Protein Quality Control by Molecular Chaperones in Neurodegeneration

PubMed Central

Ciechanover, Aaron; Kwon, Yong Tae

2017-01-01

Protein homeostasis (proteostasis) requires the timely degradation of misfolded proteins and their aggregates by protein quality control (PQC), of which molecular chaperones are an essential component. Compared with other cell types, PQC in neurons is particularly challenging because they have a unique cellular structure with long extensions. Making it worse, neurons are postmitotic, i.e., cannot dilute toxic substances by division, and, thus, are highly sensitive to misfolded proteins, especially as they age. Failure in PQC is often associated with neurodegenerative diseases, such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), and prion disease. In fact, many neurodegenerative diseases are considered to be protein misfolding disorders. To prevent the accumulation of disease-causing aggregates, neurons utilize a repertoire of chaperones that recognize misfolded proteins through exposed hydrophobic surfaces and assist their refolding. If such an effort fails, chaperones can facilitate the degradation of terminally misfolded proteins through either the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). If soluble, the substrates associated with chaperones, such as Hsp70, are ubiquitinated by Ub ligases and degraded through the proteasome complex. Some misfolded proteins carrying the KFERQ motif are recognized by the chaperone Hsc70 and delivered to the lysosomal lumen through a process called, chaperone-mediated autophagy (CMA). Aggregation-prone misfolded proteins that remain unprocessed are directed to macroautophagy in which cargoes are collected by adaptors, such as p62/SQSTM-1/Sequestosome-1, and delivered to the autophagosome for lysosomal degradation. The aggregates that have survived all these refolding/degradative processes can still be directly dissolved, i.e., disaggregated by chaperones. Studies have shown that molecular chaperones alleviate the pathogenic symptoms by

Visualizing chaperone-assisted protein folding

DOE PAGES

Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; ...

2016-05-30

We present that 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. 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 new 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 Escherichia coli chaperonemore » Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.« less

Docosahexaenoic Acid-mediated Inhibition of Heat Shock Protein 90-p23 Chaperone Complex and Downstream Client Proteins in Lung and Breast Cancer.

PubMed

Mouradian, Michael; Ma, Irvin V; Vicente, Erika D; Kikawa, Keith D; Pardini, Ronald S

2017-01-01

The molecular chaperone, heat shock protein 90 (Hsp90), is a critical regulator for the proper folding and stabilization of several client proteins, and is a major contributor to carcinogenesis. Specific Hsp90 inhibitors have been designed to target the ATP-binding site in order to prevent Hsp90 chaperone maturation. The current study investigated the effects of docosahexaenoic acid (DHA; C22:6 n-3) on Hsp90 function and downstream client protein expression. In vitro analyses of BT-474 human breast carcinoma and A549 human lung adenocarcinoma cell lines revealed dose-dependent decreases in intracellular ATP levels by DHA treatment, resulting in a significant reduction of Hsp90 and p23 association in both cell lines. Attenuation of the Hsp90-p23 complex led to the inhibition of Hsp90 client proteins, epidermal growth factor receptor 2 (ErbB2), and hypoxia-inducible factor 1α (HIF-1α). Similar results were observed when employing 2-deoxyglucose (2-DG), confirming that DHA and 2-DG, both independently and combined, can disturb Hsp90 molecular chaperone function. In vivo A549 xenograft analysis also demonstrated decreased expression levels of Hsp90-p23 association and diminished protein levels of ErbB2 and HIF-1α in mice supplemented with dietary DHA. These data support a role for dietary intervention to improve cancer therapy in tumors overexpressing Hsp90 and its client proteins.

Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20.

PubMed

Maio, N; Rouault, T A

2016-10-01

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.

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

2017-07-10

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. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Chaperone activity of human small heat shock protein-GST fusion proteins.

PubMed

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

The major protein of bovine seminal plasma, PDC-109, is a molecular chaperone.

PubMed

Sankhala, Rajeshwer Singh; Swamy, Musti J

2010-05-11

The major protein of bovine seminal plasma, PDC-109, binds to choline phospholipids on the sperm plasma membrane and induces the efflux of cholesterol and choline phospholipids, which is an important step in sperm capacitation. The high abundance, polydisperse nature and reversibility of thermal unfolding of PDC-109 suggest significant similarities to chaperone-like proteins such as spectrin, alpha-crystallin, and alpha-synuclein. In the present study, biochemical and biophysical approaches were employed to investigate the chaperone-like activity of PDC-109. The effect of various stress factors such as high temperature, chemical denaturant (urea), and acidic pH on target proteins such as lactate dehydrogenase, alcohol dehydrogenase, and insulin were studied in both the presence and absence of PDC-109. The results obtained indicate that PDC-109 exhibits chaperone-like activity, as evidenced by its ability to suppress the nonspecific aggregation of target proteins and direct them into productive folding. Atomic force microscopic studies demonstrate that PDC-109 effectively prevents the fibrillation of insulin, which is of considerable significance since amyloidogenesis has been reported to be a serious problem during sperm maturation in certain species. Binding of phosphorylcholine or high ionic strength in the medium inhibited the chaperone-like activity of PDC-109, suggesting that most likely the aggregation state of the protein is important for the chaperone function. These observations show that PDC-109 functions as a molecular chaperone in vitro, suggesting that it may assist the proper folding of proteins involved in the bovine sperm capacitation pathway. To the best of our knowledge, this is the first study reporting chaperone-like activity of a seminal plasma protein.

Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

2015-03-13

Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two othermore » 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.« less

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.

Redox-regulated chaperones.

PubMed

Kumsta, Caroline; Jakob, Ursula

2009-06-09

Redox regulation of stress proteins, such as molecular chaperones, guarantees an immediate response to oxidative stress conditions. This review focuses on the two major classes of redox-regulated chaperones, Hsp33 in bacteria and typical 2-Cys peroxiredoxins in eukaryotes. Both proteins employ redox-sensitive cysteines, whose oxidation status directly controls their affinity for unfolding proteins and therefore their chaperone function. We will first discuss Hsp33, whose oxidative stress-induced disulfide bond formation triggers the partial unfolding of the chaperone, which, in turn, leads to the exposure of a high-affinity binding site for unfolded proteins. This rapid mode of activation makes Hsp33 essential for protecting bacteria against severe oxidative stress conditions, such as hypochlorite (i.e., bleach) treatment, which leads to widespread protein unfolding and aggregation. We will compare Hsp33 to the highly abundant eukaryotic typical 2-Cys peroxiredoxin, whose oxidative stress-induced sulfinic acid formation turns the peroxidase into a molecular chaperone in vitro and presumably in vivo. These examples illustrate how proteins use reversible cysteine modifications to rapidly adjust to oxidative stress conditions and demonstrate that redox regulation plays a vital role in protecting organisms against reactive oxygen species-mediated cell death.

Allostery in the Hsp70 chaperone proteins

PubMed Central

Zuiderweg, Erik R.P.; Bertelsen, Eric B.; Rousaki, Aikaterini; Mayer, Matthias P.; Gestwicki, Jason E.; Ahmad, Atta

2013-01-01

Heat shock 70 kDa (Hsp70) chaperones are essential to in-vivo protein folding, protein transport and protein re-folding. They carry out these activities using repeated cycles of binding and release of client proteins. This process is under allosteric control of nucleotide binding and hydrolysis. X-ray crystallography, NMR spectroscopy and other biophysical techniques have contributed much to the understanding of the allosteric mechanism linking these activities and the effect of co-chaperones on this mechanism. In this chapter, these findings are critically reviewed. Studies on the allosteric mechanisms of Hsp70 have gained enhanced urgency, as recent studies have implicated this chaperone as a potential drug target in diseases such as Alzheimer's and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed. PMID:22576356

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

PubMed Central

Martínez-Alonso, Mónica; Villaverde, Antonio

2010-01-01

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

An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding.

PubMed

Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad

2017-09-01

The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Modulation of intracellular protein degradation by SSB1-SIS1 chaperon system in yeast S. cerevisiae.

PubMed

Ohba, M

1997-06-09

In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E. coli. To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S. cerevisiae. The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations. Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon. Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.

Molecular chaperones antagonize proteotoxicity by differentially modulating protein aggregation pathways

PubMed Central

Douglas, Peter M; Summers, Daniel W

2009-01-01

The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease proteins and assist in refolding or degradation pathways. Recent work suggests that chaperones may also suppress neurotoxicity by converting toxic, soluble oligomers into benign aggregates. Chaperones can therefore suppress or promote aggregation of disease proteins to ameliorate the proteotoxic accumulation of soluble, assembly intermediates. PMID:19421006

Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks

PubMed Central

Pechmann, Sebastian; Frydman, Judith

2014-01-01

Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular

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.

Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7

USDA-ARS?s Scientific Manuscript database

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...

Molecular chaperones and protein folding as therapeutic targets in Parkinson's disease and other synucleinopathies.

PubMed

Ebrahimi-Fakhari, Darius; Saidi, Laiq-Jan; Wahlster, Lara

2013-12-05

Changes in protein metabolism are key to disease onset and progression in many neurodegenerative diseases. As a prime example, in Parkinson's disease, folding, post-translational modification and recycling of the synaptic protein α-synuclein are clearly altered, leading to a progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. Altered protein folding is one of the first steps of an increasingly understood cascade in which α-synuclein forms complex oligomers and finally distinct protein aggregates, termed Lewy bodies and Lewy neurites. In neurons, an elaborated network of chaperone and co-chaperone proteins is instrumental in mediating protein folding and re-folding. In addition to their direct influence on client proteins, chaperones interact with protein degradation pathways such as the ubiquitin-proteasome-system or autophagy in order to ensure the effective removal of irreversibly misfolded and potentially pathogenic proteins. Because of the vital role of proper protein folding for protein homeostasis, a growing number of studies have evaluated the contribution of chaperone proteins to neurodegeneration. We herein review our current understanding of the involvement of chaperones, co-chaperones and chaperone-mediated autophagy in synucleinopathies with a focus on the Hsp90 and Hsp70 chaperone system. We discuss genetic and pathological studies in Parkinson's disease as well as experimental studies in models of synucleinopathies that explore molecular chaperones and protein degradation pathways as a novel therapeutic target. To this end, we examine the capacity of chaperones to prevent or modulate neurodegeneration and summarize the current progress in models of Parkinson's disease and related neurodegenerative disorders.

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

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

PubMed

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

2013-09-01

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

Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones

PubMed Central

Pausch, Patrick; Singh, Ujjwala; Ahmed, Yasar Luqman; Pillet, Benjamin; Murat, Guillaume; Altegoer, Florian; Stier, Gunter; Thoms, Matthias; Hurt, Ed; Sinning, Irmgard; Bange, Gert; Kressler, Dieter

2015-01-01

Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation. PMID:26112308

Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.

PubMed

Heikkila, John J; Kaldis, Angelo; Abdulle, Rashid

2006-01-01

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.

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

Roles of Intramolecular and Intermolecular Interactions in Functional Regulation of the Hsp70 J-protein Co-Chaperone Sis1

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

2015-04-01

Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at heir 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 interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.« less

Roles of Intramolecular and Intermolecular Interactions in Functional Regulation of the Hsp70 J-protein Co-chaperone Sis1

PubMed Central

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

2015-01-01

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. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively. PMID:25687964

Evolutionary Silence of the Acid Chaperone Protein HdeB in Enterohemorrhagic Escherichia coli O157:H7

PubMed Central

Louie, Jacqueline W.; Fagerquist, Clifton K.; Sultan, Omar; Miller, William G.; Mandrell, Robert E.

2012-01-01

The periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) in Escherichia coli and Shigella spp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less in E. coli O157:H7 strains. Deletion of hdeB did not affect the acid survival of cells, and deletion of hdeA led to less than a 5-fold decrease in survival. Sequence analysis of the hdeAB operon revealed a point mutation at the putative start codon of the hdeB gene in all 26 E. coli O157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB in E. coli O157:H7; however, the plasmid-borne O157-hdeB was able to restore partially the acid resistance in an E. coli O145ΔhdeAB mutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude that E. coli O157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms within E. coli. PMID:22179243

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

Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.

PubMed

Castiblanco, Luisa F; Triplett, Lindsay R; Sundin, George W

2018-01-01

Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora .

Spermine and spermidine act as chemical chaperones and enhance chaperone-like and membranolytic activities of major bovine seminal plasma protein, PDC-109.

PubMed

Singh, Bhanu Pratap; Saha, Ishita; Nandi, Indrani; Swamy, Musti J

2017-12-02

The major bovine seminal plasma protein, PDC-109, binds to choline phospholipids of the sperm plasma membrane and induces an efflux of cholesterol and choline phospholipids (cholesterol efflux), which is crucial for sperm capacitation. PDC-109 also exhibits chaperone-like activity and protects target proteins against various kinds of stress. Here we show that the polyamines spermine and spermidine, present in high concentration in the seminal plasma of various mammals, increase the ability of PDC-109 to perturb membrane structure as well as its chaperone-like activity. Interestingly, spermine/spermidine alone did not perturb membrane structure but exhibited chaperone-like activity by protecting target proteins against thermal and oxidative stress. When spermine/spermidine was used along with PDC-109, the observed chaperone-like activity was considerably higher than that expected for a simple additive effect, suggesting that PDC-109 and the polyamines act in a synergistic fashion. These results indicate that at the high concentrations present in the seminal plasma spermine/spermidine exhibit a positive modulatory effect on the chaperone-like activity of PDC-109 and may also function as chemical chaperones and protect other seminal plasma proteins from various kinds of stress. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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

PubMed

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

2015-04-10

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

Peroxisomal Proteostasis Involves a Lon Family Protein That Functions as Protease and Chaperone*

PubMed Central

Bartoszewska, Magdalena; Williams, Chris; Kikhney, Alexey; Opaliński, Łukasz; van Roermund, Carlo W. T.; de Boer, Rinse; Veenhuis, Marten; van der Klei, Ida J.

2012-01-01

Proteins are subject to continuous quality control for optimal proteostasis. The knowledge of peroxisome quality control systems is still in its infancy. Here we show that peroxisomes contain a member of the Lon family of proteases (Pln). We show that Pln is a heptameric protein and acts as an ATP-fueled protease and chaperone. Hence, Pln is the first chaperone identified in fungal peroxisomes. In cells of a PLN deletion strain peroxisomes contain protein aggregates, a major component of which is catalase-peroxidase. We show that this enzyme is sensitive to oxidative damage. The oxidatively damaged, but not the native protein, is a substrate of the Pln protease. Cells of the pln strain contain enhanced levels of catalase-peroxidase protein but reduced catalase-peroxidase enzyme activities. Together with the observation that Pln has chaperone activity in vitro, our data suggest that catalase-peroxidase aggregates accumulate in peroxisomes of pln cells due to the combined absence of Pln protease and chaperone activities. PMID:22733816

Chaperones and protein folding in the archaea.

PubMed

Large, Andrew T; Goldberg, Martin D; Lund, Peter A

2009-02-01

A survey of archaeal genomes for the presence of homologues of bacterial and eukaryotic chaperones reveals several interesting features. All archaea contain chaperonins, also known as Hsp60s (where Hsp is heat-shock protein). These are more similar to the type II chaperonins found in the eukaryotic cytosol than to the type I chaperonins found in bacteria, mitochondria and chloroplasts, although some archaea also contain type I chaperonin homologues, presumably acquired by horizontal gene transfer. Most archaea contain several genes for these proteins. Our studies on the type II chaperonins of the genetically tractable archaeon Haloferax volcanii have shown that only one of the three genes has to be present for the organisms to grow, but that there is some evidence for functional specialization between the different chaperonin proteins. All archaea also possess genes for prefoldin proteins and for small heat-shock proteins, but they generally lack genes for Hsp90 and Hsp100 homologues. Genes for Hsp70 (DnaK) and Hsp40 (DnaJ) homologues are only found in a subset of archaea. Thus chaperone-assisted protein folding in archaea is likely to display some unique features when compared with that in eukaryotes and bacteria, and there may be important differences in the process between euryarchaea and crenarchaea.

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

The heat-shock protein/chaperone network and multiple stress resistance.

PubMed

Jacob, Pierre; Hirt, Heribert; Bendahmane, Abdelhafid

2017-04-01

Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multistress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat-shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone 'client proteins', many are primary metabolism enzymes and signal transduction components with essential roles for the proper functioning of a cell. HSPs/chaperones are controlled by the action of diverse heat-shock factors, which are recruited under stress conditions. In this review, we give an overview of the regulation of the HSP/chaperone network with a focus on Arabidopsis thaliana. We illustrate the role of HSPs/chaperones in regulating diverse signalling pathways and discuss several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/chaperone network. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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.

Isolation and Identification of Putative Protein Substrates of the AAA+ Molecular Chaperone ClpB from the Pathogenic Spirochaete Leptospira interrogans.

PubMed

Krajewska, Joanna; Arent, Zbigniew; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

2018-04-18

Bacterial ClpB is an ATP-dependent Hsp100 chaperone that reactivates aggregated proteins in cooperation with the DnaK chaperone system and promotes survival of bacteria under stress conditions. A large number of publications also indicate that ClpB supports the virulence of bacteria, including a pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in both animals and humans. However, the exact role of ClpB in bacterial pathogenicity remains poorly characterized. It can be assumed that ClpB, due to its role as the molecular chaperone, mediates refolding of essential bacterial proteins, including the known virulence factors, which may become prone to aggregation under infection-induced stresses. In this study, we identified putative substrates of ClpB from L. interrogans (ClpB Li ). For this purpose, we used a proteomic approach combining the ClpB-Trap affinity pull-down assays, Liquid chromatography-tandem mass spectrometry (LC-MS-MS/MS), and bioinformatics analyses. Most of the identified proteins were enzymes predominantly associated with major metabolic pathways like the tricarboxylic acid (TCA) cycle, glycolysis–gluconeogenesis and amino acid and fatty acid metabolism. Based on our proteomic study, we suggest that ClpB can support the virulence of L. interrogans by protecting the conformational integrity and catalytic activity of multiple metabolic enzymes, thus maintaining energy homeostasis in pathogen cells.

The radical SAM protein HemW is a heme chaperone.

PubMed

Haskamp, Vera; Karrie, Simone; Mingers, Toni; Barthels, Stefan; Alberge, François; Magalon, Axel; Müller, Katrin; Bill, Eckhard; Lubitz, Wolfgang; Kleeberg, Kirstin; Schweyen, Peter; Bröring, Martin; Jahn, Martina; Jahn, Dieter

2018-02-16

Radical S -adenosylmethionine (SAM) enzymes exist in organisms from all kingdoms of life, and all of these proteins generate an adenosyl radical via the homolytic cleavage of the S-C(5') bond of SAM. Of particular interest are radical SAM enzymes, such as heme chaperones, that insert heme into respiratory enzymes. For example, heme chaperones insert heme into target proteins but have been studied only for the formation of cytochrome c -type hemoproteins. Here, we report that a radical SAM protein, the heme chaperone HemW from bacteria, is required for the insertion of heme b into respiratory chain enzymes. As other radical SAM proteins, HemW contains three cysteines and one SAM coordinating an [4Fe-4S] cluster, and we observed one heme per subunit of HemW. We found that an intact iron-sulfur cluster was required for HemW dimerization and HemW-catalyzed heme transfer but not for stable heme binding. A bacterial two-hybrid system screen identified bacterioferritins and the heme-containing subunit NarI of the respiratory nitrate reductase NarGHI as proteins that interact with HemW. We also noted that the bacterioferritins potentially serve as heme donors for HemW. Of note, heme that was covalently bound to HemW was actively transferred to a heme-depleted, catalytically inactive nitrate reductase, restoring its nitrate-reducing enzyme activity. Finally, the human HemW orthologue radical SAM domain-containing 1 (RSAD1) stably bound heme. In conclusion, our findings indicate that the radical SAM protein family HemW/RSAD1 is a heme chaperone catalyzing the insertion of heme into hemoproteins. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Heat Shock Proteins: A Review of the Molecular Chaperones for Plant Immunity.

PubMed

Park, Chang-Jin; Seo, Young-Su

2015-12-01

As sessile organisms, plants are exposed to persistently changing stresses and have to be able to interpret and respond to them. The stresses, drought, salinity, chemicals, cold and hot temperatures, and various pathogen attacks have interconnected effects on plants, resulting in the disruption of protein homeostasis. Maintenance of proteins in their functional native conformations and preventing aggregation of non-native proteins are important for cell survival under stress. Heat shock proteins (HSPs) functioning as molecular chaperones are the key components responsible for protein folding, assembly, translocation, and degradation under stress conditions and in many normal cellular processes. Plants respond to pathogen invasion using two different innate immune responses mediated by pattern recognition receptors (PRRs) or resistance (R) proteins. HSPs play an indispensable role as molecular chaperones in the quality control of plasma membrane-resident PRRs and intracellular R proteins against potential invaders. Here, we specifically discuss the functional involvement of cytosolic and endoplasmic reticulum (ER) HSPs/chaperones in plant immunity to obtain an integrated understanding of the immune responses in plant cells.

Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

PubMed Central

Chou, Ming-Lun; Fitzpatrick, Lynda M.; Tu, Shuh-Long; Budziszewski, Gregory; Potter-Lewis, Sharon; Akita, Mitsuru; Levin, Joshua Z.; Keegstra, Kenneth; Li, Hsou-min

2003-01-01

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane. PMID:12805212

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

Forces Driving Chaperone Action

PubMed Central

Koldewey, Philipp; Stull, Frederick; Horowitz, Scott; Martin, Raoul; Bardwell, James C. A.

2016-01-01

SUMMARY It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client’s affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins. PMID:27293188

RNA chaperone activity of human La protein is mediated by variant RNA recognition motif.

PubMed

Naeeni, Amir R; Conte, Maria R; Bayfield, Mark A

2012-02-17

La proteins are conserved factors in eukaryotes that bind and protect the 3' trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3'OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3'OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets.

RNA Chaperone Activity of Human La Protein Is Mediated by Variant RNA Recognition Motif*

PubMed Central

Naeeni, Amir R.; Conte, Maria R.; Bayfield, Mark A.

2012-01-01

La proteins are conserved factors in eukaryotes that bind and protect the 3′ trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3′OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3′OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets. PMID:22203678

Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

PubMed

Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

2017-08-04

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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

Phthalic acid chemical probes synthesized for protein-protein interaction analysis.

PubMed

Liang, Shih-Shin; Liao, Wei-Ting; Kuo, Chao-Jen; Chou, Chi-Hsien; Wu, Chin-Jen; Wang, Hui-Min

2013-06-24

Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES-SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis.

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

The chaperone-histone partnership: for the greater good of histone traffic and chromatin plasticity.

PubMed

Hondele, Maria; Ladurner, Andreas G

2011-12-01

Histones are highly positively charged proteins that wrap our genome. Their surface properties also make them prone to nonspecific interactions and aggregation. A class of proteins known as histone chaperones is dedicated to safeguard histones by aiding their proper incorporation into nucleosomes. Histone chaperones facilitate ordered nucleosome assembly and disassembly reactions through the formation of semi-stable histone-chaperone intermediates without requiring ATP, but merely providing a complementary protein surface for histones to dynamically interact with. Recurrent 'chaperoning' mechanisms involve the masking of the histone's positive charge and the direct blocking of crucial histone surface sites, including those required for H3-H4 tetramerization or the binding of nucleosomal DNA. This shielding prevents histones from engaging in premature or unwanted interactions with nucleic acids and other cellular components. In this review, we analyze recent structural studies on chaperone-histone interactions and discuss the implications of this vital partnership for nucleosome assembly and disassembly pathways. Copyright © 2011 Elsevier Ltd. All rights reserved.

N-glycan based ER molecular chaperone and protein quality control system: the calnexin binding cycle

PubMed Central

Lamriben, Lydia; Graham, Jill B.; Adams, Benjamin M.; Hebert, Daniel N.

2015-01-01

Helenius and colleagues proposed over twenty-years ago a paradigm-shifting model for how chaperone binding in the endoplasmic reticulum was mediated and controlled for a new type of molecular chaperone- the carbohydrate binding chaperones, calnexin and calreticulin. While the originally established basics for this lectin chaperone binding cycle holds true today, there has been a number of important advances that have expanded our understanding of its mechanisms of action, role in protein homeostasis, and its connection to disease states that are highlighted in this review. PMID:26676362

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.

Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

PubMed Central

James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

2015-01-01

Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

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

PubMed

Kumar, C Sudheer; Swamy, Musti J

2016-07-05

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

Visualization of a radical B 12 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

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.

NMR-monitored titration of acid-stress bacterial chaperone HdeA reveals that Asp and Glu charge neutralization produces a loosened dimer structure in preparation for protein unfolding and chaperone activation

PubMed Central

Garrison, McKinzie A; Crowhurst, Karin A

2014-01-01

HdeA is a periplasmic chaperone found in several gram-negative pathogenic bacteria that are linked to millions of cases of dysentery per year worldwide. After the protein becomes activated at low pH, it can bind to other periplasmic proteins, protecting them from aggregation when the bacteria travel through the stomach on their way to colonize the intestines. It has been argued that one of the major driving forces for HdeA activation is the protonation of aspartate and glutamate side chains. The goal for this study, therefore, was to investigate, at the atomic level, the structural impact of this charge neutralization on HdeA during the transition from near-neutral conditions to pH 3.0, in preparation for unfolding and activation of its chaperone capabilities. NMR spectroscopy was used to measure pKa values of Asp and Glu residues and monitor chemical shift changes. Measurements of R2/R1 ratios from relaxation experiments confirm that the protein maintains its dimer structure between pH 6.0 and 3.0. However, calculated correlation times and changes in amide protection from hydrogen/deuterium exchange experiments provide evidence for a loosening of the tertiary and quaternary structures of HdeA; in particular, the data indicate that the dimer structure becomes progressively weakened as the pH decreases. Taken together, these results provide insight into the process by which HdeA is primed to unfold and carry out its chaperone duties below pH 3.0, and it also demonstrates that neutralization of aspartate and glutamate residues is not likely to be the sole trigger for HdeA dissociation and unfolding. PMID:24375557

cpSRP43 Is a Novel Chaperone Specific for Light-harvesting Chlorophyll a,b-binding Proteins*

PubMed Central

Falk, Sebastian; Sinning, Irmgard

2010-01-01

The biosynthesis of most membrane proteins is directly coupled to membrane insertion, and therefore, molecular chaperones are not required. The light-harvesting chlorophyll a,b-binding proteins (LHCPs) present a prominent exception as they are synthesized in the cytoplasm, and after import into the chloroplast, they are targeted and inserted into the thylakoid membrane. Upon arrival in the stroma, LHCPs form a soluble transit complex with the chloroplast signal recognition particle (cpSRP) consisting of an SRP54 homolog and the unique cpSRP43 composed of three chromodomains and four ankyrin repeats. Here we describe that cpSRP43 alone prevents aggregation of LHCP by formation of a complex with nanomolar affinity, whereas cpSRP54 is not required for this chaperone activity. Other stromal chaperones like trigger factor cannot replace cpSRP43, which implies that LHCPs require a specific chaperone. Although cpSRP43 does not have an ATPase activity, it can dissolve aggregates of LHCPs similar to chaperones of the Hsp104/ClpB family. We show that the LHCP-cpSRP43 interaction is predominantly hydrophobic but strictly depends on an intact DPLG motif between the second and third transmembrane region. The cpSRP43 ankyrin repeats that provide the binding site for the DPLG motif are sufficient for the chaperone function, whereas the chromodomains are dispensable. Taken together, we define cpSRP43 as a highly specific chaperone for LHCPs in addition to its established function as a targeting factor for this family of membrane proteins. PMID:20498370

The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.

PubMed

Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L

2001-06-01

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

Unfolding the chaperone story

PubMed Central

Hartl, F. Ulrich

2017-01-01

Protein folding in the cell was originally assumed to be a spontaneous process, based on Anfinsen’s discovery that purified proteins can fold on their own after removal from denaturant. Consequently cell biologists showed little interest in the protein folding process. This changed only in the mid and late 1980s, when the chaperone story began to unfold. As a result, we now know that in vivo, protein folding requires assistance by a complex machinery of molecular chaperones. To ensure efficient folding, members of different chaperone classes receive the nascent protein chain emerging from the ribosome and guide it along an ordered pathway toward the native state. I was fortunate to contribute to these developments early on. In this short essay, I will describe some of the critical steps leading to the current concept of protein folding as a highly organized cellular process. PMID:29084909

Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore.

PubMed

Galigniana, Mario D; Echeverría, Pablo C; Erlejman, Alejandra G; Piwien-Pilipuk, Graciela

2010-01-01

In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR) -domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor "transformation") is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin β and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore.

Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding.

PubMed

Hageman, Jurre; Vos, Michel J; van Waarde, Maria A W H; Kampinga, Harm H

2007-11-23

Molecular chaperones are essential for cells to prevent that partially unfolded proteins form non-functional, toxic aggregates. This requirement is increased when cells experience protein unfolding stresses and such could affect all compartments in the eukaryotic cell. Whether all organelles are equipped with comparable chaperone capacities is largely unknown, mainly due to the lack of suitable reporters that allow such a comparison. Here we describe the development of fluorescent luciferase reporters that are sorted to various cellular locations (nucleus, cytoplasm, endoplasmic reticulum, and peroxisomes) and that differ minimally in their intrinsic thermal stability properties. When heating living cells, the rate of inactivation was most rapid for the nuclear-targeted luciferase, indicating that the nucleus is the most sensitive organelle toward heat-induced denaturing stress. Post-heat re-activation, however, occurred at equal kinetics irrespective of luciferase localization. Also, induction of thermotolerance by a priming heat treatment, that coordinately up-regulates all heat-inducible chaperones, resulted in a transient heat resistance of the luciferase in all organelles in a comparable manner. Overexpression of the main heat-inducible Hsp70 family member, HspA1A, protected only the cytosolic and nuclear, but not the other luciferases. Together, our data suggest that in each compartment investigated, including the peroxisome in which so far no chaperones could be detected, chaperone machines are present and can be induced with activities similar to those present in the cytosolic/nuclear compartment.

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

PubMed

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

2017-10-01

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

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

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

Molecular chaperones in Parkinson's disease--present and future.

PubMed

Ebrahimi-Fakhari, Darius; Wahlster, Lara; McLean, Pamela J

2011-01-01

Parkinson's disease, like many other neurodegenerative disorders, is characterized by the progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. The cascade by which the small synaptic protein α-synuclein misfolds to form distinctive protein aggregates, termed Lewy bodies and Lewy neurites, has been the subject of intensive research for more than a decade. Genetic and pathological studies in Parkinson's disease patients as well as experimental studies in disease models have clearly established altered protein metabolism as a key element in the pathogenesis of Parkinson's disease. Alterations in protein metabolism include misfolding and aggregation, post-translational modification and dysfunctional degradation of cytotoxic protein species. Protein folding and re-folding are both mediated by a highly conserved network of molecules, called molecular chaperones and co-chaperones. In addition to the regulatory role in protein folding, molecular chaperone function is intimately associated with pathways of protein degradation, such as the ubiquitin-proteasome system and the autophagy-lysosomal pathway, to effectively remove irreversibly misfolded proteins. Because of the central role of molecular chaperones in maintaining protein homeostasis, we herein review our current knowledge on the involvement of molecular chaperones and co-chaperones in Parkinson's disease. We further discuss the capacity of molecular chaperones to prevent or modulate neurodegeneration, an important concept for future neuroprotective strategies and summarize the current progress in preclinical studies in models of Parkinson's disease and other neurodegenerative disorders. Finally we include a discussion on the future potential of using molecular chaperones as a disease modifying therapy.

Effect of a chemical chaperone, tauroursodeoxycholic acid, on HDM-induced allergic airway disease

PubMed Central

Siddesha, Jalahalli M.; Nakada, Emily M.; Mihavics, Bethany R.; Hoffman, Sidra M.; Rattu, Gurkiranjit K.; Chamberlain, Nicolas; Cahoon, Jonathon M.; Lahue, Karolyn G.; Daphtary, Nirav; Aliyeva, Minara; Chapman, David G.; Desai, Dhimant H.; Poynter, Matthew E.

2016-01-01

Endoplasmic reticulum (ER) stress-induced unfolded protein response plays a critical role in inflammatory diseases, including allergic airway disease. However, the benefits of inhibiting ER stress in the treatment of allergic airway disease are not well known. Herein, we tested the therapeutic potential of a chemical chaperone, tauroursodeoxycholic acid (TUDCA), in combating allergic asthma, using a mouse model of house dust mite (HDM)-induced allergic airway disease. TUDCA was administered during the HDM-challenge phase (preventive regimen), after the HDM-challenge phase (therapeutic regimen), or therapeutically during a subsequent HDM rechallenge (rechallenge regimen). In the preventive regimen, TUDCA significantly decreased HDM-induced inflammation, markers of ER stress, airway hyperresponsiveness (AHR), and fibrosis. Similarly, in the therapeutic regimen, TUDCA administration efficiently decreased HDM-induced airway inflammation, mucus metaplasia, ER stress markers, and AHR, but not airway remodeling. Interestingly, TUDCA administered therapeutically in the HDM rechallenge regimen markedly attenuated HDM-induced airway inflammation, mucus metaplasia, ER stress markers, methacholine-induced AHR, and airway fibrotic remodeling. These results indicate that the inhibition of ER stress in the lungs through the administration of chemical chaperones could be a valuable strategy in the treatment of allergic airway diseases. PMID:27154200

Sugar-Terminated Nanoparticle Chaperones Are 102-105 Times Better Than Molecular Sugars in Inhibiting Protein Aggregation and Reducing Amyloidogenic Cytotoxicity.

PubMed

Pradhan, Nibedita; Shekhar, Shashi; Jana, Nihar R; Jana, Nikhil R

2017-03-29

Sugar-based osmolyte molecules are known to stabilize proteins under stress, but usually they have poor chaperone performance in inhibiting protein aggregation. Here, we show that the nanoparticle form of sugars molecule can enhance their chaperone performance typically by 10 2 -10 5 times, compared to molecular sugar. Sugar-based plate-like nanoparticles of 20-40 nm hydrodynamic size have been synthesized by simple heating of acidic aqueous solution of glucose/sucrose/maltose/trehalose. These nanoparticles have excitation-dependent green/yellow/orange emission and surface chemistry identical to the respective sugar molecule. Fibrillation of lysozyme/insulin/amyloid beta in extracellular space, aggregation of mutant huntingtin protein inside model neuronal cell, and cytotoxic effect of fibrils are investigated in the presence of these sugar nanoparticles. We found that sugar nanoparticles are 10 2 -10 5 times efficient than respective sugar molecules in inhibiting protein fibrillation and preventing cytotoxicity arising of fibrils. We propose that better performance of the nanoparticle form is linked to its stronger binding with fibril structure and enhanced cell uptake. This result suggests that nanoparticle form of osmolyte can be an attractive option in prevention and curing of protein aggregation-derived diseases.

Endoplasmic reticulum proteins SDF2 and SDF2L1 act as components of the BiP chaperone cycle to prevent protein aggregation.

PubMed

Fujimori, Tsutomu; Suno, Ryoji; Iemura, Shun-Ichiro; Natsume, Tohru; Wada, Ikuo; Hosokawa, Nobuko

2017-08-01

The folding of newly synthesized proteins in the endoplasmic reticulum (ER) is assisted by ER-resident chaperone proteins. BiP (immunoglobulin heavy-chain-binding protein), a member of the HSP70 family, plays a central role in protein quality control. The chaperone function of BiP is regulated by its intrinsic ATPase activity, which is stimulated by ER-resident proteins of the HSP40/DnaJ family, including ERdj3. Here, we report that two closely related proteins, SDF2 and SDF2L1, regulate the BiP chaperone cycle. Both are ER-resident, but SDF2 is constitutively expressed, whereas SDF2L1 expression is induced by ER stress. Both luminal proteins formed a stable complex with ERdj3 and potently inhibited the aggregation of different types of misfolded ER cargo. These proteins associated with non-native proteins, thus promoting the BiP-substrate interaction cycle. A dominant-negative ERdj3 mutant that inhibits the interaction between ERdj3 and BiP prevented the dissociation of misfolded cargo from the ERdj3-SDF2L1 complex. Our findings indicate that SDF2 and SDF2L1 associate with ERdj3 and act as components in the BiP chaperone cycle to prevent the aggregation of misfolded proteins, partly explaining the broad folding capabilities of the ER under various physiological conditions. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

A phasin with extra talents: a polyhydroxyalkanoate granule-associated protein has chaperone activity.

PubMed

Mezzina, Mariela P; Wetzler, Diana E; de Almeida, Alejandra; Dinjaski, Nina; Prieto, M Auxiliadora; Pettinari, Maria Julia

2015-05-01

Phasins are proteins associated to intracellular polyhydroxyalkanoate granules that affect polymer accumulation and the number and size of the granules. Previous work demonstrated that a phasin from Azotobacter sp FA-8 (PhaPAz ) had an unexpected growth-promoting and stress-protecting effect in Escherichia coli, suggesting it could have chaperone-like activities. In this work, in vitro and in vivo experiments were performed in order to investigate this possibility. PhaPAz was shown to prevent in vitro thermal aggregation of the model protein citrate synthase and to facilitate the refolding process of this enzyme after chemical denaturation. Microscopy techniques were used to analyse the subcellular localization of PhaPAz in E. coli strains and to study the role of PhaPAz in in vivo protein folding and aggregation. PhaPAz was shown to colocalize with inclusion bodies of PD, a protein that aggregates when overexpressed. A reduction in the number of inclusion bodies of PD was observed when it was coexpressed with PhaPAz or with the known chaperone GroELS. These results demonstrate that PhaPAz has chaperone-like functions both in vitro and in vivo in E. coli recombinants, and suggests that phasins could have a general protective role in natural polyhydroxyalkanoate producers. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Quantitative analysis of chaperone network throughput in budding yeast

PubMed Central

Brownridge, Philip; Lawless, Craig; Payapilly, Aishwarya B; Lanthaler, Karin; Holman, Stephen W; Harman, Victoria M; Grant, Christopher M; Beynon, Robert J; Hubbard, Simon J

2013-01-01

The network of molecular chaperones mediates the folding and translocation of the many proteins encoded in the genome of eukaryotic organisms, as well as a response to stress. It has been particularly well characterised in the budding yeast, Saccharomyces cerevisiae, where 63 known chaperones have been annotated and recent affinity purification and MS/MS experiments have helped characterise the attendant network of chaperone targets to a high degree. In this study, we apply our QconCAT methodology to directly quantify the set of yeast chaperones in absolute terms (copies per cell) via SRM MS. Firstly, we compare these to existing quantitative estimates of these yeast proteins, highlighting differences between approaches. Secondly, we cast the results into the context of the chaperone target network and show a distinct relationship between abundance of individual chaperones and their targets. This allows us to characterise the ‘throughput’ of protein molecules passing through individual chaperones and their groups on a proteome-wide scale in an unstressed model eukaryote for the first time. The results demonstrate specialisations of the chaperone classes, which display different overall workloads, efficiencies and preference for the sub-cellular localisation of their targets. The novel integration of the interactome data with quantification supports re-estimates of the level of protein throughout going through molecular chaperones. Additionally, although chaperones target fewer than 40% of annotated proteins we show that they mediate the folding of the majority of protein molecules (∼62% of the total protein flux in the cell), highlighting their importance. PMID:23420633

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

PubMed

Nakanishi, Kotaro

2016-09-01

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

Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem.

PubMed

Aguado, Alejandra; Fernández-Higuero, José Angel; Moro, Fernando; Muga, Arturo

2015-08-15

The oligomeric AAA+ chaperones Hsp104 in yeast and ClpB in bacteria are responsible for the reactivation of aggregated proteins, an activity essential for cell survival during severe stress. The protein disaggregase activity of these members of the Hsp100 family is linked to the activity of chaperones from the Hsp70 and Hsp40 families. The precise mechanism by which these proteins untangle protein aggregates remains unclear. Strikingly, Hsp100 proteins are not present in metazoans. This does not mean that animal cells do not have a disaggregase activity, but that this activity is performed by the Hsp70 system and a representative of the Hsp110 family instead of a Hsp100 protein. This review describes the actual view of Hsp100-mediated aggregate reactivation, including the ATP-induced conformational changes associated with their disaggregase activity, the dynamics of the oligomeric assembly that is regulated by its ATPase cycle and the DnaK system, and the tight allosteric coupling between the ATPase domains within the hexameric ring complexes. The lack of homologs of these disaggregases in metazoans has suggested that they might be used as potential targets to develop antimicrobials. The current knowledge of the human disaggregase machinery and the role of Hsp110 are also discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

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

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.

Unfolding the chaperone story.

PubMed

Hartl, F Ulrich

2017-11-01

Protein folding in the cell was originally assumed to be a spontaneous process, based on Anfinsen's discovery that purified proteins can fold on their own after removal from denaturant. Consequently cell biologists showed little interest in the protein folding process. This changed only in the mid and late 1980s, when the chaperone story began to unfold. As a result, we now know that in vivo, protein folding requires assistance by a complex machinery of molecular chaperones. To ensure efficient folding, members of different chaperone classes receive the nascent protein chain emerging from the ribosome and guide it along an ordered pathway toward the native state. I was fortunate to contribute to these developments early on. In this short essay, I will describe some of the critical steps leading to the current concept of protein folding as a highly organized cellular process. © 2017 Hartl. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

HdeB chaperone activity is coupled to its intrinsic dynamic properties

PubMed Central

Ding, Jienv; Yang, Chengfeng; Niu, Xiaogang; Hu, Yunfei; Jin, Changwen

2015-01-01

Enteric bacteria encounter extreme acidity when passing through hosts’ stomach. Since the bacterial periplasmic space quickly equilibrates with outer environment, an efficient acid resistance mechanism is essential in preventing irreversible protein denaturation/aggregation and maintaining bacteria viability. HdeB, along with its homolog HdeA, was identified as a periplasmic acid-resistant chaperone. Both proteins exist as homodimers and share similar monomeric structures under neutral pH, while showing different dimeric packing interfaces. Previous investigations show that HdeA functions through an acid-induced dimer-to-monomer transition and partial unfolding at low pH (pH 2–3), resulting in exposure of hydrophobic surfaces that bind substrate proteins. In contrast, HdeB appears to have a much higher optimal activation pH (pH 4–5), under which condition the protein maintains a well-folded dimer and the mechanism for its chaperone activity remains elusive. Herein, we present an NMR study of HdeB to investigate its dynamic properties. Our results reveal that HdeB undergoes significant micro- to milli-second timescale conformational exchanges at neutral to near-neutral pH, under the later condition it exhibits optimal activity. The current study indicates that HdeB activation is coupled to its intrinsic dynamics instead of structural changes, and therefore its functional mechanism is apparently different from HdeA. PMID:26593705

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

PubMed

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

2012-07-01

Previous evidence suggested that the stress protein grp170 can function as a highly efficient molecular chaperone, binding to large protein substrates and acting as a potent vaccine against specific tumors when purified from the same tumor. In addition, Pokemon can be found in almost all malignant tumor cells and is regarded to be a promising candidate for the treatment of tumors. However, the potential of the grp170-Pokemon chaperone complex has not been well described. In the present study, the natural chaperone complex between grp170 and the Pokemon was formed by heat shock, and its immunogenicity was detected by ELISPOT and (51)Cr-release assays in vitro and by tumor bearing models in vivo. Our results demonstrated that the grp170-Pokemon chaperone complex could elicit T cell responses as determined by ELISPOT and (51)Cr-release assays. In addition, immunized C57BL/6 mice were challenged with subcutaneous (s.c.) injection of Lewis cancer cells to induce primary tumors. Treatment of mice with the grp170-Pokemon chaperone complex also significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. Our results indicated that the grp170-Pokemon chaperone complex might represent a powerful approach to tumor immunotherapy and have significant potential for clinical application. Copyright © 2012 Elsevier GmbH. All rights reserved.

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.

The Barley Powdery Mildew Candidate Secreted Effector Protein CSEP0105 Inhibits the Chaperone Activity of a Small Heat Shock Protein1[OPEN

PubMed Central

Ahmed, Ali Abdurehim; Pedersen, Carsten; Schultz-Larsen, Torsten; Kwaaitaal, Mark; Jørgensen, Hans Jørgen Lyngs; Thordal-Christensen, Hans

2015-01-01

Pathogens secrete effector proteins to establish a successful interaction with their host. Here, we describe two barley (Hordeum vulgare) powdery mildew candidate secreted effector proteins, CSEP0105 and CSEP0162, which contribute to pathogen success and appear to be required during or after haustorial formation. Silencing of either CSEP using host-induced gene silencing significantly reduced the fungal haustorial formation rate. Interestingly, both CSEPs interact with the barley small heat shock proteins, Hsp16.9 and Hsp17.5, in a yeast two-hybrid assay. Small heat shock proteins are known to stabilize several intracellular proteins, including defense-related signaling components, through their chaperone activity. CSEP0105 and CSEP0162 localized to the cytosol and the nucleus of barley epidermal cells, whereas Hsp16.9 and Hsp17.5 are cytosolic. Intriguingly, only those specific CSEPs changed localization and became restricted to the cytosol when coexpressed with Hsp16.9 and Hsp17.5, confirming the CSEP-small heat shock protein interaction. As predicted, Hsp16.9 showed chaperone activity, as it could prevent the aggregation of Escherichia coli proteins during thermal stress. Remarkably, CSEP0105 compromised this activity. These data suggest that CSEP0105 promotes virulence by interfering with the chaperone activity of a barley small heat shock protein essential for defense and stress responses. PMID:25770154

Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin.

PubMed

Vainberg, I E; Lewis, S A; Rommelaere, H; Ampe, C; Vandekerckhove, J; Klein, H L; Cowan, N J

1998-05-29

We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins.

Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7.

PubMed

Hussain, Rawaa H; Zawawi, Mariam; Bayfield, Mark A

2013-10-01

The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3'OH-dependent trailer binding/protection and a UUU-3'OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3'OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily.

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

PubMed Central

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

2011-01-01

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

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

Redox Aspects of Chaperones in Cardiac Function

PubMed Central

Penna, Claudia; Sorge, Matteo; Femminò, Saveria; Pagliaro, Pasquale; Brancaccio, Mara

2018-01-01

Molecular chaperones are stress proteins that allow the correct folding or unfolding as well as the assembly or disassembly of macromolecular cellular components. Changes in expression and post-translational modifications of chaperones have been linked to a number of age- and stress-related diseases including cancer, neurodegeneration, and cardiovascular diseases. Redox sensible post-translational modifications, such as S-nitrosylation, glutathionylation and phosphorylation of chaperone proteins have been reported. Redox-dependent regulation of chaperones is likely to be a phenomenon involved in metabolic processes and may represent an adaptive response to several stress conditions, especially within mitochondria, where it impacts cellular bioenergetics. These post-translational modifications might underlie the mechanisms leading to cardioprotection by conditioning maneuvers as well as to ischemia/reperfusion injury. In this review, we discuss this topic and focus on two important aspects of redox-regulated chaperones, namely redox regulation of mitochondrial chaperone function and cardiac protection against ischemia/reperfusion injury. PMID:29615920

Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli.

PubMed

Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R; Liu, Yu; Powers, David L; Kelly, Jeffery W; Gierasch, Lila M; Powers, Evan T

2015-04-14

The folding fate of a protein in vivo is determined by the interplay between a protein's folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2016-01-06

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. 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 polyprotein, which is

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

Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7

PubMed Central

Hussain, Rawaa H.; Zawawi, Mariam; Bayfield, Mark A.

2013-01-01

The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3′OH-dependent trailer binding/protection and a UUU-3′OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3′OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily. PMID:23887937

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.

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

PubMed

Finka, Andrija; Goloubinoff, Pierre

2013-09-01

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10% of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

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

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

PubMed

Liang, Jingjing; Sagum, Cari A; Bedford, Mark T; Sidhu, Sachdev S; Sudol, Marius; Han, Ziying; Harty, Ronald N

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.

A Review of Acquired Thermotolerance, Heat Shock Proteins, and Molecular Chaperones in Archaea: Heat Shock in Archaea

DOE R&D Accomplishments Database

Trent, J. D.

1996-02-09

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.

A Bipartite Interaction between Hsp70 and CHIP Regulates Ubiquitination of Chaperoned Client Proteins

DOE PAGES

Zhang, Huaqun; Amick, Joseph; Chakravarti, Ritu; ...

2015-02-12

The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. Here, we demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required formore » proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.« less

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

Functional interactions of nucleocapsid protein of feline immunodeficiency virus and cellular prion protein with the viral RNA.

PubMed

Moscardini, Mila; Pistello, Mauro; Bendinelli, M; Ficheux, Damien; Miller, Jennifer T; Gabus, Caroline; Le Grice, Stuart F J; Surewicz, Witold K; Darlix, Jean-Luc

2002-04-19

All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis. Copyright 2002 Elsevier Science Ltd.

Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation.

PubMed

de Almeida, Sérgio F; Picarote, Gonçalo; Fleming, John V; Carmo-Fonseca, Maria; Azevedo, Jorge E; de Sousa, Maria

2007-09-21

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.

Preventing α-synuclein aggregation: the role of the small heat-shock molecular chaperone proteins.

PubMed

Cox, Dezerae; Carver, John A; Ecroyd, Heath

2014-09-01

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Production of 10S-hydroxy-8(E)-octadecenoic acid from oleic acid by whole recombinant Escherichia coli cells expressing 10S-dioxygenase from Nostoc punctiforme PCC 73102 with the aid of a chaperone.

PubMed

Kim, Min-Ji; Seo, Min-Ju; Shin, Kyung-Chul; Oh, Deok-Kun

2017-01-01

To increase the production of 10S-hydroxy-8(E)-octadecenoic acid from oleic acid by whole recombinant Escherichia coli cells expressing Nostoc punctiforme 10S-dioxygenase with the aid of a chaperone. The optimal conditions for 10S-hydroxy-8(E)-octadecenoic acid production by recombinant cells co-expressing chaperone plasmid were pH 9, 35 °C, 15 % (v/v) dimethyl sulfoxide, 40 g cells l -1 , and 10 g oleic acid l -1 . Under these conditions, recombinant cells co-expressing chaperone plasmid produced 7.2 g 10S-hydroxy-8(E)-octadecenoic acid l -1 within 30 min, with a conversion yield of 72 % (w/w) and a volumetric productivity of 14.4 g l -1 h -1 . The activity of recombinant cells expressing 10S-dioxygenase was increased by 200 % with the aid of a chaperone, demonstrating the first biotechnological production of 10S-hydroxy-8(E)-octadecenoic acid using recombinant cells expressing 10S-dioxygenase.

Hsp100/ClpB Chaperone Function and Mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

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/ClpBmore » 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”.« less

Super Spy variants implicate flexibility in chaperone action.

PubMed

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.

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

Chaperone-client complexes: A dynamic liaison

NASA Astrophysics Data System (ADS)

Hiller, Sebastian; Burmann, Björn M.

2018-04-01

Living cells contain molecular chaperones that are organized in intricate networks to surveil protein homeostasis by avoiding polypeptide misfolding, aggregation, and the generation of toxic species. In addition, cellular chaperones also fulfill a multitude of alternative functionalities: transport of clients towards a target location, help them fold, unfold misfolded species, resolve aggregates, or deliver clients towards proteolysis machineries. Until recently, the only available source of atomic resolution information for virtually all chaperones were crystal structures of their client-free, apo-forms. These structures were unable to explain details of the functional mechanisms underlying chaperone-client interactions. The difficulties to crystallize chaperones in complexes with clients arise from their highly dynamic nature, making solution NMR spectroscopy the method of choice for their study. With the advent of advanced solution NMR techniques, in the past few years a substantial number of structural and functional studies on chaperone-client complexes have been resolved, allowing unique insight into the chaperone-client interaction. This review summarizes the recent insights provided by advanced high-resolution NMR-spectroscopy to understand chaperone-client interaction mechanisms at the atomic scale.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choudhury, Kamalika Roy; Centre for Neuroscience, Indian Institute of Science, Bangalore 560012; Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com

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-terminalmore » 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.« less

Histone chaperones: an escort network regulating histone traffic.

PubMed

De Koning, Leanne; Corpet, Armelle; Haber, James E; Almouzni, Geneviève

2007-11-01

In eukaryotes, DNA is organized into chromatin in a dynamic manner that enables it to be accessed for processes such as transcription and repair. Histones, the chief protein component of chromatin, must be assembled, replaced or exchanged to preserve or change this organization according to cellular needs. Histone chaperones are key actors during histone metabolism. Here we classify known histone chaperones and discuss how they build a network to escort histone proteins. Molecular interactions with histones and their potential specificity or redundancy are also discussed in light of chaperone structural properties. The multiplicity of histone chaperone partners, including histone modifiers, nucleosome remodelers and cell-cycle regulators, is relevant to their coordination with key cellular processes. Given the current interest in chromatin as a source of epigenetic marks, we address the potential contributions of histone chaperones to epigenetic memory and genome stability.

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria.

PubMed

Zmijewski, Michał A; Skórko-Glonek, Joanna; Tanfani, Fabio; Banecki, Bogdan; Kotlarz, Agnieszka; Macario, Alberto J L; Lipińska, Barbara

2007-01-01

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

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.

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

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

PubMed Central

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.

2015-01-01

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 P58IPK/DnajC3, an ER luminal co-chaperone. P58IPK−/− 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 P58IPK−/− mice, suggesting P58IPK deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58IPK−/− 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. PMID:25795214

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. Copyright © 2012 Elsevier Ltd. All rights reserved.

Get3 is a holdase chaperone and moves to deposition sites for aggregated proteins when membrane targeting is blocked

PubMed Central

Powis, Katie; Schrul, Bianca; Tienson, Heather; Gostimskaya, Irina; Breker, Michal; High, Stephen; Schuldiner, Maya; Jakob, Ursula; Schwappach, Blanche

2013-01-01

Summary The endomembrane system of yeast contains different tail-anchored proteins that are post-translationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment could be hazardous in the cytosol if membrane insertion fails, resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum, where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function: promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis. PMID:23203805

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chaperone gelator for the chiral self-assembly of all proteinogenic amino acids and their enantiomers.

PubMed

Chen, Jie; Wang, Tianyu; Liu, Minghua

2016-05-04

A concept of a chaperone gelator that can assist non-gelator molecules to form gels is proposed. Such a new gelator was developed and found to tune all the proteinogenic l-amino acids, as well as their enantiomers into supramolecular gels.

Chaperone-Mediated Autophagy in the Kidney: The Road More Traveled

PubMed Central

Franch, Harold A.

2014-01-01

Summary Chaperone-mediated autophagy (CMA) is a lysosomal proteolytic pathway in which cytosolic substrate proteins contain specific chaperone recognition sequences required for degradation and are translocated directly across the lysosomal membrane for destruction. CMA proteolytic activity has a reciprocal relationship with macroautophagy: CMA is most active in cells in which macroautophagy is least active. Normal renal proximal tubular cells have low levels of macroautophagy, but high basal levels of CMA activity. CMA activity is regulated by starvation, growth factors, oxidative stress, lipids, aging, and retinoic acid signaling. The physiological consequences of changes in CMA activity depend on the substrate proteins present in a given cell type. In the proximal tubule, increased CMA results from protein or calorie starvation and from oxidative stress. Overactivity of CMA can be associated with tubular lysosomal pathology and certain cancers. Reduced CMA activity contributes to protein accumulation in renal tubular hypertrophy, but may contribute to oxidative tissue damage in diabetes and aging. Although there are more questions than answers about the role of high basal CMA activity, this remarkable feature of tubular protein metabolism appears to influence a variety of chronic diseases. PMID:24485032

From hatching to dispatching: the multiple cellular roles of the Hsp70 molecular chaperone machinery.

PubMed

Meimaridou, Eirini; Gooljar, Sakina B; Chapple, J Paul

2009-01-01

Molecular chaperones are best recognized for their roles in de novo protein folding and the cellular response to stress. However, many molecular chaperones, and in particular the Hsp70 chaperone machinery, have multiple diverse cellular functions. At the molecular level, chaperones are mediators of protein conformational change. To facilitate conformational change of client/substrate proteins, in manifold contexts, chaperone power must be closely regulated and harnessed to specific cellular locales--this is controlled by cochaperones. This review considers specialized functions of the Hsp70 chaperone machinery mediated by its cochaperones. We focus on vesicular trafficking, protein degradation and a potential role in G protein-coupled receptor processing.

Chaperone-like properties of tobacco plastid thioredoxins f and m

PubMed Central

Sanz-Barrio, Ruth; Fernández-San Millán, Alicia; Carballeda, Jon; Corral-Martínez, Patricia; Seguí-Simarro, José M.; Farran, Inmaculada

2012-01-01

Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein. PMID:21948853

Endoplasmic reticulum Chaperon Tauroursodeoxycholic Acid Alleviates Obesity-Induced Myocardial Contractile Dysfunction

PubMed Central

Ceylan-Isik, Asli F.; Sreejayan, Nair; Ren, Jun

2010-01-01

ER stress is involved in the pathophysiology of obesity although little is known about the role of ER stress on obesity-associated cardiac dysfunction. This study was designed to examine the effect of ER chaperone tauroursodeoxycholic acid (TUDCA) on obesity-induced myocardial dysfunction. Adult lean and ob/ob obese mice were treated TUDCA (50 mg/kg/d, p.o.) or vehicle for 5 wks. Oral glucose tolerance test (OGTT) was performed. Echocardiography, cardiomyocyte contractile and intracellular Ca2+ properties were assessed. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and protein expression of intracellular Ca2+ regulatory proteins were measured using 45Ca2+ uptake and Western blot analysis, respectively. Insulin signaling, ER stress markers and HSP90 were evaluated. Our results revealed that chronic TUDCA treatment lower systolic blood pressure and lessened glucose intolerance in obese mice. Obesity led to increased diastolic diameter, cardiac hypertrophy, compromised fractional shortening, cardiomyocyte contractile (peak shortening, maximal velocity of shortening/relengthening, and duration of contraction/relaxation) and intracellular Ca2+ properties, all of which were significantly attenuated by TUDCA. TUDCA reconciled obesity-associated decreased in SERCA activity and expression, and increase in serine phosphorylation of IRS, total and phosphorylated cJun, ER stress markers Bip, peIF2α and pPERK. Obesity-induced changes in phospholamban and HSP90 were unaffected by TUDCA. In vitro finding revealed that TUDCA ablated palmitic acid-induced cardiomyocyte contractile dysfunction. In summary, these data depicted a pivotal role of ER stress in obesity-associated cardiac contractile dysfunction, suggesting the therapeutic potential of ER stress as a target in the management of cardiac dysfunction in obesity. PMID:21035453

Low-power millimeter wave radiations do not alter stress-sensitive gene expression of chaperone proteins.

PubMed

Zhadobov, M; Sauleau, R; Le Coq, L; Debure, L; Thouroude, D; Michel, D; Le Dréan, Y

2007-04-01

This article reports experimental results on the influence of low-power millimeter wave (MMW) radiation at 60 GHz on a set of stress-sensitive gene expression of molecular chaperones, namely clusterin (CLU) and HSP70, in a human brain cell line. Selection of the exposure frequency is determined by its near-future applications for the new broadband civil wireless communication systems including wireless local area networks (WLAN) for domestic and professional uses. Frequencies around 60 GHz are strongly attenuated in the earth's atmosphere and such radiations represent a new environmental factor. An exposure system operating in V-band (50-75 GHz) was developed for cell exposure. U-251 MG glial cell line was sham-exposed or exposed to MMW radiation for different durations (1-33 h) and two different power densities (5.4 microW/cm(2) or 0.54 mW/cm(2)). As gene expression is a multiple-step process, we analyzed chaperone proteins induction at different levels. First, using luciferase reporter gene, we investigated potential effect of MMWs on the activation of transcription factors (TFs) and gene promoter activity. Next, using RT-PCR and Western blot assays, we verified whether MMW exposure could alter RNA accumulation, translation, or protein stability. Experimental data demonstrated the absence of significant modifications in gene transcription, mRNA, and protein amount for the considered stress-sensitive genes for the exposure durations and power densities investigated. The main results of this study suggest that low-power 60 GHz radiation does not modify stress-sensitive gene expression of chaperone proteins. (c) 2006 Wiley-Liss, Inc.

Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

PubMed

Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A

1997-03-15

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes

Heterologous Gln/Asn-Rich Proteins Impede the Propagation of Yeast Prions by Altering Chaperone Availability

PubMed Central

Yang, Zi; Hong, Joo Y.; Derkatch, Irina L.; Liebman, Susan W.

2013-01-01

Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI +] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI +] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI +]. PMID:23358669

Correlation of membrane binding and hydrophobicity to the chaperone-like activity of PDC-109, the major protein of bovine seminal plasma.

PubMed

Sankhala, Rajeshwer S; Damai, Rajani S; Swamy, Musti J

2011-03-08

The major protein of bovine seminal plasma, PDC-109 binds to choline phospholipids present on the sperm plasma membrane upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. PDC-109 also shares significant similarities with small heat shock proteins and exhibits chaperone-like activity (CLA). Although the polydisperse nature of this protein has been shown to be important for its CLA, knowledge of other factors responsible for such an activity is scarce. Since surface exposure of hydrophobic residues is known to be an important factor which modulates the CLA of chaperone proteins, in the present study we have probed the surface hydrophobicity of PDC-109 using bisANS and ANS. Further, effect of phospholipids on the structure and chaperone-like activity of PDC-109 was studied. Presence of DMPC was found to increase the CLA of PDC-109 significantly, which could be due to the considerable exposure of hydrophobic regions on the lipid-protein recombinants, which can interact productively with the nonnative structures of target proteins, resulting in their protection. However, inclusion of DMPG instead of DMPC did not significantly alter the CLA of PDC-109, which could be due to the lower specificity of PDC-109 for DMPG as compared to DMPC. Cholesterol incorporation into DMPC membranes led to a decrease in the CLA of PDC-109-lipid recombinants, which could be attributed to reduced accessibility of hydrophobic surfaces to the substrate protein(s). These results underscore the relevance of phospholipid binding and hydrophobicity to the chaperone-like activity of PDC-109.

Correlation of Membrane Binding and Hydrophobicity to the Chaperone-Like Activity of PDC-109, the Major Protein of Bovine Seminal Plasma

PubMed Central

Sankhala, Rajeshwer S.; Damai, Rajani S.; Swamy, Musti J.

2011-01-01

The major protein of bovine seminal plasma, PDC-109 binds to choline phospholipids present on the sperm plasma membrane upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. PDC-109 also shares significant similarities with small heat shock proteins and exhibits chaperone-like activity (CLA). Although the polydisperse nature of this protein has been shown to be important for its CLA, knowledge of other factors responsible for such an activity is scarce. Since surface exposure of hydrophobic residues is known to be an important factor which modulates the CLA of chaperone proteins, in the present study we have probed the surface hydrophobicity of PDC-109 using bisANS and ANS. Further, effect of phospholipids on the structure and chaperone-like activity of PDC-109 was studied. Presence of DMPC was found to increase the CLA of PDC-109 significantly, which could be due to the considerable exposure of hydrophobic regions on the lipid-protein recombinants, which can interact productively with the nonnative structures of target proteins, resulting in their protection. However, inclusion of DMPG instead of DMPC did not significantly alter the CLA of PDC-109, which could be due to the lower specificity of PDC-109 for DMPG as compared to DMPC. Cholesterol incorporation into DMPC membranes led to a decrease in the CLA of PDC-109-lipid recombinants, which could be attributed to reduced accessibility of hydrophobic surfaces to the substrate protein(s). These results underscore the relevance of phospholipid binding and hydrophobicity to the chaperone-like activity of PDC-109. PMID:21408153

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

Computational modeling of allosteric regulation in the hsp90 chaperones: a statistical ensemble analysis of protein structure networks and allosteric communications.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-06-01

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

The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone.

PubMed

Marcu, M G; Chadli, A; Bouhouche, I; Catelli, M; Neckers, L M

2000-11-24

Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases. The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitro and in vivo depletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-1, and p185(ErbB2). In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the co-chaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino- and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.

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.

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

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

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. Copyright © 2014 Mosby, Inc. All rights reserved.

Structure of Transmembrane Domain of Lysosome-associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-mediated Autophagy*

PubMed Central

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

2014-01-01

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. PMID:25342746

Expression of Mitochondrial Cytochrome C Oxidase Chaperone Gene (COX20) Improves Tolerance to Weak Acid and Oxidative Stress during Yeast Fermentation

PubMed Central

Kumar, Vinod; Hart, Andrew J.; Keerthiraju, Ethiraju R.; Waldron, Paul R.; Tucker, Gregory A.; Greetham, Darren

2015-01-01

Introduction Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid. Results A Δcox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a Δcox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress. Conclusion This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress. PMID:26427054

Crystal structure of human proteasome assembly chaperone PAC4 involved in proteasome formation.

PubMed

Kurimoto, Eiji; Satoh, Tadashi; Ito, Yuri; Ishihara, Eri; Okamoto, Kenta; Yagi-Utsumi, Maho; Tanaka, Keiji; Kato, Koichi

2017-05-01

The 26S proteasome is a large protein complex, responsible for degradation of ubiquinated proteins in eukaryotic cells. Eukaryotic proteasome formation is a highly ordered process that is assisted by several assembly chaperones. The assembly of its catalytic 20S core particle depends on at least five proteasome-specific chaperones, i.e., proteasome-assembling chaperons 1-4 (PAC1-4) and proteasome maturation protein (POMP). The orthologues of yeast assembly chaperones have been structurally characterized, whereas most mammalian assembly chaperones are not. In the present study, we determined a crystal structure of human PAC4 at 1.90-Å resolution. Our crystallographic data identify a hydrophobic surface that is surrounded by charged residues. The hydrophobic surface is complementary to that of its binding partner, PAC3. The surface also exhibits charge complementarity with the proteasomal α4-5 subunits. This will provide insights into human proteasome-assembling chaperones as potential anticancer drug targets. © 2017 The Protein Society.

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

PubMed

Kajitani, Kaori; Kato, Kohsuke; Nagata, Kyosuke

2017-04-01

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. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Contribution of chaperones to STAT pathway signaling

PubMed Central

Bocchini, Claire E; Kasembeli, Moses M; Roh, Soung-Hun; Tweardy, David J

2014-01-01

Aberrant STAT signaling is associated with the development and progression of many cancers and immune related diseases. Recent findings demonstrate that proteostasis modulators under clinical investigation for cancer therapy have a significant impact on STAT signaling, which may be critical for mediating their anti-cancer effects. Chaperones are critical for protein folding, stability and function and, thus, play an essential role in the maintenance of proteostasis. In this review we discuss the role of chaperones in STAT and tyrosine kinase (TK) protein folding, modulation of STAT and TK activity, and degradation of TKs. We highlight the important role of chaperones in STAT signaling, and how this knowledge has provided a framework for the development of new therapeutic avenues of targeting STAT signaling related pathologies. PMID:26413421

Atomistic simulations and network-based modeling of the Hsp90-Cdc37 chaperone binding with Cdk4 client protein: A mechanism of chaperoning kinase clients by exploiting weak spots of intrinsically dynamic kinase domains

PubMed Central

Czemeres, Josh; Buse, Kurt

2017-01-01

A fundamental role of the Hsp90 and Cdc37 chaperones in mediating conformational development and activation of diverse protein kinase clients is essential in signal transduction. There has been increasing evidence that the Hsp90-Cdc37 system executes its chaperoning duties by recognizing conformational instability of kinase clients and modulating their folding landscapes. The recent cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex has provided a framework for dissecting regulatory principles underlying differentiation and recruitment of protein kinase clients to the chaperone machinery. In this work, we have combined atomistic simulations with protein stability and network-based rigidity decomposition analyses to characterize dynamic factors underlying allosteric mechanism of the chaperone-kinase cycle and identify regulatory hotspots that control client recognition. Through comprehensive characterization of conformational dynamics and systematic identification of stabilization centers in the unbound and client- bound Hsp90 forms, we have simulated key stages of the allosteric mechanism, in which Hsp90 binding can induce instability and partial unfolding of Cdk4 client. Conformational landscapes of the Hsp90 and Cdk4 structures suggested that client binding can trigger coordinated dynamic changes and induce global rigidification of the Hsp90 inter-domain regions that is coupled with a concomitant increase in conformational flexibility of the kinase client. This process is allosteric in nature and can involve reciprocal dynamic exchanges that exert global effect on stability of the Hsp90 dimer, while promoting client instability. The network-based rigidity analysis and emulation of thermal unfolding of the Cdk4-cyclin D complex and Hsp90-Cdc37-Cdk4 complex revealed weak spots of kinase instability that are present in the native Cdk4 structure and are targeted by the chaperone during client recruitment. Our findings suggested that this

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

PubMed Central

Gorenberg, Erica L.; Chandra, Sreeganga S.

2017-01-01

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

Chaperoning the Cancer: The Proteostatic Functions of the Heat Shock Proteins in Cancer.

PubMed

Vahid, Sepideh; Thaper, Daksh; Zoubeidi, Amina

2017-01-01

Protein homeostasis (proteostasis) is vital for the survival of cells in physiological and pathological conditions. Particularly, cancer cells are in constant state of cellular stress due to rapid proliferation and decreased quality control in proteosynthesis and therefore, are exceedingly dependent on the homeostasis pathways. Among the complex biological mechanisms regulating proteostasis are the highly conserved molecular chaperones, heat shock proteins (HSPs). HSPs assist cell survival by catalysing the proper folding of proteins, modulation of the apoptotic machinery and finally regulating the protein degradation machinery, providing either the stability or the degradation of selected proteins under stress conditions. Inevitably, HSPs are upregulated in malignancies and participate in different hallmarks of cancer, with indispensable roles in the onset and progression of the disease. Moreover, high levels of HSPs contribute to poor prognosis and treatment resistance in various cancers. Therefore these molecular chaperones present as attractive targets for anti-cancer therapy. This review describes how HSPs regulate different hallmarks of cancer and provides an overview on the most relevant patents which have recently appeared in the literature. The patents were extracted from Google Patents (2012-2016) while the clinical trial results were mined from www.clinicaltrial.gov. Review of literature shows that the proteostatic functions of HSPs can modify different hallmarks of cancer. Moreover, targeting HSPs (notably HSP27, HSP70 and HSP90) exhibited positive results in clinical trials so far. However, more studies should be designed to optimize the efficacy of mono or combination therapy in various malignancies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

PubMed

Verma, Amit K; Diwan, Danish; Raut, Sandeep; Dobriyal, Neha; Brown, Rebecca E; Gowda, Vinita; Hines, Justin K; Sahi, Chandan

2017-06-07

Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations. Copyright © 2017 Verma et al.

RNAi-Mediated Reverse Genetic Screen Identified Drosophila Chaperones Regulating Eye and Neuromuscular Junction Morphology.

PubMed

Raut, Sandeep; Mallik, Bhagaban; Parichha, Arpan; Amrutha, Valsakumar; Sahi, Chandan; Kumar, Vimlesh

2017-07-05

Accumulation of toxic proteins in neurons has been linked with the onset of neurodegenerative diseases, which in many cases are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and the resolubilization of unfolded proteins, thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance remains largely unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C -Gal4-mediated RNAi knockdown revealed that ∼50% of the chaperones are essential in Drosophila Knocking down these genes in eyes revealed that ∼30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry, and robust behavioral assays, we identified a new set of chaperones that play critical roles in the regulation of Drosophila NMJ structural organization. Together, our data present the first classification and comprehensive analysis of Drosophila chaperones. Our screen identified a new set of chaperones that regulate eye and NMJ morphogenesis. The outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development. Copyright © 2017 Raut et al.

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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

Verma, Sharad; Singh, Amit; Mishra, Abha

2012-07-01

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

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. Copyright © 2010 Elsevier Inc. All rights reserved.

Increase in the levels of chaperone proteins by exposure to beta-estradiol, bisphenol A and 4-methoxyphenol in human cells transfected with estrogen receptor alpha cDNA.

PubMed

Kita, Kazuko; Jin, Yuan-Hu; Sun, Zhuo; Chen, Shi-Ping; Sumiya, Yoko; Hongo, Toshio; Suzuki, Nobuo

2009-06-01

We examined changes in the levels of chaperone proteins to evaluate the toxic effects of environmental chemicals in human cells in vitro. Some chaperones are up-regulated by estrogenic chemicals, but the effect is not necessarily dependent on the receptor. Thus we also investigated whether a chemical-induced change in chaperone protein expression is human estrogen receptor (hER)-dependent or not, using cultured human cell lines transfected with hERalpha cDNA or an empty vector. In the hERalpha-expressed cells, the protein levels of the heat shock protein 27 (HSP27), the glucose-regulated protein 78 (GRP78/BiP), and GRP94 increased after exposure to beta-estradiol (E(2)) (from 10(-9)M to 10(-6)M) and bisphenol A (BPA) (from 10(-6)M to 10(-5)M). On the other hand, the increase was not observed in the cells without hERalpha expression. These results suggest that the E(2)- and BPA-induced increase in the protein levels were hERalpha dependent. We next examined the effect of four phenolic chemicals similar in structure to BPA, and found that among them, 4-methoxyphenol (from 10(-6)M to 10(-5)M) increased the levels of the chaperone proteins with hERalpha dependency. Thus the human cultured cells would be suitable for evaluating whether an increase in chaperone proteins occurs upon exposure to environmental chemicals and whether the effect is ER-dependent.

Identification and characterization of a type III secretion-associated chaperone in the type III secretion system 1 of Vibrio parahaemolyticus.

PubMed

Akeda, Yukihiro; Okayama, Kanna; Kimura, Tomomi; Dryselius, Rikard; Kodama, Toshio; Oishi, Kazunori; Iida, Tetsuya; Honda, Takeshi

2009-07-01

Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus. In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30-100 amino acids and an amino terminal secretion signal encompassing the first 5-20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.

Oligomerization of a molecular chaperone modulates its activity

PubMed Central

Kawagoe, Soichiro; Ishimori, Koichiro

2018-01-01

Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining the lower affinity for protein substrates of the dimeric compared to the monomeric TF. Surprisingly, the dimeric TF associates faster with proteins and it exhibits stronger anti-aggregation and holdase activity than the monomeric TF. The structural data show that the dimer assembles in a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity, thus accounting for the observed accelerated association with unfolded proteins. Our results demonstrate how the activity of a chaperone can be modulated to provide distinct functional outcomes in the cell. PMID:29714686

The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase.

PubMed

Caldas, Thérèse; Malki, Abderrahim; Kern, Renée; Abdallah, Jad; Richarme, Gilbert

2006-05-12

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.

Interaction of the Disordered Yersinia Effector Protein YopE with Its Cognate Chaperone SycE

DTIC Science & Technology

2009-01-01

structures of YopECBD were molten globules with a hydrophobic core. Molecular dynamics (MD) simulations indi- cated that the structure remained compact at...ensembles of unfolded conformations of the Yersinia effector YopE using REMD simulations and docked them to the chaper- one SycE using a multistep protein...disordered state but transitions into an ordered state upon binding to its cognate chaperone (7). The dynamics of the disordered effector protein and

Chemical Endoplasmic Reticulum Chaperone Alleviates Doxorubicin-Induced Cardiac Dysfunction.

PubMed

Fu, Hai Ying; Sanada, Shoji; Matsuzaki, Takashi; Liao, Yulin; Okuda, Keiji; Yamato, Masaki; Tsuchida, Shota; Araki, Ryo; Asano, Yoshihiro; Asanuma, Hiroshi; Asakura, Masanori; French, Brent A; Sakata, Yasushi; Kitakaze, Masafumi; Minamino, Tetsuo

2016-03-04

Doxorubicin is an effective chemotherapeutic agent for cancer, but its use is often limited by cardiotoxicity. Doxorubicin causes endoplasmic reticulum (ER) dilation in cardiomyocytes, and we have demonstrated that ER stress plays important roles in the pathophysiology of heart failure. We evaluated the role of ER stress in doxorubicin-induced cardiotoxicity and examined whether the chemical ER chaperone could prevent doxorubicin-induced cardiac dysfunction. We confirmed that doxorubicin caused ER dilation in mouse hearts, indicating that doxorubicin may affect ER function. Doxorubicin activated an ER transmembrane stress sensor, activating transcription factor 6, in cultured cardiomyocytes and mouse hearts. However, doxorubicin suppressed the expression of genes downstream of activating transcription factor 6, including X-box binding protein 1. The decreased levels of X-box binding protein 1 resulted in a failure to induce the expression of the ER chaperone glucose-regulated protein 78 which plays a major role in adaptive responses to ER stress. In addition, doxorubicin activated caspase-12, an ER membrane-resident apoptotic molecule, which can lead to cardiomyocyte apoptosis and cardiac dysfunction. Cardiac-specific overexpression of glucose-regulated protein 78 by adeno-associated virus 9 or the administration of the chemical ER chaperone 4-phenylbutyrate attenuated caspase-12 cleavage, and alleviated cardiac apoptosis and dysfunction induced by doxorubicin. Doxorubicin activated the ER stress-initiated apoptotic response without inducing the ER chaperone glucose-regulated protein 78, further augmenting ER stress in mouse hearts. Cardiac-specific overexpression of glucose-regulated protein 78 or the administration of the chemical ER chaperone alleviated the cardiac dysfunction induced by doxorubicin and may facilitate the safe use of doxorubicin for cancer treatment. © 2016 American Heart Association, Inc.

Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90

PubMed Central

Zara, Vincenzo; Ferramosca, Alessandra; Robitaille-Foucher, Philippe; Palmieri, Ferdinando; Young, Jason C.

2016-01-01

Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment. PMID:19143589

Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90.

PubMed

Zara, Vincenzo; Ferramosca, Alessandra; Robitaille-Foucher, Philippe; Palmieri, Ferdinando; Young, Jason C

2009-04-15

Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment.

The Hsp40 proteins of Plasmodium falciparum and other apicomplexa: regulating chaperone power in the parasite and the host.

PubMed

Botha, M; Pesce, E-R; Blatch, G L

2007-01-01

Extensive structural and functional remodelling of Plasmodium falciparum (malaria)-infected erythrocytes follows the export of a range of proteins of parasite origin (exportome) across the parasitophorous vacuole into the host erythrocyte. The genome of P. falciparum encodes a diverse chaperone complement including at least 43 members of the heat shock protein 40kDa (Hsp40) family, and six members of the heat shock protein 70kDa (Hsp70) family. Nearly half of the Hsp40 proteins of P. falciparum are predicted to contain a PEXEL/HT (Plasmodium export element/host targeting signal) sequence motif, and hence are likely to be part of the exportome. In this review we critically evaluate the classification, sequence similarity and clustering, and possible interactors of the P. falciparum Hsp40 chaperone machinery. In addition to the types I, II and III Hsp40 proteins all exhibiting the signature J-domain, the P. falciparum genome also encodes a number of specialized Hsp40 proteins with a J-like domain, which we have categorized as type IV Hsp40 proteins. Analysis of the potential P. falciparum Hsp40 protein interaction network revealed connections predominantly with cytoskeletal and membrane proteins, transcriptional machinery, DNA repair and replication machinery, translational machinery, the proteasome and proteolytic enzymes, and enzymes involved in cellular physiology. Comparison of the Hsp40 proteins of P. falciparum to those of other apicomplexa reveals that most of the proteins (especially the PEXEL/HT-containing proteins) are unique to P. falciparum. Furthermore, very few of the P. falciparum Hsp40 proteins have human homologs, except for those proteins implicated in fundamental biological processes. Our analysis suggests that P. falciparum has evolved an expanded and specialized Hsp40 protein machinery to enable it successfully to invade and remodel the human erythrocyte, and we propose a model in which these proteins are involved in chaperone

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

Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

PubMed

Cooper, Colin A; Zhang, Kun; Andres, Sara N; Fang, Yuan; Kaniuk, Natalia A; Hannemann, Mandy; Brumell, John H; Foster, Leonard J; Junop, Murray S; Coombes, Brian K

2010-02-05

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

Expression of an Atriplex nummularia gene encoding a protein homologous to the bacterial molecular chaperone DnaJ.

PubMed Central

Zhu, J K; Shi, J; Bressan, R A; Hasegawa, P M

1993-01-01

DnaJ is a 36-kD heat shock protein that functions together with Dnak (Hsp70) as a molecular chaperone in Escherichia coli. We have obtained a cDNA clone from the higher plant Atriplex nummularia that encodes a 46.6-kD polypeptide (ANJ1) with an overall 35.2% amino acid sequence identity with the E. coli DnaJ. ANJ1 has 43.4% overall sequence identity with the Saccharomyces cerevisiae cytoplasmic DnaJ homolog YDJ1/MAS5. Complementation of the yeast mas5 mutation indicated that ANJ1 is a functional homolog of YDJ1/MAS5. The presence of other DnaJ homologs in A. nummularia was demonstrated by the detection of proteins that are antigenically related to the yeast mitochondrial DnaJ homolog SCJ1 and the yeast DnaJ-related protein Sec63. Expression of the ANJ1 gene was compared with that of an A. nummularia Hsp70 gene. Expression of both ANJ1 and Hsp70 transcripts was coordinately induced by heat shock. However, noncoordinate accumulation of ANJ1 and Hsp70 mRNAs occurred during the cell growth cycle and in response to NaCl stress. PMID:8467224

Expression of an Atriplex nummularia gene encoding a protein homologous to the bacterial molecular chaperone DnaJ.

PubMed

Zhu, J K; Shi, J; Bressan, R A; Hasegawa, P M

1993-03-01

DnaJ is a 36-kD heat shock protein that functions together with Dnak (Hsp70) as a molecular chaperone in Escherichia coli. We have obtained a cDNA clone from the higher plant Atriplex nummularia that encodes a 46.6-kD polypeptide (ANJ1) with an overall 35.2% amino acid sequence identity with the E. coli DnaJ. ANJ1 has 43.4% overall sequence identity with the Saccharomyces cerevisiae cytoplasmic DnaJ homolog YDJ1/MAS5. Complementation of the yeast mas5 mutation indicated that ANJ1 is a functional homolog of YDJ1/MAS5. The presence of other DnaJ homologs in A. nummularia was demonstrated by the detection of proteins that are antigenically related to the yeast mitochondrial DnaJ homolog SCJ1 and the yeast DnaJ-related protein Sec63. Expression of the ANJ1 gene was compared with that of an A. nummularia Hsp70 gene. Expression of both ANJ1 and Hsp70 transcripts was coordinately induced by heat shock. However, noncoordinate accumulation of ANJ1 and Hsp70 mRNAs occurred during the cell growth cycle and in response to NaCl stress.

Structural Basis of Interdomain Communication in the Hsc70 Chaperone

PubMed Central

Jiang, Jianwen; Prasad, Kondury; Lafer, Eileen M.; Sousa, Rui

2015-01-01

Summary Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. Were-port here the 2.6 Å structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds. PMID:16307916

Miglustat (NB-DNJ) works as a chaperone for mutated acid beta-glucosidase in cells transfected with several Gaucher disease mutations.

PubMed

Alfonso, Pilar; Pampín, Sandra; Estrada, Jorge; Rodríguez-Rey, José Carlos; Giraldo, Pilar; Sancho, Javier; Pocoví, Miguel

2005-01-01

Gaucher disease (GD) is a disorder of glycosphinglipid metabolism caused by deficiency of lysosomal acid beta-glucosidase (GC), resulting in progressive deposition of glucosylceramide in macrophages. The glucose analogue, N-butyl-deoxynojirimycin (NB-DNJ, Miglustat), is an inhibitor of the ceramide-specific glucosyltransferase (CSG) which catalyzes the first step of glycosphingolipids biosynthesis and is currently approved for the oral treatment of type 1 GD. Using site-directed mutagenesis, we constructed plasmids containing wild-type and several mutations in glucocerebrosidase (GBA) gene. The plasmids were transfected into COS-7 cells and stable transfected cell lines were obtained by geneticin (G418) selection. Cells were cultured during 6 days with medium with or without 10 microM NB-DNJ. The addition of NB-DNJ to COS-7 cell medium leads to 1.3-, 2.1-, 2.3-, 3.6-, and 9.9-fold increase in the activity of S364R, wild-type, N370S, V15M, and M123T GC, respectively. However, no significant changes were observed in the activity of the L444P, L336P, and S465del mutated proteins, but a small decrease in the rare P266L variant was observed. These results suggest that NB-DNJ, in addition to the inhibitory effect on CSG, also works as a "chemical chaperone", increasing the activity of acid beta-glucosidase of wild-type and several GC mutated proteins, including the most frequent N370S mutation. The specific location of the Miglustat binding site in GC is unknown. Potential binding sites in the enzyme have been searched for using computational molecular docking. The searching strategy identified three potential GC binding sites for Miglustat, one being the substrate-binding site of the enzyme, which was the best-ranked site by AutoDock program. Therefore, it is possible that Miglustat exerts its chaperoning activity on acid beta-glucosidase by acting as an inhibitor bound at the active site. This increase on the activity of the acid beta-glucosidase would imply that

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

Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity

PubMed Central

Sastry, M S R; Zhou, Weibin; Baneyx, François

2009-01-01

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. PMID:19517531

Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells

PubMed Central

Finka, Andrija; Mattoo, Rayees U. H.

2010-01-01

Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases. Electronic supplementary material The online version of this article (doi:10.1007/s12192-010-0216-8) contains supplementary material, which is available to authorized users. PMID:20694844

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

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

PubMed

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

2013-09-01

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 σ(S) (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.

ATP-dependent molecular chaperones in plastids--More complex than expected.

PubMed

Trösch, Raphael; Mühlhaus, Timo; Schroda, Michael; Willmund, Felix

2015-09-01

Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)*

PubMed Central

Shimamoto, Seiko; Kubota, Yasuo; Yamaguchi, Fuminori; Tokumitsu, Hiroshi; Kobayashi, Ryoji

2013-01-01

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca2+/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca2+/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca2+/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca2+-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway. PMID:23344957

Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

PubMed

Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

2014-01-01

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

Homology-based modeling of the Erwinia amylovora type III secretion chaperone DspF used to identify amino acids required for virulence and interaction with the effector DspE.

PubMed

Triplett, Lindsay R; Wedemeyer, William J; Sundin, George W

2010-09-01

The structure of DspF, a type III secretion system (T3SS) chaperone required for virulence of the fruit tree pathogen Erwinia amylovora, was modeled based on predicted structural homology to characterized T3SS chaperones. This model guided the selection of 11 amino acid residues that were individually mutated to alanine via site-directed mutagenesis. Each mutant was assessed for its effect on virulence complementation, dimerization and interaction with the N-terminal chaperone-binding site of DspE. Four amino acid residues were identified that did not complement the virulence defect of a dspF knockout mutant, and three of these residues were required for interaction with the N-terminus of DspE. This study supports the significance of the predicted beta-sheet helix-binding groove in DspF chaperone function. Copyright 2010 Elsevier Masson SAS. All rights reserved.

Membrane-associated stress proteins: more than simply chaperones.

PubMed

Horváth, Ibolya; Multhoff, Gabriele; Sonnleitner, Alois; Vígh, László

2008-01-01

The protein- and/or lipid-mediated association of chaperone proteins to membranes is a widespread phenomenon and implicated in a number of physiological and pathological events that were earlier partially or completely overlooked. A temporary association of certain HSPs with membranes can re-establish the fluidity and bilayer stability and thereby restore the membrane functionality during stress conditions. The fluidity and microdomain organization of membranes are decisive factors in the perception and transduction of stresses into signals that trigger the activation of specific HS genes. Conversely, the membrane association of HSPs may result in the inactivation of membrane-perturbing signals, thereby switch off the heat shock response. Interactions between certain HSPs and specific lipid microdomains ("rafts") might be a previously unrecognized means for the compartmentalization of HSPs to specific signaling platforms, where key signaling proteins are known to be concentrated. Any modulations of the membranes, especially the raft-lipid composition of the cells can alter the extracellular release and thus the immuno-stimulatory activity of certain HSPs. Reliable techniques, allowing mapping of the composition and dynamics of lipid microdomains and simultaneously the spatio-temporal localization of HSPs in and near the plasma membrane can provide suitable means with which to address fundamental questions, such as how HSPs are transported to and translocated through the plasma membrane. The possession of such information is critical if we are to target the membrane association principles of HSPs for successful drug development in most various diseases.

Functional cell-surface display of a lipase-specific chaperone.

PubMed

Wilhelm, Susanne; Rosenau, Frank; Becker, Stefan; Buest, Sebastian; Hausmann, Sascha; Kolmar, Harald; Jaeger, Karl-Erich

2007-01-02

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

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

PubMed

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

2015-01-01

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

Effect of glycation on α-crystallin structure and chaperone-like function

PubMed Central

Kumar, P. Anil; Kumar, M. Satish; Reddy, G. Bhanuprakash

2007-01-01

The chaperone-like activity of α-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of α-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of α-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of α-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of α-crystallin. Modification of α-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [Nϵ-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of α-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of α-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, α-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE

Active Participation of Cellular Chaperone Hsp90 in Regulating the Function of Rotavirus Nonstructural Protein 3 (NSP3)*

PubMed Central

Dutta, Dipanjan; Chattopadhyay, Shiladitya; Bagchi, Parikshit; Halder, Umesh Chandra; Nandi, Satabdi; Mukherjee, Anupam; Kobayashi, Nobumichi; Taniguchi, Koki; Chawla-Sarkar, Mamta

2011-01-01

Heat shock protein 90 (Hsp90) has been reported to positively regulate rotavirus replication by modulating virus induced PI3K/Akt and NFκB activation. Here, we report the active association of Hsp90 in the folding and stabilization of rotavirus nonstructural protein 3 (NSP3). In pCD-NSP3-transfected cells, treatment with Hsp90 inhibitor (17-N,N-dimethylethylenediamine-geldanamycin (17DMAG)) resulted in the proteasomal degradation of NSP3. Sequence analysis and deletion mutations revealed that the region spanning amino acids 225–258 within the C-terminal eIF4G-binding domain of NSP3 is a putative Hsp90 binding region. Co-immunoprecipitation and mammalian two-hybrid experiments revealed direct interaction of the C-terminal 12-kDa domain of Hsp90 (C90) with residues 225–258 of NSP3. NSP3-Hsp90 interaction is important for the formation of functionally active mature NSP3, because full-length NSP3 in the presence of the Hsp90 inhibitor or NSP3 lacking the amino acid 225–258 region did not show NSP3 dimers following in vitro coupled transcription-translation followed by chase. Disruption of residues 225–258 within NSP3 also resulted in poor RNA binding and eIF4G binding activity. In addition, inhibition of Hsp90 by 17DMAG resulted in reduced nuclear translocation of poly(A)-binding protein and translation of viral proteins. These results highlight the crucial role of Hsp90 chaperone in the regulation of assembly and functionality of a viral protein during the virus replication and propagation in host cells. PMID:21489987

Chaperones in maturation of molybdoenzymes: Why specific is better than general?

PubMed

Lemaire, Olivier N; Bouillet, Sophie; Méjean, Vincent; Iobbi-Nivol, Chantal; Genest, Olivier

2017-03-04

Molybdoenzymes play essential functions in living organisms and, as a result, in various geochemical cycles. It is thus crucial to understand how these complex proteins become highly efficient enzymes able to perform a wide range of catalytic activities. It has been established that specific chaperones are involved during their maturation process. Here, we raise the question of the involvement of general chaperones acting in concert with dedicated chaperones or not.

Functional screening of pharmacological chaperones via restoration of enzyme activity upon denaturation.

PubMed

Shanmuganathan, Meera; Britz-McKibbin, Philip

2012-10-02

Pharmacological chaperones (PCs) are small molecules that stabilize and promote protein folding. Enzyme inhibition is widely used for PC selection; however, it does not accurately reflect chaperone activity. We introduce a functional assay for characterization of PCs based on their capacity to restore enzyme activity that is abolished upon chemical denaturation. Dose-dependent activity curves were performed as a function of urea to assess the chaperone potency of various ligands to β-glucocerebrosidase as a model system. Restoration of enzyme activity upon denaturation allows direct screening of PCs for treatment of genetic disorders associated with protein deficiency, such as Gaucher disease.

Antigen Retrieval to Improve the Immunocytochemistry Detection of Sigma-1 Receptors and ER Chaperones

PubMed Central

Hayashi, Teruo; Lewis, Abasha; Hayashi, Eri; Betenbaugh, Michael J.; Su, Tsung-Ping

2011-01-01

Molecular chaperones localized at the endoplasmic reticulum (ER) lumen constitutively or cellular stress-dependently associate with a variety of proteins to promote their proper folding or to inhibit protein misfolding. ER chaperones preferentially form large complexes with co-chaperones and/or misfolded proteins in a highly crowded cellular environment that often hampers their detection by immunocytochemistry (ICC). This study establishes an antigen retrieval (AR) protocol to improve the ICC detection of ER chaperones in cultured cells using widely available antibodies against synthetic peptides. Among ten different antigen retrieval/fixation conditions, only the AR with Tris-HCl (pH 9.5) containing 6 M urea (80 °C for 10 min) significantly improved the ICC detection of the novel ER chaperone sigma-1 receptor (Sig-1R) in Chinese hamster ovary cells. Extended fixation with 4% paraformaldehyde for 1 hr effectively preserved the morphology of the ER under the AR condition. This method greatly enhanced the signal-to-noise ratio in Sig-1R ICC, thus allowing for semi-quantitative detection of protein upregulation under ER stress. The AR similarly improved the ICC detection of a series of other major ER chaperones, including BiP/GRP78, GRP94, calnexin, calreticulin, ERp57, protein disulfide isomerase, and cyclophilin B. The improved ICC methodology using the urea AR at 80°C may improve ICC of ER molecules as well as visualization of ER structure and substructures. PMID:21573736

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

PubMed

Porowińska, Dorota; Czarnecka, Joanna; Komoszyński, Michał

2014-07-01

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

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

The cardiac copper chaperone proteins Sco1 and CCS are up-regulated, but Cox 1 and Cox4 are down-regulated, by copper deficiency.

PubMed

Getz, Jean; Lin, Dingbo; Medeiros, Denis M

2011-10-01

Copper is ferried in a cell complexed to chaperone proteins, and in the heart much copper is required for cytochrome c oxidase (Cox). It is not completely understood how copper status affects the levels of these proteins. Here we determined if dietary copper deficiency could up- or down-regulate select copper chaperone proteins and Cox subunits 1 and 4 in cardiac tissue of rats. Sixteen weanling male Long-Evans rats were randomized into treatment groups, one group receiving a copper-deficient diet (<1 mg Cu/kg diet) and one group receiving a diet containing adequate copper (6 mg Cu/kg diet) for 5 weeks. Hearts were removed, weighed, and non-myofibrillar proteins separated to analyze for levels of CCS, Sco1, Ctr1, Cox17, Cox1, and Cox4 by SDS-PAGE and Western blotting. No changes were observed in the concentrations of CTR1 and Cox17 between copper-adequate and copper-deficient rats. CCS and Sco1 were up-regulated and Cox1 and Cox4 were both down-regulated as a result of copper deficiency. These data suggest that select chaperone proteins and may be up-regulated, and Cox1 and 4 down-regulated, by a dietary copper deficiency, whereas others appear not to be affected by copper status.

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

Mitochondrial Chaperones in the Brain: Safeguarding Brain Health and Metabolism?

PubMed

Castro, José Pedro; Wardelmann, Kristina; Grune, Tilman; Kleinridders, André

2018-01-01

The brain orchestrates organ function and regulates whole body metabolism by the concerted action of neurons and glia cells in the central nervous system. To do so, the brain has tremendously high energy consumption and relies mainly on glucose utilization and mitochondrial function in order to exert its function. As a consequence of high rate metabolism, mitochondria in the brain accumulate errors over time, such as mitochondrial DNA (mtDNA) mutations, reactive oxygen species, and misfolded and aggregated proteins. Thus, mitochondria need to employ specific mechanisms to avoid or ameliorate the rise of damaged proteins that contribute to aberrant mitochondrial function and oxidative stress. To maintain mitochondria homeostasis (mitostasis), cells evolved molecular chaperones that shuttle, refold, or in coordination with proteolytic systems, help to maintain a low steady-state level of misfolded/aggregated proteins. Their importance is exemplified by the occurrence of various brain diseases which exhibit reduced action of chaperones. Chaperone loss (expression and/or function) has been observed during aging, metabolic diseases such as type 2 diabetes and in neurodegenerative diseases such as Alzheimer's (AD), Parkinson's (PD) or even Huntington's (HD) diseases, where the accumulation of damage proteins is evidenced. Within this perspective, we propose that proper brain function is maintained by the joint action of mitochondrial chaperones to ensure and maintain mitostasis contributing to brain health, and that upon failure, alter brain function which can cause metabolic diseases.

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

PubMed Central

2010-01-01

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

Binding Affects the Tertiary and Quaternary Structures of the Shigella Translocator Protein IpaB and its Chaperone IpgC†

PubMed Central

Adam, Philip R.; Patil, Mrinalini K.; Dickenson, Nicholas E.; Choudhari, Shyamal; Barta, Michael; Geisbrecht, Brian V.; Picking, Wendy L.; Picking, William D.

2012-01-01

Shigella flexneri uses its type III secretion system (T3SS) to promote invasion of human intestinal epithelial cells as the first step in causing shigellosis, a life threatening form of dysentery. The Shigella type III secretion apparatus (T3SA) consists of a basal body that spans the bacterial envelope and an exposed needle that injects effector proteins into target cells. The nascent Shigella T3SA needle is topped with a pentamer of the needle tip protein invasion plasmid antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic translocator protein, IpaB, to the tip complex where it senses contact with a host membrane. In the bacterial cytoplasm, IpaB exists in a complex with its chaperone IpgC. Several structures of IpgC have been solved and we recently reported the 2.1-Å crystal structure of the N-terminal domain (IpaB74.224) of IpaB. Like IpgC, the IpaB N-terminal domain exists as a homodimer in solution. We now report that when the two are mixed, these homodimers dissociate and form heterodimers having a nanomolar dissociation constant. This is consistent with the equivalent complexes co-purified after being co-expressed in E. coli. Fluorescence data presented here also indicate that the N-terminal domain of IpaB possesses two regions that appear to contribute additively to chaperone binding. It is also likely that the IpaB N terminus adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed. PMID:22497344

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

PubMed

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

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

Screening of Neem extracts for microbial anti-chaperone activity by employing in vitro enzyme refolding assay.

PubMed

Patki, Jyoti M; Shah, Priyanka

2017-10-01

Microbial heat shock proteins (Hsps) play an important role in pathogenesis and development of resistance to existing drugs. New compounds that target microbial molecular chaperones have the potential of combating the challenge of anti-microbial resistance. The present study was aimed at assessing the employment of in vitro enzyme refolding assay to detect anti-chaperone activity of Neem ( Azadirachta indica ) extracts. Protein extracts of thermotolerant Escherichia coli cells were used as a source of Hsps or chaperones. Thermotolerance was found to be induced by pre-treating E. coli cells at 47 °C before subjecting them to a lethal temperature of 55 °C. This thermotolerance correlated with over-expression of specific proteins and reduced aggregation as evident from the SDS-PAGE profiles. Refolding assays of denatured enzymes exhibited 45% activity regain in presence of cell protein extracts containing chaperones compared to less than 5% regain in BSA negative controls. The chaperone activity was found to be ATP dependent. Addition of Neem extracts to refolding reaction mixtures distinctly reduced the activity regain (20%) in a dose dependent manner (500 and 1000 ppm). The negative influence of plant extract on refolding of the enzyme in the presence of chaperones gives evidence to its anti-chaperone activity. We propose that the employment of in vitro enzyme refolding assays will help not only to analyze the activity of known and putative chaperones but also to screen natural compounds for anti-microbial-Hsp activity.

Effect of leucine-to-methionine substitutions on the diffraction quality of histone chaperone SET/TAF-Ibeta/INHAT crystals.

PubMed

Senda, Miki; Muto, Shinsuke; Horikoshi, Masami; Senda, Toshiya

2008-10-01

One of the most frequent problems in crystallization is poor quality of the crystals. In order to overcome this obstacle several methods have been utilized, including amino-acid substitutions of the target protein. Here, an example is presented of crystal-quality improvement by leucine-to-methionine substitutions. A variant protein with three amino-acid substitutions enabled improvement of the crystal quality of the histone chaperone SET/TAF-Ibeta/INHAT when combined with optimization of the cryoconditions. This procedure improved the resolution of the SET/TAF-Ibeta/INHAT crystals from around 5.5 to 2.3 A without changing the crystallization conditions.

The RNA chaperone La promotes pre-tRNA maturation via indiscriminate binding of both native and misfolded targets.

PubMed

Vakiloroayaei, Ana; Shah, Neha S; Oeffinger, Marlene; Bayfield, Mark A

2017-11-02

Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3'OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known. La deletion in yeast is synthetically lethal when combined with the loss of tRNA modifications predicted to contribute to the native pre-tRNA fold, such as the N2, N2-dimethylation of G26 by the methyltransferase Trm1p. In this work, we identify G26 containing pre-tRNAs that misfold in the absence of Trm1p and/or La (Sla1p) in Schizosaccharomyces pombe cells, then test whether La preferentially associates with such tRNAs in vitro and in vivo. Our data suggest that La does not discriminate a native from misfolded RNA target, and highlights the potential challenges faced by RNA chaperones in preferentially binding defective substrates. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

PubMed

Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L; Southworth, Daniel R; Banerjee, Ruma

2017-10-27

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

αA-Crystallin–Derived Mini-Chaperone Modulates Stability and Function of Cataract Causing αAG98R-Crystallin

PubMed Central

Raju, Murugesan; Santhoshkumar, Puttur; Sharma, K. Krishna

2012-01-01

Background A substitution mutation in human αA-crystallin (αAG98R) is associated with autosomal dominant cataract. The recombinant mutant αAG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37°C. Our previous studies have shown that αA-crystallin–derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of αA-crystallin–derived mini-chaperone on the stability and chaperone activity of αAG98R-crystallin. Methodology/Principal Findings Recombinant αAG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with αAG98R. The rescuing of chaperone activity in mutantα-crystallin (αAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of αAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystallin. The complexes formed between mini-αA–αAG98R complex and ADH were more stable than the complexes formed between αAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin. Conclusion/Significance These results demonstrate that mini-chaperone stabilizes the mutant αA-crystallin and modulates the chaperone activity of αAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant �

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

PubMed

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

2017-12-05

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

Pharmacological chaperoning: a primer on mechanism and pharmacology.

PubMed

Leidenheimer, Nancy J; Ryder, Katelyn G

2014-05-01

Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs are considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast

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

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.

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. © FASEB.

4-Phenylbutyric acid reduces endoplasmic reticulum stress, trypsin activation, and acinar cell apoptosis while increasing secretion in rat pancreatic acini.

PubMed

Malo, Antje; Krüger, Burkhard; Göke, Burkhard; Kubisch, Constanze H

2013-01-01

Endoplasmic reticulum (ER) stress leads to misfolded proteins inside the ER and initiates unfolded protein response (UPR). Unfolded protein response components are involved in pancreatic function and activated during pancreatitis. However, the exact role of ER stress in the exocrine pancreas is unclear. The present study examined the effects of 4-phenylbutyric acid (4-PBA), an ER chaperone, on acini and UPR components. Rat acini were stimulated with cholecystokinin (10 pmol/L to 10 nmol/L) with or without preincubation of 4-PBA. The UPR components were analyzed, including chaperone-binding protein, protein kinaselike ER kinase, X-box-binding protein 1, c-Jun NH(2)-terminal kinase, CCAAT/enhancer-binding protein homologous protein, caspase 3, and apoptosis. Effects of 4-PBA were measured on secretion, calcium, and trypsin activation. 4-Phenylbutyric acid led to an increase of secretion, whereas trypsin activation with supraphysiological cholecystokinin was significantly reduced. 4-Phenylbutyric acid prevented chaperone-binding protein up-regulation, diminished protein kinaselike ER kinase, and c-Jun NH2-terminal kinase phosphorylation, prohibited X-box-binding protein 1 splicing and CCAAT/enhancer-binding protein homologous protein expression, caspase 3 activation, and apoptosis caused by supraphysiological cholecystokinin. By incubation with 4-PBA, beneficial in urea cycle deficiency, it was possible to enhance enzyme secretion to suppress trypsin activation, UPR activation, and proapoptotic pathways. The data hint new perspectives for the use of chemical chaperones in pancreatic diseases.

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. 2010 Elsevier Inc. All rights reserved.

A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

PubMed Central

van Lith, Marcel; Karala, Anna-Riikka; Bown, Dave; Gatehouse, John A.; Ruddock, Lloyd W.; Saunders, Philippa T.K.

2007-01-01

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Δ-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility. PMID:17507649

Conversion of scFv peptide-binding specificity for crystal chaperone development

PubMed Central

Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.

2011-01-01

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 Å resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a ∼52 Å 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. PMID:21217145

Characterization of the recombinant copper chaperone (CCS) from the plant Glycine (G.) max.

PubMed

Sagasti, Sara; Yruela, Inmaculada; Bernal, Maria; Lujan, Maria A; Frago, Susana; Medina, Milagros; Picorel, Rafael

2011-02-01

The goal of the present work was to characterize the recombinant copper chaperone (CCS) from soybean. Very little is known about plant copper chaperones, which makes this study of current interest, and allows for a comparison with the better known homologues from yeast and humans. To obtain sizeable amounts of pure protein suitable for spectroscopic characterization, we cloned and overexpressed the G. max CCS chaperone in E. coli in the presence of 0.5 mM CuSO(4) and 0.5 mM ZnSO(4) in the broth. A pure protein preparation was obtained by using two IMAC steps and pH gradient chromatography. Most of the proteins were obtained as apo-form, devoid of copper atoms. The chaperone showed a high content (i.e., over 40%) of loops, turns and random coil as determined both by circular dichroism and homology modelling. The homology 3-D structural model suggests the protein might fold in three structural protein domains. The 3-D model along with the primary structure and spectroscopic data may suggest that copper atoms occupy the two metal binding sites, MKCEGC and CTC, within the N-terminal domain I and C-terminal domain III, respectively. But only one Zn-binding site was obtained spectroscopically.

Molecular transformers in the cell: lessons learned from the DegP protease-chaperone.

PubMed

Sawa, Justyna; Heuck, Alexander; Ehrmann, Michael; Clausen, Tim

2010-04-01

Structure-function analysis of DegP revealed a novel mechanism for protease and chaperone regulation. Binding of unfolded proteins induces the oligomer reassembly from the resting hexamer (DegP6) into the functional protease-chaperone DegP12/24. The newly formed cage exhibits the characteristics of a proteolytic folding chamber, shredding those proteins that are severely misfolded while stabilizing and protecting proteins present in their native state. Isolation of native DegP complexes with folded outer membrane proteins (OMPs) highlights the importance of DegP in OMP biogenesis. The encapsulated OMP beta-barrel is significantly stabilized in the hydrophobic chamber of DegP12/24 and thus DegP seems to employ a reciprocal mechanism to those chaperones assisting the folding of water soluble proteins via polar interactions. In addition, we discuss in this review similarities to other complex proteolytic machines that, like DegP, are under control of a substrate-induced or stress-induced oligomer conversion.

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

Functional Diversification of Hsp40: Distinct J-Protein Functional Requirements for Two Prions Allow for Chaperone-Dependent Prion Selection

PubMed Central

Patel, Milan J.; Sporn, Zachary A.; Hines, Justin K.

2014-01-01

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

Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle

PubMed Central

Winter, Lilli; Staszewska, Ilona; Mihailovska, Eva; Fischer, Irmgard; Goldmann, Wolfgang H.; Schröder, Rolf; Wiche, Gerhard

2014-01-01

The ubiquitously expressed multifunctional cytolinker protein plectin is essential for muscle fiber integrity and myofiber cytoarchitecture. Patients suffering from plectinopathy-associated epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and mice lacking plectin in skeletal muscle display pathological desmin-positive protein aggregation and misalignment of Z-disks, which are hallmarks of myofibrillar myopathies (MFMs). Here, we developed immortalized murine myoblast cell lines to examine the pathogenesis of plectinopathies at the molecular and single cell level. Plectin-deficient myotubes, derived from myoblasts, were fully functional and mirrored the pathological features of EBS-MD myofibers, including the presence of desmin-positive protein aggregates and a concurrent disarrangement of the myofibrillar apparatus. Using this cell model, we demonstrated that plectin deficiency leads to increased intermediate filament network and sarcomere dynamics, marked upregulation of HSPs, and reduced myotube resilience following mechanical stretch. Currently, no specific therapy or treatment is available to improve plectin-related or other forms of MFMs; therefore, we assessed the therapeutic potential of chemical chaperones to relieve plectinopathies. Treatment with 4-phenylbutyrate resulted in remarkable amelioration of the pathological phenotypes in plectin-deficient myotubes as well as in plectin-deficient mice. Together, these data demonstrate the biological relevance of the MFM cell model and suggest that this model has potential use for the development of therapeutic approaches for EBS-MD. PMID:24487589

A S52P mutation in the 'α-crystallin domain' of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function.

PubMed

Nandi, Sandip K; Rehna, Elengikal A A; Panda, Alok K; Shiburaj, Sugathan; Dharmalingam, Kuppamuthu; Biswas, Ashis

2013-12-01

Mycobacterium leprae HSP18 is a small heat shock protein (sHSP). It is a major immunodominant antigen of M. leprae pathogen. Previously, we have reported the existence of two M. leprae HSP18 variants in various leprotic patients. One of the variants has serine at position 52, whereas the other one has proline at the same position. We have also reported that HSP18 having proline at position 52 (HSP18P(52)) is a nonameric protein and exhibits chaperone function. However, the structural and functional characterization of wild-type HSP18 having serine at position 52 (HSP18S(52)) is yet to be explored. Furthermore, the implications of the S52P mutation on the structure and chaperone function of HSP18 are not well understood. Therefore, we cloned and purified these two HSP18 variants. We found that HSP18S(52) is also a molecular chaperone and an oligomeric protein. Intrinsic tryptophan fluorescence and far-UV CD measurements revealed that the S52P mutation altered the tertiary and secondary structure of HSP18. This point mutation also reduced the oligomeric assembly and decreased the surface hydrophobicity of HSP18, as revealed by HPLC and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid binding studies, respectively. Mutant protein was less stable against thermal and chemical denaturation and was more susceptible towards tryptic cleavage than wild-type HSP18. HSP18P(52) had lower chaperone function and was less effective in protecting thermal killing of Escherichia coli than HSP18S(52). Taken together, our data suggest that serine 52 is important for the larger oligomerization and chaperone function of HSP18. Because both variants differ in stability and function, they may have different roles in the survival of M. leprae in infected hosts. © 2013 FEBS.

NPM1/B23: A Multifunctional Chaperone in Ribosome Biogenesis and Chromatin Remodeling

PubMed Central

Lindström, Mikael S.

2011-01-01

At a first glance, ribosome biogenesis and chromatin remodeling are quite different processes, but they share a common problem involving interactions between charged nucleic acids and small basic proteins that may result in unwanted intracellular aggregations. The multifunctional nuclear acidic chaperone NPM1 (B23/nucleophosmin) is active in several stages of ribosome biogenesis, chromatin remodeling, and mitosis as well as in DNA repair, replication and transcription. In addition, NPM1 plays an important role in the Myc-ARF-p53 pathway as well as in SUMO regulation. However, the relative importance of NPM1 in these processes remains unclear. Provided herein is an update on the expanding list of the diverse activities and interacting partners of NPM1. Mechanisms of NPM1 nuclear export functions of NPM1 in the nucleolus and at the mitotic spindle are discussed in relation to tumor development. It is argued that the suggested function of NPM1 as a histone chaperone could explain several, but not all, of the effects observed in cells following changes in NPM1 expression. A future challenge is to understand how NPM1 is activated, recruited, and controlled to carry out its functions. PMID:21152184

Kinetics and thermodynamics of the thermal inactivation and chaperone assisted folding of zebrafish dihydrofolate reductase.

PubMed

Thapliyal, Charu; Jain, Neha; Rashid, Naira; Chaudhuri Chattopadhyay, Pratima

2018-01-01

The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first-order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon. Copyright © 2017 Elsevier Inc. All rights reserved.

Chemical chaperone 4-phenylbutyric acid protects H9c2 cardiomyocytes from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis.

PubMed

Jian, Lian; Lu, Yuan; Lu, Shan; Lu, Chengzhi

2016-05-01

Myocardial ischemia/reperfusion (I/R) is a potential contributor to high rates of mortality in several cardiovascular diseases. I/R initiates the unfolded protein response and endoplasmic reticulum (ER) stress, which may lead to apoptotic pathways and exaggerate I/R injury. 4‑phenylbutyric acid (4‑PBA), a low molecular weight, terminal aromatic substituted fatty acid, has been reported to function as an ER chaperone. The aim of the present study was to investigate whether 4‑PBA is able to reduce ER stress‑induced apoptosis and prevent cardiomyocyte damage during the process of I/R in vitro. Accordingly, the rat cardiomyocyte line, H9c2, was treated with hypoxia/reoxygenation as an I/R model in vitro. Myocardium apoptosis was determined with TUNEL staining. The expression of ER stress‑related proteins were examined by western blotting. The resulting data showed that I/R activates the ER stress proteins, glucose‑regulated protein 78, activating transcription factor 6 and protein kinase RNA‑like endoplasmic reticulum kinase, which were all reduced by pretreatment with 4‑PBA. In addition, pretreatment with 4‑PBA significantly inhibited the expression levels of pro‑apoptotic proteins, C/EBP homologous protein, B cell lymphoma (Bcl‑2)‑associated X protein and phosphorylated c‑Jun N‑terminal kinase, and enhanced the expression of the anti‑apoptotic protein Bcl‑2 (n=3; P<0.05). The data demonstrated that I/R initiates ER stress‑associated apoptotic pathways, and 4‑PBA pretreatment protected the cardiomyocytes from I/R‑induced cell death. To the best of our knowledge, the present study is the first to report on the cell repair mechanism of 4‑PBA against I/R damage in cardiomyocytes based on ER stress‑associated apoptotic pathways.

Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones

PubMed Central

Shackleford, Gregory M; Ganguly, Amit; MacArthur, Craig A

2001-01-01

Background Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3. Results Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus. Conclusions Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus. PMID:11722795

Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

PubMed

Dabbaghizadeh, Afrooz; Finet, Stéphanie; Morrow, Genevieve; Moutaoufik, Mohamed Taha; Tanguay, Robert M

2017-07-01

The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

PubMed

Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

2009-10-01

Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

Thiol-based copper handling by the copper chaperone Atox1.

PubMed

Hatori, Yuta; Inouye, Sachiye; Akagi, Reiko

2017-04-01

Human antioxidant protein 1 (Atox1) plays a crucial role in cellular copper homeostasis. Atox1 captures cytosolic copper for subsequent transfer to copper pumps in trans Golgi network, thereby facilitating copper supply to various copper-dependent oxidereductases matured within the secretory vesicles. Atox1 and other copper chaperones handle cytosolic copper using Cys thiols which are ideal ligands for coordinating Cu(I). Recent studies demonstrated reversible oxidation of these Cys residues in copper chaperones, linking cellular redox state to copper homeostasis. Highlighted in this review are unique redox properties of Atox1 and other copper chaperones. Also, summarized are the redox nodes in the cytosol which potentially play dominant roles in the redox regulation of copper chaperones. © 2016 IUBMB Life, 69(4):246-254, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

TRP and rhodopsin transport depends on dual XPORT ER chaperones encoded by an operon

PubMed Central

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

2015-01-01

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

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

PubMed Central

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

2015-01-01

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

Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity.

PubMed

Jovcevski, Blagojce; Kelly, Megan A; Rote, Anthea P; Berg, Tracey; Gastall, Heidi Y; Benesch, Justin L P; Aquilina, J Andrew; Ecroyd, Heath

2015-02-19

Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function; however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27. Copyright © 2015 Elsevier Ltd. All rights reserved.

CUCURBITACIN D IS A DISRUPTOR OF THE HSP90 CHAPERONE MACHINERY

PubMed Central

Hall, Jessica A.; Seedarala, Sahithi; Rice, Nichole; Kopel, Lucas; Halaweish, Fathi; Blagg, Brian S. J.

2018-01-01

Heat shock protein 90 (Hsp90) facilitates the maturation of many newly synthesized and unfolded proteins (clients) via the Hsp90 chaperone cycle, in which Hsp90 forms a heteroprotein complex and relies upon co-chaperones, immunophilins, etc. for assistance in client folding. Hsp90 inhibition has emerged as a strategy for anticancer therapies due to the involvement of clients in many oncogenic pathways. Inhibition of chaperone function results in client ubiquitinylation and degradation via the proteasome, ultimately leading to tumor digression. Small molecule inhibitors perturb ATPase activity at the N-terminus and include derivatives of the natural product geldanamycin. However, N-terminal inhibition also leads to induction of the pro-survival heat shock response (HSR), in which displacement of the Hsp90-bound transcription factor, Heat Shock Factor-1 translocates to the nucleus and induces transcription of heat shock proteins, including Hsp90. An alternative strategy for Hsp90 inhibition is disruption of the Hsp90 heteroprotein complex. Disruption of the Hsp90 heteroprotein complex is an effective strategy to prevent client maturation without induction of the HSR. Cucurbitacin D, isolated from Cucurbita texana, and 3-epi-isocucurbitacin D prevented client maturation without induction of the HSR. Cucurbitacin D also disrupted interactions between Hsp90 and two co-chaperones, Cdc37 and p23. PMID:25756299

Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry

PubMed Central

Kriechbaumer, Verena; Tsargorodskaya, Anna; Mustafa, Mohd K.; Vinogradova, Tatiana; Lacey, Joanne; Smith, David P.; Abell, Benjamin M.; Nabok, Alexei

2011-01-01

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms. PMID:21767504

Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers.

PubMed

Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2014-01-01

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.

Thermosensitivity of growth is determined by chaperone-mediated proteome reallocation

PubMed Central

Chen, Ke; Gao, Ye; Mih, Nathan; O’Brien, Edward J.; Yang, Laurence; Palsson, Bernhard O.

2017-01-01

Maintenance of a properly folded proteome is critical for bacterial survival at notably different growth temperatures. Understanding the molecular basis of thermoadaptation has progressed in two main directions, the sequence and structural basis of protein thermostability and the mechanistic principles of protein quality control assisted by chaperones. Yet we do not fully understand how structural integrity of the entire proteome is maintained under stress and how it affects cellular fitness. To address this challenge, we reconstruct a genome-scale protein-folding network for Escherichia coli and formulate a computational model, FoldME, that provides statistical descriptions of multiscale cellular response consistent with many datasets. FoldME simulations show (i) that the chaperones act as a system when they respond to unfolding stress rather than achieving efficient folding of any single component of the proteome, (ii) how the proteome is globally balanced between chaperones for folding and the complex machinery synthesizing the proteins in response to perturbation, (iii) how this balancing determines growth rate dependence on temperature and is achieved through nonspecific regulation, and (iv) how thermal instability of the individual protein affects the overall functional state of the proteome. Overall, these results expand our view of cellular regulation, from targeted specific control mechanisms to global regulation through a web of nonspecific competing interactions that modulate the optimal reallocation of cellular resources. The methodology developed in this study enables genome-scale integration of environment-dependent protein properties and a proteome-wide study of cellular stress responses. PMID:29073085

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking

PubMed Central

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M.; Baehr, Wolfgang

2012-01-01

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d−/− mouse. PMID:22960045

Protein chaperones: a composition of matter review (2008 – 2013)

PubMed Central

Taldone, Tony; Patel, Hardik J; Bolaender, Alexander; Patel, Maulik R; Chiosis, Gabriela

2014-01-01

Introduction Heat shock proteins (Hsps) are proteins with important functions in regulating disease phenotypes. Historically, Hsp90 has first received recognition as a target in cancer, with consequent efforts extending its potential role to other diseases. Hsp70 has also attracted interest as a therapeutic target for its role as a co-chaperone to Hsp90 as well as its own anti-apoptotic roles. Areas covered Herein, patents from 2008 to 2013 are reviewed to identify those that disclose composition of matter claimed to inhibit Hsp90 or Hsp70. Expert opinion For Hsp90, there has been considerable creativity in the discovery of novel pharmacophores that fall outside the three initially discovered scaffolds (i.e., ansamycins, resorcinols and purines). Nonetheless, much of the patent literature appears to build on previously reported structure activity relationship through slight modifications of Hsp90 inhibitor space by finding weaknesses in existing patents. The major goal of future development of Hsp90 inhibitors is not necessarily identifying better molecules but rather understanding how to rationally use these agents in the clinic. The development of Hsp70 inhibitors has lagged behind. It will require a more concerted effort from the drug discovery community in order to begin to realize the potential of this target. PMID:24742089

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

PubMed

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

2018-01-01

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

Structural variants of yeast prions show conformer-specific requirements for chaperone activity

PubMed Central

Stein, Kevin C.; True, Heather L.

2016-01-01

Summary Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1, and its human ortholog Hdj1, had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone-client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation. PMID:25060529

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

2017-04-01

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.

Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates.

PubMed

Moparthi, Satish Babu; Fristedt, Rikard; Mishra, Rajesh; Almstedt, Karin; Karlsson, Martin; Hammarström, Per; Carlsson, Uno

2010-02-16

The single-domain cyclophilin 18 (Cyp18) has long been known to function as a peptidyl-prolyl cis/trans isomerase (PPI) and was proposed by us to also function as a chaperone [Freskgard, P.-O., Bergenhem, N., Jonsson, B.-H., Svensson, M., and Carlsson, U. (1992) Science 258, 466-468]. Later several multidomain PPIs were demonstrated to work as both a peptidyl-prolyl cis/trans isomerase and a chaperone. However, the chaperone ability of Cyp18 has been debated. In this work, we add additional results that show that Cyp18 can both accelerate the rate of refolding and increase the yield of native protein during the folding reaction, i.e., function as both a folding catalyst and a chaperone. Refolding experiments were performed using severely destabilized mutants of human carbonic anhydrase II under conditions where the unfolding reaction is significant and a larger fraction of a more destabilized variant populates molten globule-like intermediates during refolding. A correlation of native state protein stability of the substrate protein versus Cyp18 chaperone activity was demonstrated. The induced correction of misfolded conformations by Cyp18 likely functions through rescue from misfolding of transient molten globule intermediates. ANS binding data suggest that the interaction by Cyp18 leads to an early stage condensation of accessible hydrophobic portions of the misfolding-prone protein substrate during folding. The opposite effect was observed for GroEL known as an unfoldase at early stages of refolding. The chaperone effect of Cyp18 was also demonstrated for citrate synthase, suggesting a general chaperone effect of this PPI.

The coming of age of chaperone-mediated autophagy.

