Essentiality of Sis1, a J-domain protein Hsp70 cochaperone, can be overcome by Tti1, a specialized PIKK chaperone

J-domain protein cochaperones drive much of the functional diversity of Hsp70-based chaperone systems. Sis1 is the only essential J-domain protein of the cytosol/nucleus of Saccharomyces cerevisiae. Why it is required for cell growth is not understood, nor is how critical its role in regulation of heat shock transcription factor 1 (Hsf1). We report that single residue substitutions in Tti1, a component of the heterotrimeric TTT complex, a specialized chaperone system for phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, allow growth of cells lacking Sis1. Upon depletion of Sis1, cells become hypersensitive to rapamycin, a specific inhibitor of TORC1 kinase. In addition, levels of the three essential PIKKs (Mec1, Tra1, and Tor2), as well as Tor1, decrease upon Sis1depletion. Overexpression of Tti1 allows growth, without an increase in the other subunits of the TTT complex, Tel2 and Tti2, suggesting that it can function independent of the complex. Cells lacking Sis1, with viability supported by Tti1 suppressor, substantially upregulate some, but not all, heat shock elements activated by Hsf1. Together, our results suggest that Sis1 is required as a cochaperone of Hsp70 for the folding/maintenance of PIKKs making Sis1 an essential gene, and its requirement for Hsf1 regulation is more nuanced than generally appreciated.

A J Domain Protein Functions as a Histone Chaperone to Maintain Genome Integrity and the Response to DNA Damage in a Human Fungal Pathogen

DNA replication, gene expression, and genomic repair all require precise coordination of the many proteins that interact with DNA. This includes the histones as well as their chaperones.

Regulation of p53 and Cancer Signaling by Heat Shock Protein 40/J-Domain Protein Family Members

Heat shock proteins (HSPs) are molecular chaperones that assist diverse cellular activities including protein folding, intracellular transportation, assembly or disassembly of protein complexes, and stabilization or degradation of misfolded or aggregated proteins. HSP40, also known as J-domain proteins (JDPs), is the largest family with over fifty members and contains highly conserved J domains responsible for binding to HSP70 and stimulation of the ATPase activity as a co-chaperone. Tumor suppressor p53 (p53), the most frequently mutated gene in human cancers, is one of the proteins that functionally interact with HSP40/JDPs. The majority of p53 mutations are missense mutations, resulting in acquirement of unexpected oncogenic activities, referred to as gain of function (GOF), in addition to loss of the tumor suppressive function. Moreover, stability and levels of wild-type p53 (wtp53) and mutant p53 (mutp53) are crucial for their tumor suppressive and oncogenic activities, respectively. However, the regulatory mechanisms of wtp53 and mutp53 are not fully understood. Accumulating reports demonstrate regulation of wtp53 and mutp53 levels and/or activities by HSP40/JDPs. Here, we summarize updated knowledge related to the link of HSP40/JDPs with p53 and cancer signaling to improve our understanding of the regulation of tumor suppressive wtp53 and oncogenic mutp53 GOF activities.

Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins

Protein homeostasis is constantly being challenged with protein misfolding that leads to aggregation. Hsp70 is one of the versatile chaperones that interact with misfolded proteins and actively support their folding. Multifunctional Hsp70s are harnessed to specific roles by J-domain proteins (JDPs, also known as Hsp40s). Interaction with the J-domain of these cochaperones stimulates ATP hydrolysis in Hsp70, which stabilizes substrate binding. In eukaryotes, two classes of JDPs, Class A and Class B, engage Hsp70 in the reactivation of aggregated proteins. In most species, excluding metazoans, protein recovery also relies on an Hsp100 disaggregase. Although intensely studied, many mechanistic details of how the two JDP classes regulate protein disaggregation are still unknown. Here, we explore functional differences between the yeast Class A (Ydj1) and Class B (Sis1) JDPs at the individual stages of protein disaggregation. With real-time biochemical tools, we show that Ydj1 alone is superior to Sis1 in aggregate binding, yet it is Sis1 that recruits more Ssa1 molecules to the substrate. This advantage of Sis1 depends on its ability to bind to the EEVD motif of Hsp70, a quality specific to most of Class B JDPs. This second interaction also conditions the Hsp70-induced aggregate modification that boosts its subsequent dissolution by the Hsp104 disaggregase. Our results suggest that the Sis1-mediated chaperone assembly at the aggregate surface potentiates the entropic pulling, driven polypeptide disentanglement, while Ydj1 binding favors the refolding of the solubilized proteins. Such subspecialization of the JDPs across protein reactivation improves the robustness and efficiency of the disaggregation machinery.

