Cytosolic Hsp70 Research Articles

Exploration of the binding determinants of protein phosphatase 5 (PP5) reveals a chaperone-independent activation mechanism

The protein phosphatase 5 (PP5) is normally recruited to its substrates by the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). This interaction requires the tetratricopeptide repeat (TPR) domain of PP5, which binds to an EEVD motif at the extreme C-termini of cytosolic Hsp70 and Hsp90 isoforms. In addition to bringing PP5 into proximity with chaperone-bound substrates, this interaction also relieves auto-inhibition in PP5’s catalytic domain, promoting its phosphatase activity. To better understand the molecular determinants of this process, we screened a large, pentapeptide library for binding to PP5. This screen identified the amino acid preferences at each position, which we validated by showing that the optimal sequences bind 4- to 7-fold tighter than the natural EEVD motifs and stimulate PP5’s enzymatic activity. The enhanced affinity for PP5’s TPR domain was confirmed using a protein-adaptive differential scanning fluorimetry (paDSF) assay. Using this increased knowledge of structure-activity relationships, we re-examined affinity proteomics results to look for potential EEVD-like motifs in the C-termini of known PP5-binding partners. This search identified elongator acetyltransferase complex subunit 1 (ELP1/IKBKAP) as a putative partner and, indeed, we found that its C-terminal sequence, LSLLD, binds directly to PP5’s TPR domain in vitro. Consistent with this idea, mutation of ELP1’s terminal aspartate was sufficient to interrupt the interaction with PP5 in vitro and in cells. Together, these findings reveal the sequence preferences of PP5’s TPR domain and expand the scope of PP5’s functions to include chaperone-independent complexes.

Autorepression of yeast Hsp70 cochaperones by intramolecular interactions involving their J-domains.

The 70kDa heat shock protein (Hsp70) chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) coevolved with Hsp70s to trigger ATP hydrolysis and catalytically upload various substrate polypeptides in need to be structurally modified by the chaperone. Here, we measured the protein disaggregation and refolding activities of the main yeast cytosolic Hsp70, Ssa1, in the presence of its most abundant JDPs, Sis1 and Ydj1, and two swap mutants, in which the J-domains have been interchanged. The observed differences by which the four constructs differently cooperate with Ssa1 and cooperate with each other, as well as their observed intrinsic ability to bind misfolded substrates and trigger Ssa1's ATPase, indicate the presence of yet uncharacterized intramolecular dynamic interactions between the J-domains and the remaining C-terminal segments of these proteins. Taken together, the data suggest an autoregulatory role to these intramolecular interactions within both type A and B JDPs, which might have evolved to reduce energy-costly ATPase cycles by the Ssa1-4 chaperones that are the most abundant Hsp70s in the yeast cytosol.

Abstract 1814: Development and evaluation of Hsp90β-selective inhibitors to overcome the safety challenges associated with pan-Hsp90 inhibitors

Abstract The 90 kD heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding of select proteins, many of which are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. More than 20 small-molecule inhibitors of Hsp90 have entered clinical trials for the treatment of cancer, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms (Hsp90a, Hsp90β, Grp94 and TRAP-1). Most of these clinical trials have failed as pan-Inhibition of Hsp90 appears to be detrimental with toxicities reported alongside induction of the pro-survival heat shock response, which leads to additional dosing challenges/toxicities. The cytosolic Hsp90 isoforms Hsp90a and Hsp90β are the major contributors to cancer growth/survival. It has been established that inhibition of Hsp90a is associated with cardiac toxicity, retinal toxicity, and induction of the heat shock response. Therefore, the development of Hsp90β -selective inhibitors represents an alternative approach for the treatment of cancer with limited side effects. Towards this goal, Hsp90β-selective inhibitors that exhibit >150-fold selectivity over Hsp90a have been developed, which exhibit efficacy against select cancers while overcoming the safety challenges associated with previous Hsp90 inhibitors. The results from such studies will be presented. Citation Format: Sanket Mishra, Tyelor Reynolds, Brian Blagg. Development and evaluation of Hsp90β-selective inhibitors to overcome the safety challenges associated with pan-Hsp90 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1814.

Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells

HSP90 has emerged as an appealing anti-cancer target. However, HSP90 inhibitors (HSP90i) are characterized by limited clinical utility, primarily due to the resistance acquisition via heat shock response (HSR) induction. Understanding the roles of abundantly expressed cytosolic HSP90 isoforms (α and β) in sustaining malignant cells’ growth and the mechanisms of resistance to HSP90i is crucial for exploiting their clinical potential. Utilizing multi-omics approaches, we identified that ablation of the HSP90β isoform induces the overexpression of HSP90α and extracellular-secreted HSP90α (eHSP90α). Notably, we found that the absence of HSP90α causes downregulation of PTPRC (or CD45) expression and restricts in vivo growth of BCR-ABL1+ leukemia cells. Subsequently, chronic long-term exposure to the clinically advanced HSP90i PU-H71 (Zelavespib) led to copy number gain and mutation (p.S164F) of the HSP90AA1 gene, and HSP90α overexpression. In contrast, acquired resistance toward other tested HSP90i (Tanespimycin and Coumermycin A1) was attained by MDR1 efflux pump overexpression. Remarkably, combined CDK7 and HSP90 inhibition display synergistic activity against therapy-resistant BCR-ABL1+ patient leukemia cells via blocking pro-survival HSR and HSP90α overexpression, providing a novel strategy to avoid the emergence of resistance against treatment with HSP90i alone.

Insertion of GGMP repeat residues of Plasmodium falciparum Hsp70-1 in the lid of DnaK adversely impacts client recognition

Although Hsp70 is a conserved molecular chaperone, it exhibits some degree of functional specialisation across species. Features of Hsp70 regulating its functional specialisation remain to be fully established. We previously demonstrated that E. coli Hsp70 (DnaK) exhibits functional features that distinguishes it from PfHsp70-1, a canonical cytosolic Hsp70 of Plasmodium falciparum. One of the defining features of PfHsp70-1 is that it possesses GGMP repeat residues located in its C-terminal lid segment, while DnaK lacks this motif. Previously, we demonstrated that the insertion of GGMP repeat residues of PfHsp70-1 into E. coli DnaK abrogates the chaperone activity of DnaK. However, the role of the GGMP motif in regulating Hsp70 function remains to be fully understood. To explore the function of this motif, we expressed recombinant forms of wild type DnaK and its GGMP insertion motif, DnaK-G and systematically characterised the structure-function features of the two proteins using in silico analysis, biophysical approaches and an in cellulo complementation assay. Our findings demonstrated that the GGMP inserted in DnaK compromised various functional features such as nucleotide binding, allostery, substrate binding affinity and cellular proteome client selectivity. These findings thus, highlight the GGMP motif of Hsp70 as an important functional module.

Exploration of the truncated cytosolic Hsp70 in plants - unveiling the diverse T1 lineage and the conserved T2 lineage.

The 70-kDa heat shock proteins (Hsp70s) are chaperone proteins involved in protein folding processes. Truncated Hsp70 (Hsp70T) refers to the variant lacking a conserved C-terminal motif, which is crucial for co-chaperone interactions or protein retention. Despite their significance, the characteristics of Hsp70Ts in plants remain largely unexplored. In this study, we performed a comprehensive genome-wide analysis of 192 sequenced plant and green algae genomes to investigate the distribution and features of Hsp70Ts. Our findings unveil the widespread occurrence of Hsp70Ts across all four Hsp70 forms, including cytosolic, endoplasmic reticulum, mitochondrial, and chloroplast Hsp70s, with cytosolic Hsp70T being the most prevalent and abundant subtype. Cytosolic Hsp70T is characterized by two distinct lineages, referred to as T1 and T2. Among the investigated plant and green algae species, T1 genes were identified in approximately 60% of cases, showcasing a variable gene count ranging from one to several dozens. In contrast, T2 genes were prevalent across the majority of plant genomes, usually occurring in fewer than five gene copies per species. Sequence analysis highlights that the putative T1 proteins exhibit higher similarity to full-length cytosolic Hsp70s in comparison to T2 proteins. Intriguingly, the T2 lineage demonstrates a higher level of conservation within their protein sequences, whereas the T1 lineage presents a diverse range in the C-terminal and SBDα region, leading to categorization into four distinct subtypes. Furthermore, we have observed that T1-rich species characterized by the possession of 15 or more T1 genes exhibit an expansion of T1 genes into tandem gene clusters. The T1 gene clusters identified within the Laurales order display synteny with clusters found in a species of the Chloranthales order and another species within basal angiosperms, suggesting a conserved evolutionary relationship of T1 gene clusters among these plants. Additionally, T2 genes demonstrate distinct expression patterns in seeds and under heat stress, implying their potential roles in seed development and stress response.