PubMed

Kaushik, Susmita; Cuervo, Ana Maria

2018-06-01

Chaperone-mediated autophagy (CMA) was the first studied process that indicated that degradation of intracellular components by the lysosome can be selective - a concept that is now well accepted for other forms of autophagy. Lysosomes can degrade cellular cytosol in a nonspecific manner but can also discriminate what to target for degradation with the involvement of a degradation tag, a chaperone and a sophisticated mechanism to make the selected proteins cross the lysosomal membrane through a dedicated translocation complex. Recent studies modulating CMA activity in vivo using transgenic mouse models have demonstrated that selectivity confers on CMA the ability to participate in the regulation of multiple cellular functions. Timely degradation of specific cellular proteins by CMA modulates, for example, glucose and lipid metabolism, DNA repair, cellular reprograming and the cellular response to stress. These findings expand the physiological relevance of CMA beyond its originally identified role in protein quality control and reveal that CMA failure with age may aggravate diseases, such as ageing-associated neurodegeneration and cancer.

Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1α via Chaperone-Mediated Autophagy.

PubMed

Matsui, Chieko; Deng, Lin; Minami, Nanae; Abe, Takayuki; Koike, Kazuhiko; Shoji, Ikuo

2018-07-01

Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1α caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1α in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1α matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1α was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1α disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1α is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1α with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1α protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1α protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1α. Immunofluorescence study revealed colocalization of NS5A and HNF-1α in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1α, thereby promoting the lysosomal degradation of HNF-1α via CMA. IMPORTANCE Many viruses use a protein degradation system, such as

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

PubMed

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

2000-12-01

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

A subclass of plant heat shock cognate 70 chaperones carries a motif that facilitates trafficking through plasmodesmata

PubMed Central

Aoki, Koh; Kragler, Friedrich; Xoconostle-Cázares, Beatriz; Lucas, William J.

2002-01-01

Plasmodesmata establish a pathway for the trafficking of non-cell-autonomously acting proteins and ribonucleoprotein complexes. Plasmodesmal enriched cell fractions and the contents of enucleate sieve elements, in the form of phloem sap, were used to isolate and characterize heat shock cognate 70 (Hsc70) chaperones associated with this cell-to-cell transport pathway. Three Cucurbita maxima Hsc70 chaperones were cloned and functional and sequence analysis led to the identification of a previously uncharacterized subclass of non-cell-autonomous chaperones. The highly conserved nature of the heat shock protein 70 (Hsp70) family, in conjunction with mutant analysis, permitted the characterization of a motif that allows these Hsc70 chaperones to engage the plasmodesmal non-cell-autonomous translocation machinery. Proof of concept that this motif is necessary for Hsp70 gain-of-movement function was obtained through the engineering of a human Hsp70 that acquired the capacity to traffic through plasmodesmata. These results are discussed in terms of the roles likely played by this subclass of Hsc70 chaperones in the trafficking of non-cell-autonomous proteins. PMID:12456884

Myeloid Leukemia Factor Acts in a Chaperone Complex to Regulate Transcription Factor Stability and Gene Expression.

PubMed

Dyer, Jamie O; Dutta, Arnob; Gogol, Madelaine; Weake, Vikki M; Dialynas, George; Wu, Xilan; Seidel, Christopher; Zhang, Ying; Florens, Laurence; Washburn, Michael P; Abmayr, Susan M; Workman, Jerry L

2017-06-30

Mutations that affect myelodysplasia/myeloid leukemia factor (MLF) proteins are associated with leukemia and several other cancers. However, with no strong homology to other proteins of known function, the role of MLF proteins in the cell has remained elusive. Here, we describe a proteomics approach that identifies MLF as a member of a nuclear chaperone complex containing a DnaJ protein, BCL2-associated anthanogene 2, and Hsc70. This complex associates with chromatin and regulates the expression of target genes. The MLF complex is bound to sites of nucleosome depletion and sites containing active chromatin marks (e.g., H3K4me3 and H3K4me1). Hence, MLF binding is enriched at promoters and enhancers. Additionally, the MLF-chaperone complex functions to regulate transcription factor stability, including the RUNX transcription factor involved in hematopoiesis. Although Hsc70 and other co-chaperones have been shown to play a role in nuclear translocation of a variety of proteins including transcription factors, our findings suggest that MLF and the associated co-chaperones play a direct role in modulating gene transcription. Copyright © 2016 Elsevier Ltd. All rights reserved.

Counteraction of urea-induced protein denaturation by trimethylamine N-oxide: a chemical chaperone at atomic resolution.

PubMed

Bennion, Brian J; Daggett, Valerie

2004-04-27

Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea and 4 M TMAO/8 M urea solutions, in addition to other control simulations, to investigate this effect at the atomic level. In 8 M urea, the protein unfolds, and urea acts in both a direct and indirect manner to achieve this effect. In contrast, introduction of 4 M TMAO counteracts the effect of urea and the protein remains well structured. TMAO makes few direct interactions with the protein. Instead, it prevents unfolding of the protein by structuring the solvent. In particular, TMAO orders the solvent and discourages it from competing with intraprotein H bonds and breaking up the hydrophobic core of the protein.

Study of receptor-chaperone interactions using the optical technique of spectroscopic ellipsometry.

PubMed

Kriechbaumer, Verena; Tsargorodskaya, Anna; Mustafa, Mohd K; Vinogradova, Tatiana; Lacey, Joanne; Smith, David P; Abell, Benjamin M; Nabok, Alexei

2011-07-20

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Inhibition of Mitochondrial Matrix Chaperones and Antiapoptotic Bcl-2 Family Proteins Empower Antitumor Therapeutic Responses.

PubMed

Karpel-Massler, Georg; Ishida, Chiaki Tsuge; Bianchetti, Elena; Shu, Chang; Perez-Lorenzo, Rolando; Horst, Basil; Banu, Matei; Roth, Kevin A; Bruce, Jeffrey N; Canoll, Peter; Altieri, Dario C; Siegelin, Markus D

2017-07-01

Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof of concept that targeting the mitochondrial Hsp90 chaperone network (mtHsp90) and inhibition of Bcl-2, Bcl-xL, and Mcl-1 is sufficient to elicit synthetic lethality in tumors recalcitrant to therapy. Our analyses focused on BH3 mimetics that are broad acting (ABT263 and obatoclax) or selective (ABT199, WEHI-539, and A1210477), along with the established mitochondrial matrix chaperone inhibitor gamitrinib-TPP. Drug combinations were tested in various therapy-resistant tumors in vitro and in vivo in murine model systems of melanoma, triple-negative breast cancer, and patient-derived orthotopic xenografts (PDX) of human glioblastoma. We found that combining BH3 mimetics and gamitrinib-TPP blunted cellular proliferation in a synergistic manner by massive activation of intrinsic apoptosis. In like manner, suppressing either Bcl-2, Bcl-xL, or Mcl-1 recapitulated the effects of BH3 mimetics and enhanced the effects of gamitrinib-TPP. Mechanistic investigations revealed that gamitrinib-TPP activated a PERK-dependent integrated stress response, which activated the proapoptotic BH3 protein Noxa and its downstream targets Usp9X and Mcl-1. Notably, in the PDX glioblastoma and BRAFi-resistant melanoma models, this drug combination safely and significantly extended host survival. Our results show how combining mitochondrial chaperone and Bcl-2 family inhibitors can synergize to safely degrade the growth of tumors recalcitrant to other treatments. Cancer Res; 77(13); 3513-26. ©2017 AACR . ©2017 American Association for Cancer Research.

The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors

PubMed Central

Demand, Jens; Lüders, Jens; Höhfeld, Jörg

1998-01-01

The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70’s cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell. PMID:9528774

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking.

PubMed

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d(-/-) mouse. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro reconstitution of chaperone-mediated human RISC assembly.

PubMed

Naruse, Ken; Matsuura-Suzuki, Eriko; Watanabe, Mariko; Iwasaki, Shintaro; Tomari, Yukihide

2018-01-01

To silence target mRNAs, small RNAs and Argonaute (Ago) proteins need to be assembled into RNA-induced silencing complexes (RISCs). Although the assembly of Drosophila melanogaster RISC was recently reconstituted by Ago2, the Dicer-2/R2D2 heterodimer, and five chaperone proteins, the absence of a reconstitution system for mammalian RISC assembly has posed analytical challenges. Here we describe reconstitution of human RISC assembly using Ago2 and five recombinant chaperone proteins: Hsp90β, Hsc70, Hop, Dnaja2, and p23. Our data show that ATP hydrolysis by both Hsp90β and Hsc70 is required for RISC assembly of small RNA duplexes but not for that of single-stranded RNAs. The reconstitution system lays the groundwork for further studies of small RNA-mediated gene silencing in mammals. © 2018 Naruse et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Probing Molecular Mechanisms of the Hsp90 Chaperone: Biophysical Modeling Identifies Key Regulators of Functional Dynamics

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2012-01-01

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

The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein.

PubMed

Gabus, C; Auxilien, S; Péchoux, C; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W; Nandi, P; Darlix, J L

2001-04-06

The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand transfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo. Copyright 2001 Academic Press.

GP96 is a GARP chaperone and controls regulatory T cell functions.

PubMed

Zhang, Yongliang; Wu, Bill X; Metelli, Alessandra; Thaxton, Jessica E; Hong, Feng; Rachidi, Saleh; Ansa-Addo, Ephraim; Sun, Shaoli; Vasu, Chenthamarakshan; Yang, Yi; Liu, Bei; Li, Zihai

2015-02-01

Molecular chaperones control a multitude of cellular functions via folding chaperone-specific client proteins. CD4+FOXP3+ Tregs play key roles in maintaining peripheral tolerance, which is subject to regulation by multiple molecular switches, including mTOR and hypoxia-inducible factor. It is not clear whether GP96 (also known as GRP94), which is a master TLR and integrin chaperone, controls Treg function. Using murine genetic models, we demonstrated that GP96 is required for Treg maintenance and function, as loss of GP96 resulted in instability of the Treg lineage and impairment of suppressive functions in vivo. In the absence of GP96, Tregs were unable to maintain FOXP3 expression levels, resulting in systemic accumulation of pathogenic IFN-γ-producing and IL-17-producing T cells. We determined that GP96 serves as an essential chaperone for the cell-surface protein glycoprotein A repetitions predominant (GARP), which is a docking receptor for latent membrane-associated TGF-β (mLTGF-β). The loss of both GARP and integrins on GP96-deficient Tregs prevented expression of mLTGF-β and resulted in inefficient production of active TGF-β. Our work demonstrates that GP96 regulates multiple facets of Treg biology, thereby placing Treg stability and immunosuppressive functions strategically under the control of a major stress chaperone.

Targeting Hsp90 and its co-chaperones to treat Alzheimer’s disease

PubMed Central

Blair, Laura J.; Sabbagh, Jonathan J.; Dickey, Chad A.

2015-01-01

Introduction Alzheimer’s disease (AD), characterized by the accumulation of hyperphosphorylated tau and beta amyloid (Aβ), currently lacks effective treatment. Chaperone proteins, such as the heat shock protein (Hsp) 90, form macromolecular complexes with co-chaperones, which can regulate tau metabolism and Aβ processing. While small molecule inhibitors of Hsp90 have been successful at ameliorating tau and Aβ burden, their development into drugs to treat disease has been slow due to the off- and on-target effects of this approach as well as challenges with the pharmacology of current scaffolds. Thus, other approaches are being developed to improve these compounds and to target co-chaperones of Hsp90 in an effort to limit these liabilities. Areas Covered This article discusses the most current developments in Hsp90 inhibitors including advances in blood-brain barrier permeability, decreased toxicity, and homolog-specific small molecule inhibitors. In addition, we discuss current strategies targeting Hsp90 co-chaperones rather than Hsp90 itself to reduce off-target effects. Expert Opinion While Hsp90 inhibitors have proven their efficacy at reducing tau pathology, they have yet to meet with success in the clinic. The development of Hsp90/tau complex specific inhibitors and further development of Hsp90 co-chaperone specific drugs should yield more potent, less toxic therapeutics. PMID:25069659

A passive physical model for DnaK chaperoning

NASA Astrophysics Data System (ADS)

Uhl, Lionel; Dumont, Audrey; Dukan, Sam

2018-03-01

Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction-diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in Escherichia coli cells during transient proteotoxic collapse characterized by the accumulation of insoluble proteins. In the model, misfolded (‘abnormal’) proteins are produced during alcoholic stress and have the propensity to aggregate with a polymerization-like kinetics. When aggregates diffuse more slowly they grow larger. According to Michaelis-Menten-type kinetics, DnaK has the propensity to bind with misfolded proteins or aggregates in order to catalyse refolding. To match experimental fluorescence microscopy data showing clusters of DnaK-GFP localized in multiple foci, the model includes spatial zones with local reduced diffusion rates to generate spontaneous assemblies of DnaK called ‘foci’. Numerical simulations of our model succeed in reproducing the kinetics of DnaK localization experimentally observed. DnaK starts from foci, moves to large aggregates during acute stress, resolves those aggregates during recovery and finally returns to its initial punctate localization pattern. Finally, we compare real biological events with hypothetical repartitions of the protein aggregates or DnaK. We then notice that DnaK action is more efficient on protein aggregates than on protein homogeneously distributed.

Chaperone substrate provides missing link for cancer drug discovery.

PubMed

Byrd, Katherine M; Blagg, Brian S J

2018-02-16

Both Hsp70 and Hsp90 chaperones are overexpressed in cancer, making them relevant targets for the development of cancer chemotherapeutics, but a lack of biomolecular readouts for Hsp70 inhibition has limited the pursuit of specific inhibitors for this enzyme. A new study from Cesa et al. identifies two inhibitors of apoptosis proteins (IAPs) as specific client substrates of Hsp70. These results establish biomarkers that can be utilized to monitor Hsp70 inhibition and provide a framework for future efforts to deconvolute chaperone networks. © 2018 Byrd and Blagg.

Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

PubMed Central

Thomson, Nicholas M.; Saika, Azusa; Ushimaru, Kazunori; Sangiambut, Smith; Tsuge, Takeharu; Summers, David K.

2013-01-01

The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter−1), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter−1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer. PMID:23335776

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

PubMed

Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup

2015-11-13

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

Metal chaperones prevent zinc-mediated cognitive decline.

PubMed

Adlard, Paul A; Parncutt, Jacqui; Lal, Varsha; James, Simon; Hare, Dominic; Doble, Philip; Finkelstein, David I; Bush, Ashley I

2015-09-01

Zinc transporter-3 (ZnT3) protein is responsible for loading zinc into presynaptic vesicles and consequently controls the availability of zinc at the glutamatergic synapse. ZnT3 has been shown to decline with age and in Alzheimer's disease (AD) and is crucially involved in learning and memory. In this study, we utilised whole animal behavioural analyses in the ZnT3 KO mouse line, together with electrophysiological analysis of long-term potentiation in brain slices from ZnT3 KO mice, to show that metal chaperones (clioquinol, 30 mg/kg/day for 6weeks) can prevent the age-dependent cognitive phenotype that characterises these animals. This likely occurs as a result of a homeostatic restoration of synaptic protein expression, as clioquinol significantly restored levels of various pre- and postsynaptic proteins that are critical for normal cognition, including PSD-95; AMPAR and NMDAR2b. We hypothesised that this clioquinol-mediated restoration of synaptic health resulted from a selective increase in synaptic zinc content within the hippocampus. While we demonstrated a small regional increase in hippocampal zinc content using synchrotron x-ray fluorescence microscopy, further sub-region analyses are required to determine whether this effect is seen in other regions of the hippocampal formation that are more closely linked to the synaptic plasticity effects observed in this study. These data support our recent report on the use of a different metal chaperone (PBT2) to prevent normal age-related cognitive decline and demonstrate that metal chaperones are efficacious in preventing the zinc-mediated cognitive decline that characterises ageing and disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD.

PubMed

Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E; Birket, Susan; Field, Terry; Picking, William D; Blocker, Ariel J; Galyov, Edouard E; Picking, Wendy L; Lea, Susan M

2007-02-09

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.

Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape.

PubMed

Ferreon, Allan Chris M; Moosa, Mahdi Muhammad; Gambin, Yann; Deniz, Ashok A

2012-10-30

Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson's disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 21 [urea][TMAO] ratio has a net neutral effect on the protein's dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments.

The mechanism of Cu+ transport ATPases: interaction with CU+ chaperones and the role of transient metal-binding sites.

PubMed

Padilla-Benavides, Teresita; McCann, Courtney J; Argüello, José M

2013-01-04

Cu(+)-ATPases are membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic Cu(+). In cells, soluble chaperone proteins bind and distribute cytoplasmic Cu(+), delivering the ion to the transmembrane metal-binding sites in the ATPase. The structure of Legionella pneumophila Cu(+)-ATPase (Gourdon, P., Liu, X. Y., Skjørringe, T., Morth, J. P., Møller, L. B., Pedersen, B. P., and Nissen, P. (2011) Nature 475, 59-64) shows that a kinked transmembrane segment forms a "platform" exposed to the cytoplasm. In addition, neighboring invariant Met, Asp, and Glu are located at the "entrance" of the ion path. Mutations of amino acids in these regions of the Archaeoglobus fulgidus Cu(+)-ATPase CopA do not affect ATPase activity in the presence of Cu(+) free in solution. However, Cu(+) bound to the corresponding chaperone (CopZ) could not activate the mutated ATPases, and in parallel experiments, CopZ was unable to transfer Cu(+) to CopA. Furthermore, mutation of a specific electronegative patch on the CopZ surface abolishes the ATPase activation and Cu(+) transference, indicating that the region is required for the CopZ-CopA interaction. Moreover, the data suggest that the interaction is driven by the complementation of the electropositive platform in the ATPase and the electronegative Cu(+) chaperone. This docking likely places the Cu(+) proximal to the conserved carboxyl and thiol groups in the entrance site that induce metal release from the chaperone via ligand exchange. The initial interaction of Cu(+) with the pump is transient because Cu(+) is transferred from the entrance site to transmembrane metal-binding sites involved in transmembrane translocation.

A thermodynamic assay to test pharmacological chaperones for Fabry disease.

PubMed

Andreotti, Giuseppina; Citro, Valentina; Correra, Antonella; Cubellis, Maria Vittoria

2014-03-01

The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs. The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells. Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape

PubMed Central

Ferreon, Allan Chris M.; Moosa, Mahdi Muhammad; Deniz, Ashok A.

2012-01-01

Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson’s disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 2∶1 [urea]∶[TMAO] ratio has a net neutral effect on the protein’s dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments. PMID:22826265

Role and mechanism of the Hsp70 molecular chaperone machines in bacterial pathogens.

PubMed

Ghazaei, Ciamak

2017-03-01

Heat shock proteins are highly conserved, stress-inducible, ubiquitous proteins that maintain homeostasis in both eukaryotes and prokaryotes. Hsp70 proteins belong to the heat shock protein family and enhance bacterial survival in hostile environments. Hsp70, known as DnaK in prokaryotes, supports numerous processes such as the assembly and disassembly of protein complexes, the refolding of misfolded and clustered proteins, membrane translocation and the regulation of regulatory proteins. The chaperone-based activity of Hsp70 depends on dynamic interactions between its two domains, known as the ATPase domain and the substrate-binding domain. It also depends on interactions between these domains and other co-chaperone molecules such as the Hsp40 protein family member DnaJ and nucleotide exchange factors. DnaJ is the primary chaperone that interacts with nascent polypeptide chains and functions to prevent their premature release from the ribosome and misfolding before it is targeted by DnaK. Adhesion of bacteria to host cells is mediated by both host and bacterial Hsp70. Following infection of the host, bacterial Hsp70 (DnaK) is in a position to initiate bacterial survival processes and trigger an immune response by the host. Any mutations in the dnaK gene have been shown to decrease the viability of bacteria inside the host. This review will give insights into the structure and mechanism of Hsp70 and its role in regulating the protein activity that contributes to pathogenesis.

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress

PubMed Central

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

2017-01-01

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity. PMID:28724793

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

PubMed

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

2017-07-20

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

Self-Chaperoning of the Type III Secretion System needle tip proteins IpaD and BipD

PubMed Central

Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E.; Birket, Susan; Field, Terry; Picking, William D.; Blocker, Ariel; Galyov, Edouard E.; Picking, Wendy L.; Lea, Susan M.

2007-01-01

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence; IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intra-molecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure. PMID:17077085

Interaction of HIV-1 Gag protein components with single DNA molecules

NASA Astrophysics Data System (ADS)

Cruceanu, Margareta; Gorelick, Robert J.; Williams, Mark C.

2003-03-01

The Gag protein of the HIV-1 retrovirus is cleaved into three major proteins as part of viral maturation: nucleocapsid (NC), capsid, and matrix. NC is the first of these proteins to be cleaved, and it is cleaved in three stages into NCp15, followed by NCp9, and finally NCp7. In this study, we use optical tweezers to investigate the capability of these NC proteins to alter the helix-coil transition of single DNA molecules. We have previously shown that the capability to alter the DNA helix-coil transition is an excellent probe of the nucleic acid chaperone activity of NC proteins, in which the secondary structure of nucleic acids is rearranged to facilitate reverse transcription. By examining the capability of NCp15, NCp9, and NCp7 to alter DNA stretching, the current studies will test the role of proteolytic cleavage of Gag in regulating the nucleic acid chaperone activity of NC. Whereas binding studies suggest that NCp9 and NCp15 bind more strongly to DNA than NCp7, our DNA stretching results indicate that these proteins all have similar effects on DNA stretching.

The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans

PubMed Central

Morton, Elizabeth A.; Cornwell, Adam B.; Crick, Beresford; Lamitina, Todd; Douglas, Peter M.

2017-01-01

An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show

Saturated fatty acid palmitate negatively regulates autophagy by promoting ATG5 protein degradation in meniscus cells.

PubMed

Mallik, Aritra; Yammani, Raghunatha R

2018-07-20

Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Temperature-dependent subunit exchange and chaperone-like activities of Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis.

PubMed

Fu, Xinmiao; Chang, Zengyi

2004-04-02

Small heat shock proteins (sHsps) usually exist as oligomers that undergo dynamic oligomeric dissociation/re-association, with the dissociated oligomers as active forms to bind substrate proteins under heat shock conditions. In this study, however, we found that Hsp16.3, one sHsp from Mycobacterium tuberculosis, is able to sensitively modulate its chaperone-like activity in a range of physiological temperatures (from 25 to 37.5 degrees C) while its native oligomeric size is still maintained. Further analysis demonstrated that Hsp16.3 exposes higher hydrophobic surfaces upon temperatures increasing and that a large soluble complex between Hsp16.3 and substrate is formed only in the condition of heating temperature up to 35 and 37.5 degrees C. Structural analysis by fluorescence anisotropy showed that Hsp16.3 nonameric structure becomes more dynamic and variable at elevated temperatures. Moreover, subunit exchange between Hsp16.3 oligomers was found to occur faster upon temperatures increasing as revealed by fluorescence energy resonance transfer. These observations indicate that Hsp16.3 is able to modulate its chaperone activity by adjusting the dynamics of oligomeric dissociation/re-association process while maintaining its static oligomeric size unchangeable. A kinetic model is therefore proposed to explain the mechanism of sHsps-binding substrate proteins through oligomeric dissociation. The present study also implied that Hsp16.3 is at least capable of binding non-native proteins in vivo while expressing in the host organism that survives at 37 degrees C.

Influence of Xylella fastidiosa cold shock proteins on pathogenesis in grapevine.

USDA-ARS?s Scientific Manuscript database

Cold shock proteins (CSPs), a family of nucleic acid binding proteins are an essential part of microbial adaptation to temperature changes. Bacterial CSPs are often expressed in a temperature-dependent manner, and act as chaperones, facilitating translation at low temperature by stabilizing mRNA. In...

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

Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

PubMed

Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

2010-07-30

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

Isoprenylation of the plant molecular chaperone ANJ1 facilitates membrane association and function at high temperature.

PubMed

Zhu, J K; Bressan, R A; Hasegawa, P M

1993-09-15

We demonstrate that ANJ1, a higher plant homolog of the bacterial molecular chaperone DnaJ, is a substrate in vitro for protein farnesyl- and geranylgeranyl-transferase activities present in cell extracts of the plant Atriplex nummularia and yeast Saccharomyces cerevisiae. Isoprenylation did not occur when cysteine was replaced by serine in the CAQQ motif at the carboxyl terminus of ANJ1, indicating that this sequence functions as a CaaX consensus sequence for polyisoprenylation (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue). Substitution of leucine for the terminal glutamine did not result in the expected geranylgeranylation as occurs with mammalian proteins containing a carboxyl-terminal leucine. Unlike the wild-type ANJ1, neither of the proteins containing these amino acid substitutions could functionally complement the yeast temperature-sensitive mutant mas5. Farnesylation enhanced the association of ANJ1 with A. nummularia microsomal membranes. Electrophoretic mobility of ANJ1 from the plant indicated that the protein is isoprenylated in vivo.

Isoprenylation of the plant molecular chaperone ANJ1 facilitates membrane association and function at high temperature.

PubMed Central

Zhu, J K; Bressan, R A; Hasegawa, P M

1993-01-01

We demonstrate that ANJ1, a higher plant homolog of the bacterial molecular chaperone DnaJ, is a substrate in vitro for protein farnesyl- and geranylgeranyl-transferase activities present in cell extracts of the plant Atriplex nummularia and yeast Saccharomyces cerevisiae. Isoprenylation did not occur when cysteine was replaced by serine in the CAQQ motif at the carboxyl terminus of ANJ1, indicating that this sequence functions as a CaaX consensus sequence for polyisoprenylation (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue). Substitution of leucine for the terminal glutamine did not result in the expected geranylgeranylation as occurs with mammalian proteins containing a carboxyl-terminal leucine. Unlike the wild-type ANJ1, neither of the proteins containing these amino acid substitutions could functionally complement the yeast temperature-sensitive mutant mas5. Farnesylation enhanced the association of ANJ1 with A. nummularia microsomal membranes. Electrophoretic mobility of ANJ1 from the plant indicated that the protein is isoprenylated in vivo. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:8378331

Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress.

PubMed

Voloshin, Olga; Gocheva, Yana; Gutnick, Marina; Movshovich, Natalia; Bakhrat, Anya; Baranes-Bachar, Keren; Bar-Zvi, Dudy; Parvari, Ruti; Gheber, Larisa; Raveh, Dina

2010-06-01

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds alpha-tubulin and promotes alpha/beta dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds alpha-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Delta mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Delta mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.

Chaperone-mediated autophagy degrades mutant p53

PubMed Central

Vakifahmetoglu-Norberg, Helin; Kim, Minsu; Xia, Hong-guang; Iwanicki, Marcin P.; Ofengeim, Dimitry; Coloff, Jonathan L.; Pan, Lifeng; Ince, Tan A.; Kroemer, Guido; Brugge, Joan S.; Yuan, Junying

2013-01-01

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells. PMID:23913924

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. Copyright © 2011 Elsevier Inc. All rights reserved.

Dynamics of Linker Residues Modulate the Nucleic Acid Binding Properties of the HIV-1 Nucleocapsid Protein Zinc Fingers

PubMed Central

Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2014-01-01

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity. PMID:25029439

Relationship between the structure of SET/TAF-Ibeta/INHAT and its histone chaperone activity.

PubMed

Muto, Shinsuke; Senda, Miki; Akai, Yusuke; Sato, Lui; Suzuki, Toru; Nagai, Ryozo; Senda, Toshiya; Horikoshi, Masami

2007-03-13

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Ibeta/INHAT at a resolution of 2.3 A. We found that SET/TAF-Ibeta/INHAT formed a dimer that assumed a "headphone"-like structure. Each subunit of the SET/TAF-Ibeta/INHAT dimer consisted of an N terminus, a backbone helix, and an "earmuff" domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Ibeta/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by approximately 40 degrees . Our biochemical analyses of mutants revealed that the region of SET/TAF-Ibeta/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Ibeta/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.

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

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 ERmore » 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.« less

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.

Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warren, Christopher; Matsui, Tsutomu; Karp, Jerome M.

Here, nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowedmore » us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.« less

Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release

DOE PAGES

Warren, Christopher; Matsui, Tsutomu; Karp, Jerome M.; ...

2017-12-20

Here, nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowedmore » us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.« less

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

Quantitative proteomics and network analysis of SSA1 and SSB1 deletion mutants reveals robustness of chaperone HSP70 network in Saccharomyces cerevisiae

PubMed Central

Jarnuczak, Andrew F.; Eyers, Claire E.; Schwartz, Jean‐Marc; Grant, Christopher M.

2015-01-01

Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC‐based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs. PMID:25689132

Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation.

PubMed

Geller, Ron; Pechmann, Sebastian; Acevedo, Ashley; Andino, Raul; Frydman, Judith

2018-05-03

Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.

Multi-chaperone function modulation and association with cytoskeletal proteins are key features of the function of AIP in the pituitary gland

PubMed Central

Hernández-Ramírez, Laura C.; Morgan, Rhodri M.L.; Barry, Sayka; D’Acquisto, Fulvio; Prodromou, Chrisostomos; Korbonits, Márta

2018-01-01

Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-specific function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant findings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was confirmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/stability of multiple chaperone client proteins could be perturbed by a deficient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identified. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our findings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP. PMID:29507682

Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis

PubMed Central

Yoshida, Soichiro; Tsutsumi, Shinji; Muhlebach, Guillaume; Sourbier, Carole; Lee, Min-Jung; Lee, Sunmin; Vartholomaiou, Evangelia; Tatokoro, Manabu; Beebe, Kristin; Miyajima, Naoto; Mohney, Robert P.; Chen, Yang; Hasumi, Hisashi; Xu, Wanping; Fukushima, Hiroshi; Nakamura, Ken; Koga, Fumitaka; Kihara, Kazunori; Trepel, Jane; Picard, Didier; Neckers, Leonard

2013-01-01

TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor. PMID:23564345

Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

PubMed Central

Veisi, Kamal; Farajnia, Safar; Zarghami, Nosratollah; Khoram Khorshid, Hamid Reza; Samadi, Nasser; Ahdi Khosroshahi, Shiva; Zarei Jaliani, Hossein

2015-01-01

Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies. PMID:26793607

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

PubMed Central

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

2000-01-01

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

Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

PubMed Central

Bender, Tom; Lewrenz, Ilka; Franken, Sebastian; Baitzel, Catherina; Voos, Wolfgang

2011-01-01

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria. PMID:21209324

Asp 58 modulates lens αA-crystallin oligomer formation and chaperone function.

PubMed

Takata, Takumi; Nakamura-Hirota, Tooru; Inoue, Rintaro; Morishima, Ken; Sato, Nobuhiro; Sugiyama, Masaaki; Fujii, Noriko

2018-06-01

Senile cataract onset is caused by insolubilization of lens proteins. The lens crystallin protein family correctly orders the formation of homo- or hetero-oligomers in lens fiber cells. Because lens fiber cells do not divide, covalent post-translational modifications, such as isomerization of aspartate residues, accumulate with aging. Although many isomerization sites of αA-crystallin have been reported, their structural and functional contributions have never been identified. In this study, αA-crystallin was extracted from aged human lens and separated into each oligomeric state by size exclusion chromatography and electrophoresis. The novel combination methodology of in-solution/gel tryptic digestion with liquid chromatography equipped with mass spectrometry (LC-MS/MS) was used to evaluate the isomerization of Asp 58. The contributions of isomerization to assembly, solubility, and chaperone functions of αA-crystallin were estimated using a series of mutations of Asp 58 in αA-crystallin. The results indicated that the isomerization of Asp 58 depended on the oligomer size and age of the lens. The substitution of Asp 58 for hydrophobic residues increased αA-crystallin oligomer size and decreased solubility. All substitutions decreased the chaperone function of αA-crystallin for aggregates of bovine βL-crystallin and alcohol dehydrogenase. The data indicated that Asp 58 in αA-crystallin was critical for intermolecular interactions in the lens. Our results also suggested that LC-MS/MS-based isomerization analyses of in-gel-digested products could be useful for investigating the isomerization of Asp residues in oligomeric states. This method could also be used to analyze d/l ratios of amino acid residues in soluble protein aggregates. © 2018 Federation of European Biochemical Societies.

Quantitative proteomics and network analysis of SSA1 and SSB1 deletion mutants reveals robustness of chaperone HSP70 network in Saccharomyces cerevisiae.

PubMed

Jarnuczak, Andrew F; Eyers, Claire E; Schwartz, Jean-Marc; Grant, Christopher M; Hubbard, Simon J

2015-09-01

Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC-based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs. © 2015 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pea Chaperones under Centrifugation

NASA Astrophysics Data System (ADS)

Talalaiev, Oleksandr

2008-06-01

Etiolated Pisum sativum seedlings were subjected to altered g-forces by centrifugation (3-14g). By using semiquantitative RT-PCR, we studied transcripts of pea genes coding for chaperones that are representatives of small heat shock proteins (sHsps) family. Four members from the different classes of sHsps: cytosolic Hsp17.7 and Hsp18.1 (class I and class II accordingly), chloroplast Hsp21 (class III) and endoplasmic reticulum Hsp22.7 (class IV) were investigated. We conclude that exposure to 3, 7, 10 and 14g for 1h did not affect the level of sHsp transcripts.

The histone shuffle: histone chaperones in an energetic dance

PubMed Central

Das, Chandrima; Tyler, Jessica K.; Churchill, Mair E.A.

2014-01-01

Our genetic information is tightly packaged into a rather ingenious nucleoprotein complex called chromatin in a manner that enables it to be rapidly accessed during genomic processes. Formation of the nucleosome, which is the fundamental unit of chromatin, occurs via a stepwise process that is reversed to enable the disassembly of nucleosomes. Histone chaperone proteins have prominent roles in facilitating these processes as well as in replacing old histones with new canonical histones or histone variants during the process of histone exchange. Recent structural, biophysical and biochemical studies have begun to shed light on the molecular mechanisms whereby histone chaperones promote chromatin assembly, disassembly and histone exchange to facilitate DNA replication, repair and transcription. PMID:20444609

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…

Structure and function of Hip, an attenuator of the Hsp70 chaperone cycle.

PubMed

Li, Zhuo; Hartl, F Ulrich; Bracher, Andreas

2013-08-01

The Hsp70-interacting protein, Hip, cooperates with the chaperone Hsp70 in protein folding and prevention of aggregation. Hsp70 interacts with non-native protein substrates in an ATP-dependent reaction cycle regulated by J-domain proteins and nucleotide exchange factors (NEFs). Hip is thought to delay substrate release by slowing ADP dissociation from Hsp70. Here we present crystal structures of the dimerization domain and the tetratricopeptide repeat (TPR) domain of rat Hip. As shown in a cocrystal structure, the TPR core of Hip interacts with the Hsp70 ATPase domain through an extensive interface, to form a bracket that locks ADP in the binding cleft. Hip and NEF binding to Hsp70 are mutually exclusive, and thus Hip attenuates active cycling of Hsp70-substrate complexes. This mechanism explains how Hip enhances aggregation prevention by Hsp70 and facilitates transfer of specific proteins to downstream chaperones or the proteasome.

Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import

PubMed Central

Banci, Lucia; Bertini, Ivano; Cefaro, Chiara; Cenacchi, Lucia; Ciofi-Baffoni, Simone; Felli, Isabella Caterina; Gallo, Angelo; Gonnelli, Leonardo; Luchinat, Enrico; Sideris, Dionisia; Tokatlidis, Kostas

2010-01-01

Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation. PMID:21059946

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bakkouri, Majida El; Pow, Andre; Mulichak, Anne

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, weremore » 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.« less

Regulation of the copper chaperone CCS by XIAP-mediated ubiquitination.

PubMed

Brady, Graham F; Galbán, Stefanie; Liu, Xuwen; Basrur, Venkatesha; Gitlin, Jonathan D; Elenitoba-Johnson, Kojo S J; Wilson, Thomas E; Duckett, Colin S

2010-04-01

In order to balance the cellular requirements for copper with its toxic properties, an elegant set of mechanisms has evolved to regulate and buffer intracellular copper. The X-linked inhibitor of apoptosis (XIAP) protein was recently identified as a copper-binding protein and regulator of copper homeostasis, although the mechanism by which XIAP binds copper in the cytosol is unclear. Here we describe the identification of the copper chaperone for superoxide dismutase (CCS) as a mediator of copper delivery to XIAP in cells. We also find that CCS is a target of the E3 ubiquitin ligase activity of XIAP, although interestingly, ubiquitination of CCS by XIAP was found to lead to enhancement of its chaperone activity toward its physiologic target, superoxide dismutase 1, rather than proteasomal degradation. Collectively, our results reveal novel links among apoptosis, copper metabolism, and redox regulation through the XIAP-CCS complex.

Can foreign proteins imported into yeast mitochondria interfere with PIM1p protease and/or chaperone function?

PubMed

Saveliev, A S; Kovaleva, I E; Novikova, L A; Isaeva, L V; Luzikov, V N

1999-03-15

When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pim1p protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pim1p, respectively. These data imply that chaperones and Pim1p protease prefer their natural targets in mitochondria to imported foreign proteins. Copyright 1999 Academic Press.

A small molecule chemical chaperone optimizes its unfolded state contraction and denaturant like properties

NASA Astrophysics Data System (ADS)

Sharma, Sunny; Sarkar, Suparna; Paul, Simanta Sarani; Roy, Syamal; Chattopadhyay, Krishnananda

2013-12-01

Protein aggregation is believed to occur through the formation of misfolded conformations. It is expected that, in order to minimize aggregation, an effective small molecule chaperone would destabilize these intermediates. To study the mechanism of a chemical chaperone, we have designed a series of mutant proteins in which a tryptophan residue experiences different local environments and solvent exposures. We show that these mutants correspond to a series of conformationally altered proteins with varying degree of misfolding stress and aggregation propensities. Using arginine as a model small molecule, we show that a combination of unfolded state contraction and denaturant like properties results in selective targeting and destabilization of the partially folded proteins. In comparison, the effect of arginine towards the folded like control mutant, which is not aggregation prone, is significantly less. Other small molecules, lacking either of the above two properties, do not offer any specificity towards the misfolded proteins.

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, whichmore » 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.« less

A PQM-1-Mediated Response Triggers Transcellular Chaperone Signaling and Regulates Organismal Proteostasis.

PubMed

O'Brien, Daniel; Jones, Laura M; Good, Sarah; Miles, Jo; Vijayabaskar, M S; Aston, Rebecca; Smith, Catrin E; Westhead, David R; van Oosten-Hawle, Patricija

2018-06-26

In metazoans, tissues experiencing proteotoxic stress induce "transcellular chaperone signaling" (TCS) that activates molecular chaperones, such as hsp-90, in distal tissues. How this form of inter-tissue communication is mediated to upregulate systemic chaperone expression and whether it can be utilized to protect against protein misfolding diseases remain open questions. Using C. elegans, we identified key components of a systemic stress signaling pathway that links the innate immune response with proteostasis maintenance. We show that mild perturbation of proteostasis in the neurons or the intestine activates TCS via the GATA zinc-finger transcription factor PQM-1. PQM-1 coordinates neuron-activated TCS via the innate immunity-associated transmembrane protein CLEC-41, whereas intestine-activated TCS depends on the aspartic protease ASP-12. Both TCS pathways can induce hsp-90 in muscle cells and facilitate amelioration of Aβ 3-42 -associated toxicity. This may have powerful implications for the treatment of diseases related to proteostasis dysfunction. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

Thermally induced disintegration of the oligomeric structure of alphaB-crystallin mutant F28S is associated with diminished chaperone activity.

PubMed

Kelley, Patrick B; Abraham, Edathara C

2003-10-01

alphaB-crystallin, a member of the small heat-shock protein (hsp) family of proteins, is able to function as a molecular chaperone by protecting other proteins from stress-induced aggregation by recognizing and binding to partially unfolded species of damaged proteins. The present work has investigated the role of phenylalanine-28 (F28) of the 22RLFDQFF28 region of alphaB-crystallin in maintaining chaperone function and oligomeric structure under physiological condition and under thermal stress. Bovine alphaB-crystallin was cloned for the first time and the cDNA sequence revealed greater than 90% homology to that of human, rat and mouse alphaB-crystallins. F28 was mutated to a serine followed by expression of the mutant F28S and the wild-type alphaB (alphaB-wt) in E. coli and subsequent purification of the protein by size-exclusion chromatography. Secondary and tertiary structure analyses showed some structural changes in the mutant. Chaperone activity and oligomeric size of the mutant was unchanged at 37 degrees C whereas at 58 degrees C the chaperone activity was significantly decreased and the oligomeric size ranged from low molecular weight to high molecular weight showing disintegration of the oligomeric structure. The data support the idea that the participation of large oligomeric structure rather than smaller units is required to have optimal chaperone activity and the hydrophobic F28 residue is needed for maintaining the native oligomeric structure under thermal stress.

The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sarowar, Samema; Hu, Olivia J.; Werneburg, Glenn T.

ABSTRACT The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains ofEscherichia coliuse 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 themore » 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. IMPORTANCEPili are critical virulence factors for many bacterial pathogens. UropathogenicE. colirelies 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

A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor σ32 of Escherichia coli

PubMed Central

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 σ32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ32 facilitates RB43 bacteriophage proliferation is discussed. PMID:23133404

Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

NASA Astrophysics Data System (ADS)

Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

2007-12-01

A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

Structure and function of archaeal prefoldin, a co-chaperone of group II chaperonin.

PubMed

Ohtaki, Akashi; Noguchi, Keiichi; Yohda, Masafumi

2010-01-01

Molecular chaperones are key cellular components involved in the maintenance of protein homeostasis and other unrelated functions. Prefoldin is a chaperone that acts as a co-factor of group II chaperonins in eukaryotes and archaea. It assists proper folding of protein by capturing nonnative proteins and delivering it to the group II chaperonin. Eukaryotic prefoldin is a multiple subunit complex composed of six different polypeptide chains. Archaeal prefoldin, on the other hand, is a heterohexameric complex composed of two alpha and four beta subunits, and forms a double beta barrel assembly with six long coiled coils protruding from it like a jellyfish with six tentacles. Based on the structural information of the archaeal prefoldin, substrate recognition and prefoldin-chaperonin binding mechanisms have been investigated. In this paper, we review a series of studies on the molecular mechanisms of archaeal PFD function. Particular emphasis will be placed on the molecular structures revealed by X-ray crystallography and molecular dynamics induced by binding to nonnative protein substrates.

Malfunctioning of Chaperone-Mediated Autophagy in Parkinson's Disease: Feats, Constraints, and Flaws of Modulators.

PubMed

Tripathi, Manish Kumar; Rajput, Charul; Mishra, Saumya; Rasheed, Mohd Sami Ur; Singh, Mahendra Pratap

2018-06-11

Homeostatic regulation of class II programmed cell death/autophagy for the degradation and elimination of substandard organelles and defective proteins is decisive for the survival of dopaminergic neurons. Chaperone-mediated autophagy (CMA), one of the most highly dedicated self-sacrificing events, is accountable for the partial elimination of redundant soluble cytoplasmic proteins in Parkinson's disease (PD). CMA is characterized by the selective delivery of superfluous protein containing lysine-phenylalanine-glutamate-arginine-glutamine (KFERQ)/KFERQ-like motif to the lysosome through molecular chaperones, such as heat shock cognate-70 (Hsc-70). KFERQ/KFERQ-like motif present in the poor quality cytoplasmic substrate protein and Hsc-70 complex is recognized by a janitor protein, which is referred to as the lysosome-associated membrane protein-2A (LAMP-2A). This protein is known to facilitate an entry of substrate-chaperone complex in the lumen for hydrolytic cleavage of substrate and elimination of end-products. Impaired CMA is repeatedly blamed for an accumulation of surplus soluble proteins. However, it is still an enigma if CMA is a bonus or curse for PD. Case-control studies and cellular and animal models have deciphered the contribution of impaired CMA in PD. Current article updates the role of CMA in toxicant models and recapitulates the evidences that have highlighted a link between impaired CMA and PD. Although PD is an irreversible happening and CMA is a dual edging phenomenon, it is anticipated that fine-tuning of the latter encounters the former to a certain extent. Besides, the truth, embellishment, and propaganda regarding the issue are also emphasized in the final segment of the article.

System level mechanisms of adaptation, learning, memory formation and evolvability: the role of chaperone and other networks.

PubMed

Gyurko, David M; Soti, Csaba; Stetak, Attila; Csermely, Peter

2014-05-01

During the last decade, network approaches became a powerful tool to describe protein structure and dynamics. Here, we describe first the protein structure networks of molecular chaperones, then characterize chaperone containing sub-networks of interactomes called as chaperone-networks or chaperomes. We review the role of molecular chaperones in short-term adaptation of cellular networks in response to stress, and in long-term adaptation discussing their putative functions in the regulation of evolvability. We provide a general overview of possible network mechanisms of adaptation, learning and memory formation. We propose that changes of network rigidity play a key role in learning and memory formation processes. Flexible network topology provides ' learning-competent' state. Here, networks may have much less modular boundaries than locally rigid, highly modular networks, where the learnt information has already been consolidated in a memory formation process. Since modular boundaries are efficient filters of information, in the 'learning-competent' state information filtering may be much smaller, than after memory formation. This mechanism restricts high information transfer to the 'learning competent' state. After memory formation, modular boundary-induced segregation and information filtering protect the stored information. The flexible networks of young organisms are generally in a 'learning competent' state. On the contrary, locally rigid networks of old organisms have lost their 'learning competent' state, but store and protect their learnt information efficiently. We anticipate that the above mechanism may operate at the level of both protein-protein interaction and neuronal networks.

Serine/Threonine Kinase Unc-51-like Kinase-1 (Ulk1) Phosphorylates the Co-chaperone Cell Division Cycle Protein 37 (Cdc37) and Thereby Disrupts the Stability of Cdc37 Client Proteins.

PubMed

Li, Ran; Yuan, Fengjie; Fu, Wan; Zhang, Luyao; Zhang, Nan; Wang, Yanan; Ma, Ke; Li, Xue; Wang, Lina; Zhu, Wei-Guo; Zhao, Ying

2017-02-17

The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (Cdc37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in Cdc37 compromised the recruitment of client kinases to a complex comprising Cdc37 and heat shock protein 90 (Hsp90) but only modestly affected Cdc37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of Cdc37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-Cdc37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

PubMed

Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

2003-08-01

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

The groESL Chaperone Operon of Lactobacillus johnsonii†

PubMed Central

Walker, D. Carey; Girgis, Hany S.; Klaenhammer, Todd R.

1999-01-01

The Lactobacillus johnsonii VPI 11088 groESL operon was localized on the chromosome near the insertion element IS1223. The operon was initially cloned as a series of three overlapping PCR fragments, which were sequenced and used to design primers to amplify the entire operon. The amplified fragment was used as a probe to recover the chromosomal copy of the groESL operon from a partial library of L. johnsonii VPI 11088 (NCK88) DNA, cloned in the shuttle vector pTRKH2. The 2,253-bp groESL fragment contained three putative open reading frames, two of which encoded the ubiquitous GroES and GroEL chaperone proteins. Analysis of the groESL promoter region revealed three transcription initiation sites, as well as three sets of inverted repeats (IR) positioned between the transcription and translation start sites. Two of the three IR sets bore significant homology to the CIRCE elements, implicated in negative regulation of the heat shock response in many bacteria. Northern analysis and primer extension revealed that multiple temperature-sensitive promoters preceded the groESL chaperone operon, suggesting that stress protein production in L. johnsonii is strongly regulated. Maximum groESL transcription activity was observed following a shift to 55°C, and a 15 to 30-min exposure of log-phase cells to this temperature increased the recovery of freeze-thawed L. johnsonii VPI 11088. These results suggest that a brief, preconditioning heat shock can be used to trigger increased chaperone production and provide significant cross-protection from the stresses imposed during the production of frozen culture concentrates. PMID:10388700

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao, Jiahui; Petrova, Kseniya; Ron, David

2010-05-25

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

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

PubMed Central

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

2017-01-01

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2–associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure. PMID:28737513

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

PubMed

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

2017-08-01

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

The Endoplasmic Reticulum Chaperone GRP170: From Immunobiology to Cancer Therapeutics

PubMed Central

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

2014-01-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grover, Abhinav; Shandilya, Ashutosh; Agrawal, Vibhuti

2011-01-07

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

Allosteric mechanism controls traffic in the chaperone/usher pathway.

PubMed

Di Yu, Xiao; Dubnovitsky, Anatoly; Pudney, Alex F; Macintyre, Sheila; Knight, Stefan D; Zavialov, Anton V

2012-11-07

Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a "proline lock" that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of the molecular chaperone calnexin in the channel catfish, Ictalurus punctatus, and its association with MHC class II molecules.