Nematode CDC-37 and DNJ-13 form complexes and can interact with HSP-90

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.

Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.

Pathway of Hsp70 interactions at the ribosome

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb’s ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

Dnj1 Promotes Virulence in Cryptococcus neoformans by Maintaining Robust Endoplasmic Reticulum Homeostasis Under Temperature Stress

The capacity of opportunistic fungal pathogens such as Cryptococcus neoformans to cause disease is dependent on their ability to overcome an onslaught of stresses including elevated temperature under mammalian host conditions. Protein chaperones and co-chaperones play key roles in thermotolerance. In this study, we characterized the role of the endoplasmic reticulum (ER) J-domain containing co-chaperone, Dnj1, in the virulence of C. neoformans. A strain expressing a Dnj1-GFP fusion protein was used to confirm localization to the ER, and a dnj1∆ deletion mutant was shown to be hypersensitive to the ER stress caused by tunicamycin (TM) or 4μ8C. Dnj1 and another ER chaperone, calnexin were found to coordinately maintain ER homeostasis and contribute to maintenance of cell wall architecture. Dnj1 also contributed to thermotolerance and increased in abundance at elevated temperatures representative of febrile patients (e.g., 39°C) thus highlighting its role as a temperature-responsive J domain protein. The elaboration of virulence factors such as the polysaccharide capsule and extracellular urease activity were also markedly impaired in the dnj1∆ mutant when induced at human body temperature (i.e., 37°C). These virulence factors are immunomodulatory and, indeed, infection with the dnj1∆ mutant revealed impaired induction of the cytokines IL-6, IL-10, and MCP-1 in the lungs of mice compared to infection with wild type or complemented strains. The dnj1∆ mutant also had attenuated virulence in an intranasal murine model of cryptococcosis. Altogether, our data indicate that Dnj1 is crucial for survival and virulence factor production at elevated temperatures. The characterization of this co-chaperone also highlights the importance of maintaining homeostasis in the ER for the pathogenesis of C. neoformans.

DnaJC7 binds natively folded structural elements in tau to inhibit amyloid formation

Molecular chaperones, including Hsp70/J-domain protein (JDP) families, play central roles in binding substrates to prevent their aggregation. How JDPs select different conformations of substrates remains poorly understood. Here, we report an interaction between the JDP DnaJC7 and tau that efficiently suppresses tau aggregation in vitro and in cells. DnaJC7 binds preferentially to natively folded wild-type tau, but disease-associated mutants in tau reduce chaperone binding affinity. We identify that DnaJC7 uses a single TPR domain to recognize a β-turn structural element in tau that contains the 275VQIINK280 amyloid motif. Wild-type tau, but not mutant, β-turn structural elements can block full-length tau binding to DnaJC7. These data suggest DnaJC7 preferentially binds and stabilizes natively folded conformations of tau to prevent tau conversion into amyloids. Our work identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention.

Abnormal triaging of misfolded proteins by adult neuronal ceroid lipofuscinosis-associated CSPα mutants causes lipofuscin accumulation

Mutations in DNAJC5 (encoding the J domain-containing HSP70 co-chaperone CSPα) are associated with adult neuronal ceroid lipofuscinosis (ANCL), a dominant-inherited neurodegenerative disease featuring lysosome-derived autofluorescent storage material (AFSM) termed lipofuscin. Functionally, CSPα has been implicated in chaperoning synaptic proteins and in misfolding-associated protein secretion (MAPS), but how CSPα dysfunction causes lipofuscinosis and neurodegeneration is unclear. Here we report two distinct protein quality control functions of CSPα at endolysosomes and perinuclear vesicles, respectively. We show that the endolysosome-associated CSPα promotes microautophagy of misfolded clients, but is dispensable for MAPS. By contrast, the perinuclear-localized CSPα, regulated by a previously unknown CSPα interactor named CD98hc, is critical for MAPS but unneeded for microautophagy. Importantly, these processes are coupled by CSPα in a J-domain regulated manner. Uncoupling these two processes, as seen in cells lacking CD98hc or expressing ANCL-associated CSPα mutants, generates CSPα-containing AFSMs resembling NCL patient-derived lipofuscin, and also induces neurodegeneration in a Drosophila ANCL model. These findings suggest that blocking MAPS while allowing CSPα-mediated microautophagy disrupts lysosome homeostasis, causing CSPα-associated lipofuscinosis and neurodegeneration.