A cytosolic surveillance mechanism activates the mitochondrial UPR

The mitochondrial unfolded protein response (UPRmt) is essential to safeguard mitochondria from proteotoxic damage by activating a dedicated transcriptional response in the nucleus to restore proteostasis1,2. Yet, it remains unclear how the information on mitochondria misfolding stress (MMS) is signalled to the nucleus as part of the human UPRmt (refs. 3,4). Here, we show that UPRmt signalling is driven by the release of two individual signals in the cytosol—mitochondrial reactive oxygen species (mtROS) and accumulation of mitochondrial protein precursors in the cytosol (c-mtProt). Combining proteomics and genetic approaches, we identified that MMS causes the release of mtROS into the cytosol. In parallel, MMS leads to mitochondrial protein import defects causing c-mtProt accumulation. Both signals integrate to activate the UPRmt; released mtROS oxidize the cytosolic HSP40 protein DNAJA1, which leads to enhanced recruitment of cytosolic HSP70 to c-mtProt. Consequently, HSP70 releases HSF1, which translocates to the nucleus and activates transcription of UPRmt genes. Together, we identify a highly controlled cytosolic surveillance mechanism that integrates independent mitochondrial stress signals to initiate the UPRmt. These observations reveal a link between mitochondrial and cytosolic proteostasis and provide molecular insight into UPRmt signalling in human cells.

Hsp90 mutants with distinct defects provide novel insights into cochaperone regulation of the folding cycle.

Molecular chaperones play a key role in maintaining proteostasis and cellular health. The abundant, essential, cytosolic Hsp90 (Heat shock protein, 90 kDa) facilitates the folding and activation of hundreds of newly synthesized or misfolded client proteins in an ATP-dependent folding pathway. In a simplified model, Hsp70 first helps load client onto Hsp90, ATP binding results in conformational changes in Hsp90 that result in the closed complex, and then less defined events result in nucleotide hydrolysis, client release and return to the open state. Cochaperones bind and assist Hsp90 during this process. We previously identified a series of yeast Hsp90 mutants that appear to disrupt either the 'loading', 'closing' or 'reopening' events, and showed that the mutants had differing effects on activity of some clients. Here we used those mutants to dissect Hsp90 and cochaperone interactions. Overexpression or deletion of HCH1 had dramatically opposing effects on the growth of cells expressing different mutants, with a phenotypic shift coinciding with formation of the closed conformation. Hch1 appears to destabilize Hsp90-nucleotide interaction, hindering formation of the closed conformation, whereas Cpr6 counters the effects of Hch1 by stabilizing the closed conformation. Hch1 and the homologous Aha1 share some functions, but the role of Hch1 in inhibiting progression through the early stages of the folding cycle is unique. Sensitivity to the Hsp90 inhibitor NVP-AUY922 also correlates with the conformational cycle, with mutants defective in the loading phase being most sensitive and those defective in the reopening phase being most resistant to the drug. Overall, our results indicate that the timing of transition into and out of the closed conformation is tightly regulated by cochaperones. Further analysis will help elucidate additional steps required for progression through the Hsp90 folding cycle and may lead to new strategies for modulating Hsp90 function.