PubMed

Fuller, James R; Pitzer, Joshua E; Godwin, Ulla; Albertino, Mark; Machon, Benjamin D; Kearse, Kelly P; McConnell, Thomas J

2004-05-17

Folding and assembly of MHC molecules in mammals occurs in the endoplasmic reticulum (ER), but has not been studied in teleosts. Calnexin (CNX) is an ER chaperone that associates with glycoproteins bearing a monoglucosylated N-linked oligosaccharide side chain. Here we report the first identification and characterization of a full-length CNX cDNA clone in a teleost, and the association of the CNX chaperone with MHC class II in a channel catfish T cell line. The 1.8 kb CNX clone encodes a protein of 607 amino acids that is 72% identical to the consensus sequence of mammalian CNXs. The association of CNX with class II is of particular interest because the native MHC class II alpha chain of Ictalurus punctatus does not bear any N-linked oligosaccharide consensus glycosylation sequences. Thus the assembly of class II molecules in the catfish probably proceeds via different steps than occurs in mammals. Copyright 2003 Elsevier Ltd.

Conformational heterogeneity in the Hsp70 chaperone-substrate ensemble identified from analysis of NMR-detected titration data.

PubMed

Sekhar, Ashok; Nagesh, Jayashree; Rosenzweig, Rina; Kay, Lewis E

2017-11-01

The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes. © 2017 The Protein Society.

Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

PubMed

Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J; Gottesman, Susan; Woodson, Sarah A

2015-11-06

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Aspartic acid functions as carbonyl trapper to inhibit the formation of advanced glycation end products by chemical chaperone activity.

PubMed

Prasanna, Govindarajan; Saraswathi, N T

2016-05-01

Advanced glycation end products (AGEs) were implicated in pathology of numerous diseases. In this study, we present the bioactivity of aspartic acid (Asp) to inhibit the AGEs. Hemoglobin and bovine serum albumin (BSA) were glycated with glucose, fructose, and ribose in the presence and absence of Asp (100-200 μM). HbA1c inhibition was investigated using human blood and characterized by micro-column ion exchange chromatography. The effect of methyl glyoxal (MG) on hemoglobin and BSA was evaluated by fluorescence spectroscopy and gel electrophoresis. The effect of MG on red blood cells morphology was characterized by scanning electron micrographs. Molecular docking was performed on BSA with Asp. Asp is capable of inhibiting the formation of fluorescent AGEs by reacting with the reducing sugars. The presence of Asp as supplement in whole blood reduced the HbA1c% from 8.8 to 6.1. The presence of MG showed an increase in fluorescence and the presence of Asp inhibited the glycation thereby the fluorescence was quenched. MG also affected the electrophoretic mobility of hemoglobin and BSA by forming high molecular weight aggregates. Normal RBCs showed typical biconcave shape. MG modified RBCs showed twisted and elongated shape whereas the presence of ASP tends to protect RBC from twisting. Asp interacted with arginine residues of bovine serum albumin particularly ARG 194, ARG 198, and ARG 217 thereby stabilized the protein complex. We conclude that Asp has dual functions as a chemical chaperone to stabilize protein and as a dicarbonyl trapper, and thereby it can prevent the complications caused by glycation.

Effects of heat, cold, acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1.

PubMed

Chen, Ming-Ju; Tang, Hsin-Yu; Chiang, Ming-Lun

2017-09-01

Lactobacillus kefiranofaciens M1 is a probiotic strain isolated from Taiwanese kefir grains. The present study evaluated the effects of heat, cold, acid and bile salt adaptations on the stress tolerance of L. kefiranofaciens M1. The regulation of protein expression of L. kefiranofaciens M1 under these adaptation conditions was also investigated. The results showed that adaptation of L. kefiranofaciens M1 to heat, cold, acid and bile salts induced homologous tolerance and cross-protection against heterologous challenge. The extent of induced tolerance varied depending on the type and condition of stress. Proteomic analysis revealed that 27 proteins exhibited differences in expression between non-adapted and stress-adapted L. kefiranofaciens M1 cells. Among these proteins, three proteins involved in carbohydrate metabolism (triosephosphate isomerase, enolase and NAD-dependent glycerol-3-phosphate dehydrogenase), two proteins involved in pH homeostasis (ATP synthase subunits AtpA and AtpB), two stress response proteins (chaperones DnaK and GroEL) and one translation-related protein (30S ribosomal protein S2) were up-regulated by three of the four adaptation treatments examined. The increased synthesis of these stress proteins might play a critical protective role in the cellular defense against heat, cold, acid and bile salt stresses. Copyright © 2017 Elsevier Ltd. All rights reserved.

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 Central

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

2014-01-01

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

The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

PubMed

Cellini, Barbara; Montioli, Riccardo; Oppici, Elisa; Astegno, Alessandra; Voltattorni, Carla Borri

2014-02-01

The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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

PubMed

Olive, Joshua A; Cowan, J A

2018-07-01

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

DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography.

PubMed

Batyuk, Alexander; Wu, Yufan; Honegger, Annemarie; Heberling, Matthew M; Plückthun, Andreas

2016-04-24

DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 β-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Histone Chaperone NRP1 Interacts with WEREWOLF to Activate GLABRA2 in Arabidopsis Root Hair Development

PubMed Central

Rong, Liang; Luo, Qiang; Wang, Baihui; Zhou, Nana; Zhang, Chi; Feng, Haiyang

2017-01-01

NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) defines an evolutionarily conserved family of histone chaperones and loss of function of the Arabidopsis thaliana NAP1 family genes NAP1-RELATED PROTEIN1 (NRP1) and NRP2 causes abnormal root hair formation. Yet, the underlying molecular mechanisms remain unclear. Here, we show that NRP1 interacts with the transcription factor WEREWOLF (WER) in vitro and in vivo and enriches at the GLABRA2 (GL2) promoter in a WER-dependent manner. Crystallographic analysis indicates that NRP1 forms a dimer via its N-terminal α-helix. Mutants of NRP1 that either disrupt the α-helix dimerization or remove the C-terminal acidic tail, impair its binding to histones and WER and concomitantly lead to failure to activate GL2 transcription and to rescue the nrp1-1 nrp2-1 mutant phenotype. Our results further demonstrate that WER-dependent enrichment of NRP1 at the GL2 promoter is involved in local histone eviction and nucleosome loss in vivo. Biochemical competition assays imply that the association between NRP1 and histones may counteract the inhibitory effect of histones on the WER-DNA interaction. Collectively, our study provides important insight into the molecular mechanisms by which histone chaperones are recruited to target chromatin via interaction with a gene-specific transcription factor to moderate chromatin structure for proper root hair development. PMID:28138017

Characterization of the Grp94/OS-9 chaperone-lectin complex

PubMed Central

Seidler, Paul M.; Shinsky, Stephen A.; Hong, Feng; Li, Zihai; Cosgrove, Michael S.; Gewirth, Daniel T.

2014-01-01

Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER associated degradation (ERAD) quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins (TMPs). To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal MRH lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains ‘mammalian-specific insets’ that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate, and additionally hints at a plausible biological role for the Grp94/OS-9 complex. PMID:25193139

Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiao, Yongqin; D'Haeseleer, Patrik M; Dill, Brian

2011-01-01

In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80%more » of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by 2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as -N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.« less

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

PubMed

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

2012-03-22

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

In Vivo Substrates of the Lens Molecular Chaperones αA-Crystallin and αB-Crystallin

PubMed Central

Andley, Usha P.; Malone, James P.; Townsend, R. Reid

2014-01-01

αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin. PMID:24760011

Trigger Factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli

PubMed Central

Ullers, Ronald S.; Ang, Debbie; Schwager, Françoise; Georgopoulos, Costa; Genevaux, Pierre

2007-01-01

Polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytoplasmic, integral membrane, or exported proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF), and DnaK are key players in this process. Here, we report that, as with dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive (Cs) phenotype. Through suppressor analyses, we found that inactivating mutations in the tig gene encoding TF fully relieve both the Cs phenotype and protein aggregation observed in the absence of SecB. This antagonistic effect of TF depends on its ribosome-binding and chaperone activities but unrelated to its peptidyl-prolyl cis/trans isomerase (PPIase) activity. Furthermore, in contrast to the previously known synergistic action of TF and DnaK/DnaJ above 30°C, a tig null mutation partially suppresses the Cs phenotype exhibited by a compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF is further exemplified by the fact that the secB dnaJ double mutant is viable only in the absence of TF. Finally, we show that, in the absence of TF, more SecA and ribosomes are associated with the inner membrane, suggesting that the presence of TF directly or indirectly interferes with the process of cotranslational protein targeting to the Sec translocon. PMID:17360615

A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii.

PubMed

Whitehead, Timothy A; Boonyaratanakornkit, Boonchai B; Höllrigl, Volker; Clark, Douglas S

2007-04-01

Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. Previously, our group identified a putative single subunit of prefoldin, gamma PFD, that was up-regulated in response to heat stress in the hyperthermophilic archaeon Methanocaldococcus jannaschii. In order to characterize this protein, we subcloned and expressed it and the other two prefoldin subunits from M. jannaschii, alpha and beta PFD, into Eschericia coli and characterized the proteins. Whereas alpha and beta PFD readily assembled into the expected hexamer, gamma PFD would not assemble with either protein. Instead, gamma PFD forms long filaments of defined dimensions measuring 8.5 nm x 1.7-3.5 nm and lengths exceeding 1 microm. Filamentous gamma PFD acts as a molecular chaperone through in vitro assays, in a manner comparable to PFD. A possible molecular model for filament assembly is discussed.

A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii

PubMed Central

Whitehead, Timothy A.; Boonyaratanakornkit, Boonchai B.; Höllrigl, Volker; Clark, Douglas S.

2007-01-01

Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. Previously, our group identified a putative single subunit of prefoldin, γ PFD, that was up-regulated in response to heat stress in the hyperthermophilic archaeon Methanocaldococcus jannaschii. In order to characterize this protein, we subcloned and expressed it and the other two prefoldin subunits from M. jannaschii, α and β PFD, into Eschericia coli and characterized the proteins. Whereas α and β PFD readily assembled into the expected hexamer, γ PFD would not assemble with either protein. Instead, γ PFD forms long filaments of defined dimensions measuring 8.5 nm × 1.7–3.5 nm and lengths exceeding 1 μm. Filamentous γ PFD acts as a molecular chaperone through in vitro assays, in a manner comparable to PFD. A possible molecular model for filament assembly is discussed. PMID:17384227

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family.

PubMed

Shaner, Lance; Trott, Amy; Goeckeler, Jennifer L; Brodsky, Jeffrey L; Morano, Kevin A

2004-05-21

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.

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

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

PubMed

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

2004-12-24

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

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 I; Wernersson, Sven; Respondek, Michal; Akke, Mikael; Højrup, Peter; Koeck, Philip J B; Söderberg, Christopher A G; Emanuelsson, Cecilia

2017-05-12

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, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal 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, cross-linking 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 I X V X I 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 I X V X I 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. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and function of small heat shock/alpha-crystallin proteins: established concepts and emerging ideas.

PubMed

MacRae, T H

2000-06-01

Small heat shock/alpha-crystallin proteins are defined by conserved sequence of approximately 90 amino acid residues, termed the alpha-crystallin domain, which is bounded by variable amino- and carboxy-terminal extensions. These proteins form oligomers, most of uncertain quaternary structure, and oligomerization is prerequisite to their function as molecular chaperones. Sequence modelling and physical analyses show that the secondary structure of small heat shock/alpha-crystallin proteins is predominately beta-pleated sheet. Crystallography, site-directed spin-labelling and yeast two-hybrid selection demonstrate regions of secondary structure within the alpha-crystallin domain that interact during oligomer assembly, a process also dependent on the amino terminus. Oligomers are dynamic, exhibiting subunit exchange and organizational plasticity, perhaps leading to functional diversity. Exposure of hydrophobic residues by structural modification facilitates chaperoning where denaturing proteins in the molten globule state associate with oligomers. The flexible carboxy-terminal extension contributes to chaperone activity by enhancing the solubility of small heat shock/alpha-crystallin proteins. Site-directed mutagenesis has yielded proteins where the effect of the change on structure and function depends upon the residue modified, the organism under study and the analytical techniques used. Most revealing, substitution of a conserved arginine residue within the alpha-crystallin domain has a major impact on quaternary structure and chaperone action probably through realignment of beta-sheets. These mutations are linked to inherited diseases. Oligomer size is regulated by a stress-responsive cascade including MAPKAP kinase 2/3 and p38. Phosphorylation of small heat shock/alpha-crystallin proteins has important consequences within stressed cells, especially for microfilaments.

Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation

PubMed Central

Gao, Wei-wei; Xiao, Rong-quan; Peng, Bing-ling; Xu, Huan-teng; Shen, Hai-feng; Huang, Ming-feng; Shi, Tao-tao; Yi, Jia; Zhang, Wen-juan; Wu, Xiao-nan; Gao, Xiang; Lin, Xiang-zhi; Dorrestein, Pieter C.; Rosenfeld, Michael G.; Liu, Wen

2015-01-01

Although “histone” methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain–containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor β2 (RARβ2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70’s function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control. PMID:26080448

Real-time monitoring of artemin in vivo chaperone activity using luciferase as an intracellular reporter.

PubMed

Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Asghari, S Mohsen

2016-11-15

Artemin is an abundant thermostable protein in Artemia encysted embryos and considered as a stress protein, as its highly regulated expression is associated with stress resistance. Artemin cDNA was previously isolated and cloned from Artemia urmiana and artemin was found as an efficient molecular chaperone in vitro. Here, co-transformation of E. coli was performed with two expression vectors containing artemin and firefly luciferase for in vivo studies. The time-course of luciferase inactivation at low and elevated temperatures showed that luciferase was rapidly inactivated in control cells, but it was found that luciferase was protected significantly in artemin expressing cells. More interestingly, luciferase activity was completely regained in heat treated artemin expressing cells at room temperature. In addition, in both stress conditions, similar to residual activity of luciferase, cell viability in induced cultures over-expressing artemin was significantly higher than non-expressed artemin cells. It can be suggested that artemin confers impressive resistance in stressful conditions when introduced into E. coli cells, which is due to that it protects proteins against aggregation. Such luciferase co-expression system can be used as a real-time reporter to investigate the activity of chaperone proteins in vivo and provide a rapid and simple test for molecular chaperones. Copyright © 2016 Elsevier Inc. All rights reserved.

Leptospiral outer membrane protein LipL41 is not essential for acute leptospirosis but requires a small chaperone protein, lep, for stable expression.

PubMed

King, Amy M; Bartpho, Thanatchaporn; Sermswan, Rasana W; Bulach, Dieter M; Eshghi, Azad; Picardeau, Mathieu; Adler, Ben; Murray, Gerald L

2013-08-01

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp., but knowledge of leptospiral pathogenesis remains limited. However, the development of mutagenesis systems has allowed the investigation of putative virulence factors and their involvement in leptospirosis. LipL41 is the third most abundant lipoprotein found in the outer membranes of pathogenic leptospires and has been considered a putative virulence factor. LipL41 is encoded on the large chromosome 28 bp upstream of a small open reading frame encoding a hypothetical protein of unknown function. This gene was named lep, for LipL41 expression partner. In this study, lipL41 was found to be cotranscribed with lep. Two transposon mutants were characterized: a lipL41 mutant and a lep mutant. In the lep mutant, LipL41 protein levels were reduced by approximately 90%. Lep was shown through cross-linking and coexpression experiments to bind to LipL41. Lep is proposed to be a molecular chaperone essential for the stable expression of LipL41. The roles of LipL41 and Lep in the pathogenesis of Leptospira interrogans were investigated; surprisingly, neither of these two unique proteins was essential for acute leptospirosis.

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

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.

EML4-ALK fusions: propelling cancer but creating exploitable chaperone dependence.

PubMed

Workman, Paul; van Montfort, Rob

2014-06-01

The crystal structure of a conserved tubulin-binding region of the EML1 protein reveals a highly atypical fold in one of its β-propeller domains. Disruption of the EML1 core region domain in many of the oncogenic EML4-ALK fusion protein variants that drive non-small cell lung cancer explains their dependence on the HSP90 molecular chaperone, provides a basis to allow more precise patient stratification for therapy, and suggests a more general model for other oncogenic fusion proteins. ©2014 American Association for Cancer Research.

Chemical Chaperones Reduce ER Stress and Restore Glucose Homeostasis in a Mouse Model of Type 2 Diabetes

NASA Astrophysics Data System (ADS)

Özcan, Umut; Yilmaz, Erkan; Özcan, Lale; Furuhashi, Masato; Vaillancourt, Eric; Smith, Ross O.; Görgün, Cem Z.; Hotamisligil, Gökhan S.

2006-08-01

Endoplasmic reticulum (ER) stress is a key link between obesity, insulin resistance, and type 2 diabetes. Here, we provide evidence that this mechanistic link can be exploited for therapeutic purposes with orally active chemical chaperones. 4-Phenyl butyric acid and taurine-conjugated ursodeoxycholic acid alleviated ER stress in cells and whole animals. Treatment of obese and diabetic mice with these compounds resulted in normalization of hyperglycemia, restoration of systemic insulin sensitivity, resolution of fatty liver disease, and enhancement of insulin action in liver, muscle, and adipose tissues. Our results demonstrate that chemical chaperones enhance the adaptive capacity of the ER and act as potent antidiabetic modalities with potential application in the treatment of type 2 diabetes.

The role of fibrinogen glycation in ATTR: evidence for chaperone activity loss in disease.

PubMed

Fonseca, Daniel; Gilberto, Samuel; Ribeiro-Silva, Cristina; Ribeiro, Raquel; Guinote, Inês Batista; Saraiva, Susana; Gomes, Ricardo A; Mateus, Élia; Viana, Ana; Barroso, Eduardo; Freire, Ana Ponces; Freire, Patrick; Cordeiro, Carlos; da Costa, Gonçalo

2016-07-15

Transthyretin amyloidosis (ATTR) belongs to a class of disorders caused by protein misfolding and aggregation. ATTR is a disabling disorder of autosomal dominant trait, where transthyretin (TTR) forms amyloid deposits in different organs, causing dysfunction of the peripheral nervous system. We previously discovered that amyloid fibrils from ATTR patients are glycated by methylglyoxal. Even though no consensus has been reached about the actual role of methylglyoxal-derived advanced glycation end-products in amyloid diseases, evidence collected so far points to a role for protein glycation in conformational abnormalities, being ubiquitously found in amyloid deposits in Alzheimer's disease, dialysis-related amyloidosis and Parkinson's diseases. Human fibrinogen, an extracellular chaperone, was reported to specifically interact with a wide spectrum of stressed proteins and suppress their aggregation, being an interacting protein with TTR. Fibrinogen is differentially glycated in ATTR, leading to its chaperone activity loss. Here we show the existence of a proteostasis imbalance in ATTR linked to fibrinogen glycation by methylglyoxal. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Ensemble-based modeling and rigidity decomposition of allosteric interaction networks and communication pathways in cyclin-dependent kinases: Differentiating kinase clients of the Hsp90-Cdc37 chaperone

PubMed Central

Stetz, Gabrielle; Tse, Amanda

2017-01-01

The overarching goal of delineating molecular principles underlying differentiation of protein kinase clients and chaperone-based modulation of kinase activity is fundamental to understanding activity of many oncogenic kinases that require chaperoning of Hsp70 and Hsp90 systems to attain a functionally competent active form. Despite structural similarities and common activation mechanisms shared by cyclin-dependent kinase (CDK) proteins, members of this family can exhibit vastly different chaperone preferences. The molecular determinants underlying chaperone dependencies of protein kinases are not fully understood as structurally similar kinases may often elicit distinct regulatory responses to the chaperone. The regulatory divergences observed for members of CDK family are of particular interest as functional diversification among these kinases may be related to variations in chaperone dependencies and can be exploited in drug discovery of personalized therapeutic agents. In this work, we report the results of a computational investigation of several members of CDK family (CDK5, CDK6, CDK9) that represented a broad repertoire of chaperone dependencies—from nonclient CDK5, to weak client CDK6, and strong client CDK9. By using molecular simulations of multiple crystal structures we characterized conformational ensembles and collective dynamics of CDK proteins. We found that the elevated dynamics of CDK9 can trigger imbalances in cooperative collective motions and reduce stability of the active fold, thus creating a cascade of favorable conditions for chaperone intervention. The ensemble-based modeling of residue interaction networks and community analysis determined how differences in modularity of allosteric networks and topography of communication pathways can be linked with the client status of CDK proteins. This analysis unveiled depleted modularity of the allosteric network in CDK9 that alters distribution of communication pathways and leads to impaired

Induced ER-chaperones regulate a novel receptor-like kinase to mediate a viral innate immune response

PubMed Central

Caplan, Jeffrey L.; Zhu, Xiaohong; Mamillapalli, Padmavathi; Marathe, Rajendra; Anandalakshmi, Radhamani; Dinesh-Kumar, S. P.

2009-01-01

Summary The plant innate immune response requires a rapid, global reprogramming of cellular processes. Here we employed two complementary proteomic methods, two-dimensional differential in-gel electrophoresis (2D-DIGE) and iTRAQ, to identify differentially regulated proteins early during a defense response. Besides defense-related proteins, the constituents of the largest category of up-regulated proteins were cytoplasmic- and endoplasmic reticulum (ER)-residing molecular chaperones. Silencing of ER-resident protein disulfide isomerases, NbERp57 and NbP5, and the calreticulins, NbCRT2 and NbCRT3, lead to a partial loss of N immune receptor-mediated defense against Tobacco mosaic virus (TMV). Furthermore, NbCRT2 and NbCRT3 are required for the expression of a novel induced receptor-like kinase (IRK). IRK is a plasma membrane-localized protein required for the N-mediated hypersensitive response programmed cell death (HR-PCD) and resistance to TMV. These data support a model in which ER-resident chaperones are required for the accumulation of membrane bound or secreted proteins that are necessary for innate immunity. PMID:19917500

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

PubMed

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

2015-01-01

The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90. © 2014 FEBS.

Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin

PubMed Central

Lundin, Victor F.; Stirling, Peter C.; Gomez-Reino, Juan; Mwenifumbo, Jill C.; Obst, Jennifer M.; Valpuesta, José M.; Leroux, Michel R.

2004-01-01

Prefoldin (PFD) is a jellyfish-shaped molecular chaperone that has been proposed to play a general role in de novo protein folding in archaea and is known to assist the biogenesis of actins, tubulins, and potentially other proteins in eukaryotes. Using point mutants, chimeras, and intradomain swap variants, we show that the six coiledcoil tentacles of archaeal PFD act in concert to bind and stabilize nonnative proteins near the opening of the cavity they form. Importantly, the interaction between chaperone and substrate depends on the mostly buried interhelical hydrophobic residues of the coiled coils. We also show by electron microscopy that the tentacles can undergo an en bloc movement to accommodate an unfolded substrate. Our data reveal how archael PFD uses its unique architecture and intrinsic coiled-coil properties to interact with nonnative polypeptides. PMID:15070724

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

PubMed Central

Bencomo, Alberto; Lara-Martínez, Reyna; Rivera-Marrero, Suchitil; Domínguez, Guadalupe; Pérez-Perera, Rafaela; Jiménez-García, Luis Felipe; Altamirano-Bustamante, Nelly F.; Diaz-Delgado, Massiel; Vedrenne, Fernand; Rivillas-Acevedo, Lina; Pasten-Hidalgo, Karina; Segura-Valdez, María de Lourdes; Islas-Andrade, Sergio; Garrido-Magaña, Eulalia; Perera-Pintado, Alejandro; Prats-Capote, Anaís; Rodríguez-Tanty, Chryslaine; Altamirano-Bustamante, Myriam M.

2015-01-01

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

Oligomerization and chaperone-like activity of Drosophila melanogaster small heat shock protein DmHsp27 and three arginine mutants in the alpha-crystallin domain.

PubMed

Moutaoufik, Mohamed Taha; Morrow, Geneviève; Maaroufi, Halim; Férard, Céline; Finet, Stéphanie; Tanguay, Robert M

2017-07-01

The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.

Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro.

PubMed

Petzoldt, Svenja; Kahra, Dana; Kovermann, Michael; Dingeldein, Artur P G; Niemiec, Moritz S; Ådén, Jörgen; Wittung-Stafshede, Pernilla

2015-06-01

After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

Mutant glucocerebrosidase in Gaucher disease recruits Hsp27 to the Hsp90 chaperone complex for proteasomal degradation

PubMed Central

Yang, Chunzhang; Wang, Herui; Zhu, Dongwang; Hong, Christopher S.; Dmitriev, Pauline; Zhang, Chao; Li, Yan; Ikejiri, Barbara; Brady, Roscoe O.; Zhuang, Zhengping

2015-01-01

Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation. PMID:25583479

Mutant glucocerebrosidase in Gaucher disease recruits Hsp27 to the Hsp90 chaperone complex for proteasomal degradation.

PubMed

Yang, Chunzhang; Wang, Herui; Zhu, Dongwang; Hong, Christopher S; Dmitriev, Pauline; Zhang, Chao; Li, Yan; Ikejiri, Barbara; Brady, Roscoe O; Zhuang, Zhengping

2015-01-27

Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation.

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

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.