaperone immunoglobulin heavy-chain-binding protein (BiP) as limiting Ca2+ leakage via the open Sec61 channel by facilitating channel closing. This BiP activity involves binding of BiP to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344. Of note, the Y344H mutation destroys the BiP binding site and causes pancreatic β-cell apoptosis and diabetes in mice. Here, we systematically depleted HeLa cells of the BiP co-chaperones by siRNA-mediated gene silencing and used live cell Ca2+ imaging to monitor the effects on ER Ca2+ leakage. Depletion of either one of the ER lumenal BiP co-chaperones, ERj3 and ERj6, but not the ER membrane-resident co-chaperones (such as Sec63 protein, which assists BiP in Sec61 channel opening) led to increased Ca2+ leakage via Sec6 complex, thereby phenocopying the effect of BiP depletion. Thus, BiP facilitates Sec61 channel closure (i.e. limits ER Ca2+ leakage) via the Sec61 channel with the help of ERj3 and ERj6. Interestingly, deletion of ERj6 causes pancreatic β-cell failure and diabetes in mice and humans. We suggest that co-chaperone-controlled gating of the Sec61 channel by BiP is particularly important for cells, which are highly active in protein secretion, and that breakdown of this regulatory mechanism can cause apoptosis and disease. PMID:26085089

  5. The selective inhibition of serpin aggregation by the molecular chaperone, alpha-crystallin, indicates a nucleation-dependent specificity.

    PubMed

    Devlin, Glyn L; Carver, John A; Bottomley, Stephen P

    2003-12-05

    Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that prevent the misfolding and aggregation of proteins. However, specific details about their substrate specificity and mechanism of chaperone action are lacking. alpha1-Antichymotrypsin (ACT) and alpha1-antitrypsin (alpha1-AT) are two closely related members of the serpin superfamily that aggregate through nucleation-dependent and nucleation-independent pathways, respectively. The sHsp alpha-crystallin was unable to prevent the nucleation-independent aggregation of alpha1-AT, whereas alpha-crystallin inhibited ACT aggregation in a dose-dependent manner. This selective inhibition of ACT aggregation coincided with the formation of a stable high molecular weight alpha-crystallin-ACT complex with a stoichiometry of 1 on a molar subunit basis. The kinetics of this interaction occur at the same rate as the loss of ACT monomer, suggesting that the monomeric species is bound by the chaperone. 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) binding and far-UV circular dichroism data suggest that alpha-crystallin interacts specifically with a non-native conformation of ACT. The finding that alpha-crystallin does not interact with alpha1-AT under these conditions suggests that alpha-crystallin displays a specificity for proteins that aggregate through a nucleation-dependent pathway, implying that the dynamic nature of both the chaperone and its substrate protein is a crucial factor in the chaperone action of alpha-crystallin and other sHsps.

  6. Evaluation of structure, chaperone-like activity and protective ability of peroxynitrite modified human α-Crystallin subunits against copper-mediated ascorbic acid oxidation.

    PubMed

    Ghahramani, Maryam; Yousefi, Reza; Khoshaman, Kazem; Moghadam, Sogand Sasan; Kurganov, Boris I

    2016-06-01

    The copper-catalyzed oxidation of ascorbic acid (ASA) to dehydroascorbate (DHA) and hydrogen peroxide plays a central role in pathology of cataract diseases during ageing and in diabetic patients. In the current study, the structural feature, chaperone-like activity and protective ability of peroxynitrite (PON) modified αA- and αB-Crystallin (Cry) against copper-mediated ASA oxidation were studied using different spectroscopic measurements and gel mobility shift assay. Upon PON modification, additional to protein structural alteration, the contents of nitrotyrosine, nitrotryptophan, dityrosine and carbonyl groups were significantly increased. Moreover, αB-Cry demonstrates significantly larger capacity for PON modification than αA-Cry. Also, based on the extent of PON modification, these proteins may display an improved chaperone-like activity and enhanced protective ability against copper-mediated ASA oxidation. In the presence of copper ions, chaperone-like activity of both native and PON-modified α-Cry subunits were appreciably improved. Additionally, binding of copper ions to native and PON-modified proteins results in the significant reduction of their solvent exposed hydrophobic patches. Overall, the increase in chaperone-like activity/ASA protective ability of PON-modified α-Cry and additional enhancement of its chaperoning action with copper ions appear to be an important defense mechanism offered by this protein.

  7. Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones.

    PubMed

    Lyupina, Yulia V; Orlova, Olga V; Abaturova, Svetlana B; Beljelarskaya, Svetlana N; Lavrov, Andrey N; Mikhailov, Victor S

    2014-11-04

    The induction of heat shock proteins in baculovirus infected cells is well documented. However a role of these chaperones in infection cycle remains unknown. The observation that HSP70s are associated with virions of different baculoviruses reported by several researchers suggests that HSPs might be structural components of viruses or involved in virion assembly. These hypotheses were examined by using a novel inhibitor of the ATPase and chaperoning activity of HSP/HSC70s, VER-155008. When VER-155008 was added early in infection, the synthesis of viral proteins, genome replication and the production of budded virions (BV) were markedly inhibited indicating the dependence of virus reproduction on host chaperones. However, BV production was unaffected when VER-155008 was added in the mid-replication phase which is after accumulation of products required for completion of the viral DNA replication. These results suggest that the final stages in assembly of BV and their egress from cells do not depend on chaperone activity of host HSP/HSC70s.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  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. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. FrnE, a Cadmium-Inducible Protein in Deinococcus radiodurans, Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo

    PubMed Central

    Khairnar, Nivedita P.; Joe, Min-Ho; Misra, H. S.; Lim, Sang-Yong

    2013-01-01

    Deinococcus radiodurans R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including frnE (drfrnE) and some of those involved in DNA repair and oxidative stress tolerance. The drfrnE::nptII mutant of D. radiodurans showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ∼15- and ∼3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ∼2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In trans expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the dsbA mutant of Escherichia coli. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42°C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase in vitro and has a role in oxidative stress tolerance of D. radiodurans possibly by protecting the damaged cellular proteins from inactivation. PMID:23603741

  2. Characterization and Structure of a Novel Zn2+ and [2Fe-2S]-Containing Copper Chaperone from Archaeoglobus fulgidus

    SciTech Connect

    Sazinsky,M.; LeMoine, B.; Orofino, M.; Davydo, R.; Bencze, K.; Stemmler, T.; Hoffman, B.; Arguello, J.; Rosenzweig, A.

    2007-01-01

    Bacterial CopZ proteins deliver copper to P{sub 1B}-type Cu{sup +}-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8{angstrom} resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu{sup +} to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu{sup 2+} to Cu{sup +}. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms