ABSTRACT
The Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J-domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J-domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2-Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class-specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position.
Subject(s)
HSC70 Heat-Shock Proteins , HSP40 Heat-Shock Proteins , Protein Domains , Protein Folding , Humans , HSC70 Heat-Shock Proteins/metabolism , HSC70 Heat-Shock Proteins/chemistry , HSC70 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , Models, Molecular , PhosphorylationABSTRACT
Polypeptide chains experience mechanical tension while translocating through cellular tunnels, which are subsequently folded by molecular chaperones. However, interactions between tunnel-associated chaperones and these emerging polypeptides under force is not completely understood. Our investigation focused on mechanical chaperone activity of two tunnel-associated chaperones, BiP and ERdj3 both with and without mechanical constraints and comparing them with their cytoplasmic homologs: DnaK and DnaJ. While BiP/ERdj3 have been observed to exhibit robust foldase activity under force, DnaK/DnaJ showed holdase function. Importantly, the tunnel-associated chaperones (BiP/ERdj3) transitioned to a holdase state in the absence of force, indicating a force-dependent chaperone behavior. This chaperone-driven folding event in the tunnel generated an additional mechanical energy of up to 54 zJ, potentially aiding protein translocation. Our findings align with strain theory, where chaperones with higher intrinsic deformability act as mechanical foldases (BiP, ERdj3), while those with lower deformability serve as holdases (DnaK and DnaJ). This study thus elucidates the differential mechanically regulated chaperoning activity and introduces a novel perspective on co-translocational protein folding.
Subject(s)
Escherichia coli Proteins , HSP40 Heat-Shock Proteins , HSP70 Heat-Shock Proteins , Heat-Shock Proteins , HSP40 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Heat-Shock Proteins/metabolism , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Protein Folding , Escherichia coli/genetics , Escherichia coli/metabolism , Endoplasmic Reticulum Chaperone BiP/metabolism , Molecular Chaperones/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/geneticsABSTRACT
J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8 that drives self-assembly through π-π stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity.
Subject(s)
HSP40 Heat-Shock Proteins , Protein Multimerization , Humans , HSP40 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , Models, Molecular , Amino Acid Motifs , Crystallography, X-Ray , Protein Binding , tau Proteins/metabolism , tau Proteins/chemistry , tau Proteins/genetics , Molecular Chaperones/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Mutation , Protein FoldingABSTRACT
J-domain proteins are critical Hsp70 co-chaperones. A and B types have a poorly understood glycine-rich region (Grich) adjacent to their N-terminal J-domain (Jdom). We analyzed the ability of Jdom/Grich segments of yeast Class B Sis1 and a suppressor variant of Class A, Ydj1, to rescue the inviability of sis1-∆. In each, we identified a cluster of Grich residues required for rescue. Both contain conserved hydrophobic and acidic residues and are predicted to form helices. While, as expected, the Sis1 segment docks on its J-domain, that of Ydj1 does not. However, data suggest both interact with Hsp70. We speculate that the Grich-Hsp70 interaction of Classes A and B J-domain proteins can fine tune the activity of Hsp70, thus being particularly important for the function of Class B.
Subject(s)
Glycine , HSP70 Heat-Shock Proteins , Protein Domains , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Glycine/metabolism , Glycine/chemistry , HSP70 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , HSP40 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , Amino Acid Sequence , Protein Binding , Molecular Chaperones/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/chemistry , Models, MolecularABSTRACT
Plasmodium falciparum renovates the host erythrocyte to survive during intraerythrocytic development. This renovation requires many parasite proteins to unfold and move outside the parasitophorous vacuolar membrane, and chaperone-regulated protein folding becomes essential for the exported proteins to function. We report on a type-IV J domain protein (JDP), PF3D7_1401100, which we found to be processed before export and trafficked inside the lumen of parasite-derived structures known as J-dots. We found this protein to have holdase activity, as well as stimulate the ATPase and aggregation suppression activity of the human HSP70 chaperone HsHSPA8; thus, we named it "HSPA8-interacting J protein" (A8iJp). Moreover, we found a subset of HsHSPA8 to co-localize with A8iJp inside the infected human erythrocyte. Our results suggest that A8iJp modulates HsHSPA8 chaperone activity and may play an important role in host erythrocyte renovation.
Subject(s)
HSP40 Heat-Shock Proteins , Plasmodium falciparum , Humans , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/metabolism , Protein Binding , Protozoan Proteins/metabolism , Molecular Chaperones/metabolism , Erythrocytes , Protein Folding , HSC70 Heat-Shock Proteins/metabolismABSTRACT
BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder with clinical presentations of progressive cognitive and memory deterioration. The pathologic hallmarks of AD include tau neurofibrillary tangles and amyloid plaque depositions in the hippocampus and associated neocortex. The neuronal aggregated tau observed in AD cells suggests that the protein folding problem is a major cause of AD. J-domain-containing proteins (JDPs) are the largest family of cochaperones, which play a vital role in specifying and directing HSP70 chaperone functions. JDPs bind substrates and deliver them to HSP70. The association of JDP and HSP70 opens the substrate-binding domain of HSP70 to help the loading of the clients. However, in the initial HSP70 cycle, which JDP delivers tau to the HSP70 system in neuronal cells remains unclear. RESULTS: We screened the requirement of a diverse panel of JDPs for preventing tau aggregation in the human neuroblastoma cell line SH-SY5Y by a filter retardation method. Interestingly, knockdown of DNAJB6, one of the JDPs, displayed tau aggregation and overexpression of DNAJB6b, one of the isoforms generated from the DNAJB6 gene by alternative splicing, reduced tau aggregation. Further, the tau bimolecular fluorescence complementation assay confirmed the DNAJB6b-dependent tau clearance. The co-immunoprecipitation and the proximity ligation assay demonstrated the protein-protein interaction between tau and the chaperone-cochaperone complex. The J-domain of DNAJB6b was critical for preventing tau aggregation. Moreover, reduced DNAJB6 expression and increased tau aggregation were detected in an age-dependent manner in immunohistochemical analysis of the hippocampus tissues of a mouse model of tau pathology. CONCLUSIONS: In summary, downregulation of DNAJB6b increases the insoluble form of tau, while overexpression of DNAJB6b reduces tau aggregation. Moreover, DNAJB6b associates with tau. Therefore, this study reveals that DNAJB6b is a direct sensor for its client tau in the HSP70 folding system in neuronal cells, thus helping to prevent AD.
Subject(s)
Alzheimer Disease , HSP40 Heat-Shock Proteins , Molecular Chaperones , Nerve Tissue Proteins , Neuroblastoma , Animals , Humans , Mice , Alternative Splicing , Alzheimer Disease/genetics , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Nerve Tissue Proteins/genetics , Protein Folding , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
The DNAJB1-PRKACA oncogenic gene fusion results in an active kinase enzyme, J-PKAcα, that has been identified as an attractive antitumor target for fibrolamellar hepatocellular carcinoma (FLHCC). A high-throughput assay was used to identify inhibitors of J-PKAcα catalytic activity by screening the NCI Program for Natural Product Discovery (NPNPD) prefractionated natural product library. Purification of the active agent from a single fraction of an Aplidium sp. marine tunicate led to the discovery of two unprecedented alkaloids, aplithianines A (1) and B (2). Aplithianine A (1) showed potent inhibition against J-PKAcα with an IC50 of â¼1 µM in the primary screening assay. In kinome screening, 1 inhibited wild-type PKA with an IC50 of 84 nM. Further mechanistic studies including cocrystallization and X-ray diffraction experiments revealed that 1 inhibited PKAcα catalytic activity by competitively binding to the ATP pocket. Human kinome profiling of 1 against a panel of 370 kinases revealed potent inhibition of select serine/threonine kinases in the CLK and PKG families with IC50 values in the range â¼11-90 nM. An efficient, four-step total synthesis of 1 has been accomplished, enabling further evaluation of aplithianines as biologically relevant kinase inhibitors.