The Distinct Assignments for Hsp90α and Hsp90β: More Than Skin Deep.

For decades, the undisputable definition of the cytosolic Hsp90α and hsp90β proteins being evolutionarily conserved, ATP-driven chaperones has ruled basic research and clinical trials. The results of recent studies, however, have fundamentally challenged this paradigm, not to mention the spectacular failures of the paradigm-based clinical trials in cancer and beyond. We now know that Hsp90α and Hsp90β are both ubiquitously expressed in all cell types but assigned for distinct and irreplaceable functions. Hsp90β is essential during mouse development and Hsp90α only maintains male reproductivity in adult mice. Neither Hsp90β nor Hsp90α could substitute each other under these biological processes. Hsp90β alone maintains cell survival in culture and Hsp90α cannot substitute it. Hsp90α also has extracellular functions under stress and Hsp90β does not. The dramatic difference in the steady-state expression of Hsp90 in different mouse organs is due to the variable expressions of Hsp90α. The lowest expression of Hsp90 is less than 2% and the highest expression of Hsp90 is 9% among non-transformed cell lines. The two linker regions only take up less than 5% of the Hsp90 proteins, but harbor 21% of the total amino acid substitutions, i.e., 40% in comparison to the 86% overall amino acid homology. A full understanding of the distinctions between Hsp90α and Hsp90β could lead to new, safe and effective therapeutics targeting Hsp90 in human disorders such as cancer. This is the first comprehensive review of a comparison between the two cytosolic Hsp90 isoforms.

Translational reprogramming in response to accumulating stressors ensures critical threshold levels of Hsp90 for mammalian life

The cytosolic molecular chaperone Hsp90 is essential for eukaryotic life. Although reduced Hsp90 levels correlate with aging, it was unknown whether eukaryotic cells and organisms can tune the basal Hsp90 levels to alleviate physiologically accumulated stress. We have investigated whether and how mice adapt to the deletion of three out of four alleles of the two genes encoding cytosolic Hsp90, with one Hsp90β allele being the only remaining one. While the vast majority of such mouse embryos die during gestation, survivors apparently manage to increase their Hsp90β protein to at least wild-type levels. Our studies reveal an internal ribosome entry site in the 5’ untranslated region of the Hsp90β mRNA allowing translational reprogramming to compensate for the genetic loss of Hsp90 alleles and in response to stress. We find that the minimum amount of total Hsp90 required to support viability of mammalian cells and organisms is 50–70% of what is normally there. Those that fail to maintain a threshold level are subject to accelerated senescence, proteostatic collapse, and ultimately death. Therefore, considering that Hsp90 levels can be reduced ≥100-fold in the unicellular budding yeast, critical threshold levels of Hsp90 have markedly increased during eukaryotic evolution.

A novel probe to assess cytosolic entry of exogenous proteins

Dendritic cells use a specialized pathway called cross-presentation to activate CD8+ T cells by presenting peptides from exogenous protein antigens on major histocompatibility complex class I molecules. Considerable evidence suggests that internalized antigens cross endocytic membranes to access cytosolic proteasomes for processing. The mechanism of protein dislocation represents a major unsolved problem. Here we describe the development of a sensitive reporter substrate, an N-glycosylated variant of Renilla luciferase fused to the Fc region of human IgG1. The luciferase variant is designed to be enzymatically inactive when glycosylated, but active after the asparagine to aspartic acid conversion that occurs upon deglycosylation by the cytosolic enzyme N-glycanase-1. The generation of cytosolic luminescence depends on internalization, deglycosylation, the cytosolic AAA-ATPase VCP/p97, and the cytosolic chaperone HSP90. By incorporating a T cell epitope into the fusion protein, we demonstrate that antigen dislocation into the cytosol is the rate-limiting step in cross-presentation. Currently, we are using this novel probe to determine the efficiency of the cytosolic entry of nanoparticles that co-encapsulate ddRLuc-Fc and mRNA. It will provide us with important information about mRNA delivery by those nanoparticles, potentially leading to a breakthrough in mRNA vaccine development.

Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.

Cytosolic Hsp90 Isoform-Specific Functions and Clinical Significance.

The heat shock protein 90 (Hsp90) is a molecular chaperone and a key regulator of proteostasis under both physiological and stress conditions. In mammals, there are two cytosolic Hsp90 isoforms: Hsp90α and Hsp90β. These two isoforms are 85% identical and encoded by two different genes. Hsp90β is constitutively expressed and essential for early mouse development, while Hsp90α is stress-inducible and not necessary for survivability. These two isoforms are known to have largely overlapping functions and to interact with a large fraction of the proteome. To what extent there are isoform-specific functions at the protein level has only relatively recently begun to emerge. There are studies indicating that one isoform is more involved in the functionality of a specific tissue or cell type. Moreover, in many diseases, functionally altered cells appear to be more dependent on one particular isoform. This leaves space for designing therapeutic strategies in an isoform-specific way, which may overcome the unfavorable outcome of pan-Hsp90 inhibition encountered in previous clinical trials. For this to succeed, isoform-specific functions must be understood in more detail. In this review, we summarize the available information on isoform-specific functions of mammalian Hsp90 and connect it to possible clinical applications.

The APE2 Exonuclease Is a Client of the Hsp70-Hsp90 Axis in Yeast and Mammalian Cells.

Molecular chaperones such as Hsp70 and Hsp90 help fold and activate proteins in important signal transduction pathways that include DNA damage response (DDR). Previous studies have suggested that the levels of the mammalian APE2 exonuclease, a protein critical for DNA repair, may be dependent on chaperone activity. In this study, we demonstrate that the budding yeast Apn2 exonuclease interacts with molecular chaperones Ssa1 and Hsp82 and the co-chaperone Ydj1. Although Apn2 does not display a binding preference for any specific cytosolic Hsp70 or Hsp90 paralog, Ssa1 is unable to support Apn2 stability when present as the sole Ssa in the cell. Demonstrating conservation of this mechanism, the exonuclease APE2 also binds to Hsp70 and Hsp90 in mammalian cells. Inhibition of chaperone function via specific small molecule inhibitors results in a rapid loss of APE2 in a range of cancer cell lines. Taken together, these data identify APE2 and Apn2 as clients of the chaperone system in yeast and mammalian cells and suggest that chaperone inhibition may form the basis of novel anticancer therapies that target APE2-mediated processes.

TRAP1 Chaperones the Metabolic Switch in Cancer.

Mitochondrial function is dependent on molecular chaperones, primarily due to their necessity in the formation of respiratory complexes and clearance of misfolded proteins. Heat shock proteins (Hsps) are a subset of molecular chaperones that function in all subcellular compartments, both constitutively and in response to stress. The Hsp90 chaperone TNF-receptor-associated protein-1 (TRAP1) is primarily localized to the mitochondria and controls both cellular metabolic reprogramming and mitochondrial apoptosis. TRAP1 upregulation facilitates the growth and progression of many cancers by promoting glycolytic metabolism and antagonizing the mitochondrial permeability transition that precedes multiple cell death pathways. TRAP1 attenuation induces apoptosis in cellular models of cancer, identifying TRAP1 as a potential therapeutic target in cancer. Similar to cytosolic Hsp90 proteins, TRAP1 is also subject to post-translational modifications (PTM) that regulate its function and mediate its impact on downstream effectors, or ‘clients’. However, few effectors have been identified to date. Here, we will discuss the consequence of TRAP1 deregulation in cancer and the impact of post-translational modification on the known functions of TRAP1.

HSP70 and HSP90 in Cancer: Cytosolic, Endoplasmic Reticulum and Mitochondrial Chaperones of Tumorigenesis.