Subject(s)
Biological Products , Carcinoma, Hepatocellular , Humans , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases , Carcinoma, Hepatocellular/pathology , Serine , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolismABSTRACT
Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.
Subject(s)
Escherichia coli Proteins , HSP70 Heat-Shock Proteins , HSP90 Heat-Shock Proteins , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Protein DomainsABSTRACT
The imbalance in metal homeostasis can be associated with several human diseases, and exposure to increasing concentrations of metals promotes cell stress and toxicity. Therefore, understanding the cytotoxic effect of metal imbalance is important to unravel the biochemical mechanism of homeostasis and the action of potential protective proteins against metal toxicity. Several studies, including gene deletion in yeast, provide evidence indicating the possible indirect involvement of cochaperones from the Hsp40/DNAJA family in metal homeostasis, possibly through modulating the activity of Hsp 70.This work first investigated the effect of zinc and copper on the conformation and function of the human Hsp40 cochaperone DNAJA1, a zinc-binding protein. DNAJA1 was capable to complement the phenotype of a yeast strain deleted of the ydj1 gene, which was more sensitive to the presence of zinc and copper than the wild-type strain. To gain further insight about the role of the DNAJA family in metal binding, the recombinant human DNAJA1 protein was studied. Zinc removal from DNAJA1 affected both its stability and ability to act as a chaperone, i.e., to protect other proteins from aggregation. The reintroduction of zinc restored the native properties of DNAJA1 and, surprisingly, the addition of copper partially restored the native properties.
Subject(s)
Copper , Saccharomyces cerevisiae , Humans , Saccharomyces cerevisiae/metabolism , Zinc/pharmacology , HSP40 Heat-Shock Proteins/chemistry , Molecular Chaperones/genetics , HSP70 Heat-Shock Proteins/metabolismABSTRACT
Cellular homeostasis and stress survival requires maintenance of the proteome and suppression of proteotoxicity. Molecular chaperones promote cell survival through repair of misfolded proteins and cooperation with protein degradation machines to discard terminally damaged proteins. Hsp70 family members play an essential role in cellular protein metabolism by binding and releasing non-native proteins to facilitate protein folding, refolding, and degradation. Hsp40 (DnaJ-like proteins) family members are Hsp70 co-chaperones that determine the fate of Hsp70 clients by facilitating protein folding, assembly, and degradation. Hsp40s select substrates for Hsp70 via use of an intrinsic chaperone activity to bind non-native regions of proteins. During delivery of bound cargo Hsp40s employ a conserved J-domain to stimulate Hsp70 ATPase activity and thereby stabilize complexes between Hsp70 and non-native proteins. This review describes the mechanisms by which different Hsp40s use specialized sub-domains to direct clients of Hsp70 for triage between folding versus degradation.
Subject(s)
HSP40 Heat-Shock Proteins , HSP70 Heat-Shock Proteins , Protein Folding , Proteolysis , Humans , Homeostasis , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein BindingABSTRACT
The J-domain proteins (JDP) form the largest protein family among cellular chaperones. In cooperation with the Hsp70 chaperone system, these co-chaperones orchestrate a plethora of distinct functions, including those that help maintain cellular proteostasis and development. JDPs evolved largely through the fusion of a J-domain with other protein subdomains. The highly conserved J-domain facilitates the binding and activation of Hsp70s. How JDPs (re)wire Hsp70 chaperone circuits and promote functional diversity remains insufficiently explained. Here, we discuss recent advances in our understanding of the JDP family with a focus on the regulation built around J-domains to ensure correct pairing and assembly of JDP-Hsp70 machineries that operate on different clientele under various cellular growth conditions.
Subject(s)
HSP40 Heat-Shock Proteins , Proteostasis , Humans , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , HSP70 Heat-Shock Proteins/metabolism , Protein BindingABSTRACT
Abrupt environmental changes are faced by Leishmania parasites during transmission from a poikilothermic insect vector to a warm-blooded host. Adaptation to harsh environmental conditions, such as nutrient deprivation, hypoxia, oxidative stress and heat shock needs to be accomplished by rapid reconfiguration of gene expression and remodeling of protein interaction networks. Chaperones play a central role in the maintenance of cellular homeostasis, and they are responsible for crucial tasks such as correct folding of nascent proteins, protein translocation across different subcellular compartments, avoiding protein aggregates and elimination of damaged proteins. Nearly one percent of the gene content in the Leishmania genome corresponds to members of the HSP40 family, a group of proteins that assist HSP70s in a variety of cellular functions. Despite their expected relevance in the parasite biology and infectivity, little is known about their functions or partnership with the different Leishmania HSP70s. Here, we summarize the structural features of the 72 HSP40 proteins encoded in the Leishmania infantum genome and their classification into four categories. A review of proteomic data, together with orthology analyses, allow us to postulate cellular locations and possible functional roles for some of them. A detailed study of the members of this family would provide valuable information and opportunities for drug discovery and improvement of current treatments against leishmaniasis.
Subject(s)
HSP40 Heat-Shock Proteins , Leishmania infantum , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Leishmania infantum/genetics , Leishmania infantum/metabolism , Molecular Chaperones , ProteomicsABSTRACT
The glucocorticoid receptor (GR) is an important transcription factor and drug target linked to a variety of biological functions and diseases. It is one of the most stringent physiological clients of the Hsp90/Hsp70/Hsp40 chaperone system. In this study, we used single-molecule force spectroscopy by optical tweezers to observe the interaction of the GR's ligand-binding domain (GR-LBD) with the Hsp70/Hsp40 chaperone system (Hsp70/40). We show in real time that Hsp70/40 can unfold the complete GR-LBD in a stepwise manner. Each unfolding step involves binding of an Hsp70 to the GR-LBD and subsequent adenosine triphosphate (ATP) hydrolysis, stimulated by Hsp40. The kinetics of chaperone-mediated unfolding depend on chaperone concentrations as well as the presence of the nucleotide exchange factor BAG1. We find that Hsp70/40 can stabilize new unfolding intermediates, showing that Hsp70/40 can directly interact with the folded core of the protein when working as an unfoldase. Our results support an unfolding mechanism where Hsp70 can directly bind to folded protein structures and unfold them upon ATP hydrolysis. These results provide important insights into the regulation of GR by Hsp70/40.
Subject(s)
HSP40 Heat-Shock Proteins , HSP70 Heat-Shock Proteins , Receptors, Glucocorticoid , Adenosine Triphosphate/chemistry , HSP40 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/chemistry , Hydrolysis , Optical Tweezers , Protein Binding , Protein Domains , Protein Folding , Receptors, Glucocorticoid/chemistry , Single Molecule ImagingABSTRACT
KEY MESSAGE: J-like proteins (JLPs) are emerging as ancillaries to the cellular chaperone network. They modulate functions of Hsp70:J-domain protein (JDP) systems in novel ways thereby having key roles in diverse plant processes. J-domain proteins (JDPs) form an obligate co-chaperone partnership with Hsp70s with their highly conserved J-domain to steer protein quality control processes in the cell. The HPD motif between helix II and helix III of the J-domain is crucial for JDP's interaction with Hsp70s. According to the most recent classification, J-like proteins (JLPs) form an extended class of the JDP family possessing a degenerate J-domain with the HPD motif non-conservatively replaced by other amino acid residues and hence are not able to interact with Hsp70s. Considering this most updated and acceptable JLP classification, we identified 21 JLPs in Arabidopsis thaliana that share a structurally conserved J-like domain (JLD), but lack the HPD motif. Analysis of publicly available gene expression data as well as real-time quantitative PCR performed for a few selected JLPs implicated some of these proteins in growth, development and stress response. Here, we summarize the current state of knowledge on plant JLPs and their involvement in vital plant cellular/metabolic processes, including chloroplast division, mitochondrial protein import and flowering. Finally, we propose possible modes of action for these highly elusive proteins and other DnaJ-related proteins (DNAJRs) in regulating the Hsp70 chaperone network.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolismABSTRACT
The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein "as is" with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.
Subject(s)
HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/isolation & purification , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/isolation & purification , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Protein Engineering/methods , Ammonium Sulfate/chemistry , Amyloidogenic Proteins/chemistry , Amyloidogenic Proteins/metabolism , Chemical Precipitation , Chromatography, Gel , Escherichia coli/genetics , Fluorescent Dyes/chemistry , HSP40 Heat-Shock Proteins/metabolism , Humans , Hydrogen-Ion Concentration , Molecular Chaperones/metabolism , Nerve Tissue Proteins/metabolism , Protein Denaturation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Rhodamines/chemistry , Solubility , Sulfonic Acids/chemistryABSTRACT
Protein aggregation is a hallmark of neurodegeneration. Here, we find that Huntington's disease-related HTT-polyQ aggregation induces a cellular proteotoxic stress response, while ALS-related mutant FUS (mutFUS) aggregation leads to deteriorated proteostasis. Further exploring chaperone function as potential modifiers of pathological aggregation in these contexts, we reveal divergent effects of naturally-occurring chaperone isoforms on different aggregate types. We identify a complex of the full-length (FL) DNAJB14 and DNAJB12, that substantially protects from mutFUS aggregation, in an HSP70-dependent manner. Their naturally-occurring short isoforms, however, do not form a complex, and lose their ability to preclude mutFUS aggregation. In contrast, DNAJB12-short alleviates, while DNAJB12-FL aggravates, HTT-polyQ aggregation. DNAJB14-FL expression increases the mobility of mutFUS aggregates, and restores the deteriorated proteostasis in mutFUS aggregate-containing cells and primary neurons. Our results highlight a maladaptive cellular response to pathological aggregation, and reveal a layer of chaperone network complexity conferred by DNAJ isoforms, in regulation of different aggregate types.
Subject(s)
HSP40 Heat-Shock Proteins/metabolism , Huntingtin Protein/metabolism , Huntington Disease/metabolism , Molecular Chaperones/metabolism , Peptides/metabolism , Protein Aggregates , RNA-Binding Protein FUS/metabolism , HEK293 Cells , HSP40 Heat-Shock Proteins/chemistry , Humans , Molecular Chaperones/chemistry , Neurons/metabolism , Optical Imaging , Protein Isoforms/metabolism , ProteostasisABSTRACT
[PSI+] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI+] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI+] prions typically affect viability only modestly, so [PSI+] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI+] propagation, yet [PSI+] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI+] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI+] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI+] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI+] prions.
Subject(s)
HSP40 Heat-Shock Proteins/metabolism , Peptide Termination Factors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , Peptide Termination Factors/genetics , Protein Domains , Proteostasis , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C. elegans, does not bind to CDC-37 in a similar manner. Analytical ultracentrifugation is employed to show that one CDC-37 molecule binds to a dimeric DNJ-13 protein with low micromolar affinity. We perform cross-linking studies with mass spectrometry to identify the interaction site and obtain specific cross-links connecting the N-terminal J-domain of DNJ-13 with the N-terminal domain of CDC-37. Further AUC experiments reveal that both, the N-terminal part of CDC-37 and the C-terminal domain of CDC-37, are required for efficient interaction. Furthermore, the presence of DNJ-13 strengthens the complex formation between CDC-37 and HSP-90 and modulates the nucleotide-dependent effects. These findings on the interaction between Hsp40 proteins and Hsp90-cofactors provide evidence for a more intricate interaction between the two chaperone systems during client processing.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Cell Cycle Proteins/metabolism , HSP40 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Animals , Caenorhabditis elegans/chemistry , Caenorhabditis elegans Proteins/chemistry , Cell Cycle Proteins/chemistry , HSP40 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/chemistry , Models, Molecular , Protein Binding , Protein Folding , Protein Interaction MapsABSTRACT
Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.
Subject(s)
Adenosine Diphosphate/metabolism , Chaetomium/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/metabolism , Chaetomium/chemistry , Cryoelectron Microscopy , Crystallography, X-Ray , Fungal Proteins/chemistry , Microscopy, Atomic Force , Models, Molecular , Protein Aggregates , Protein Binding , Protein Conformation , Protein Domains , Protein MultimerizationABSTRACT
The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.