HSP70 and HSP90 are two powerful chaperone machineries involved in survival and proliferation of tumor cells. Residing in various cellular compartments, HSP70 and HSP90 perform specific functions. Concurrently, HSP70 and HSP90 homologs may also translocate from their primary site under various stress conditions. Herein, we address the current literature on the role of HSP70 and HSP90 chaperone networks in cancer. The goal is to provide a comprehensive review on the functions of cytosolic, mitochondrial and endoplasmic reticulum HSP70 and HSP90 homologs in cancer. Given that high expression of HSP70 and HSP90 enhances tumor development and associates with tumor aggressiveness, further understanding of HSP70 and HSP90 chaperone networks may provide clues for the discoveries of novel anti-cancer therapies.

Designing HSP90 inhibitor drugs against mutations profiles of cancer patients

Background: Effective drug binding to its specific receptor determines the therapeutic effect. Tumor cells employ Heat Shock Protein 90 (Hsp90) to survive as Hsp90 prevents apoptosis. The protein is the top expressed protein among others. This expression rate further increases in tumor cells and provides a fine target for drug design. However, mutations and copy number variations, single nucleotide polymorphisms of the target gene as well as strong binding of therapeutic agents to the target provide adverse effects. Aims: This work investigates HSP90 interaction with common inhibitor geldanamycin through key mutations to understand possible effects. Methods: In silico analysis by YASARA software was employed to reveal interactions between HSP90 WT and its mutant along with geldanamycin. Results: One key inhibitor-resistant critical residue interaction was monitored for each cytosolic HSP90 isoform. The residues are at critical positions during conformational changes and at the mutated regions of the protein. However, finely designed geldanamycin still tolerates its binding to HSP90. Conclusion: HSP has no introns and may seem non-mutable, but designing inhibitors by considering structural alterations increases the effectiveness of the drug candidates and limits side effects through in silico experiments.

Cytosolic and Mitochondrial Hsp90 in Cytokinesis, Mitochondrial DNA Replication, and Drug Action in Trypanosoma brucei.

Trypanosoma brucei subspecies cause African sleeping sickness in humans, an infection that is commonly fatal if not treated, and available therapies are limited. Previous studies have shown that heat shock protein 90 (Hsp90) inhibitors have potent and vivid activity against bloodstream-form trypanosomes. Hsp90s are phylogenetically conserved and essential catalysts that function at the crux of cell biology, where they ensure the proper folding of proteins and their assembly into multicomponent complexes. To assess the specificity of Hsp90 inhibitors and further define the role of Hsp90s in African trypanosomes, we used RNA interference (RNAi) to knock down cytosolic and mitochondrial Hsp90s (HSP83 and HSP84, respectively). Loss of either protein led to cell death, but the phenotypes were distinctly different. Depletion of cytosolic HSP83 closely mimicked the consequences of chemically depleting Hsp90 activity with inhibitor 17-AAG. In these cells, cytokinesis was severely disrupted, and segregation of the kinetoplast (the massive mitochondrial DNA structure unique to this family of eukaryotic pathogens) was impaired, leading to cells with abnormal kinetoplast DNA (kDNA) structures. Quite differently, knockdown of mitochondrial HSP84 did not impair cytokinesis but halted the initiation of new kDNA synthesis, generating cells without kDNA. These findings highlight the central role of Hsp90s in chaperoning cell cycle regulators in trypanosomes, reveal their unique function in kinetoplast replication, and reinforce their specificity and value as drug targets.

The TPR- and J-domain-containing proteins DJC31 and DJC62 are involved in abiotic stress responses in Arabidopsis thaliana.

Molecular chaperones play an important role during the response to different stresses. Since plants are sessile organisms, they need to be able to adapt quickly to different conditions. To do so, plants possess a complex chaperone machinery, composed of HSP70, HSP90, J proteins and other factors. In this study we characterized DJC31 (also known as TPR16) and DJC62 (also known as TPR15) of Arabidopsis thaliana, two J proteins that additionally carry clamp-type tetratricopeptide repeat domains. Using cell fractionation and split GFP, we could show that both proteins are attached to the cytosolic side of the endoplasmic reticulum membrane. Moreover, an interaction with cytosolic HSP70.1 and HSP90.2 could be shown using bimolecular fluorescence complementation. Knockout of both DJC31 and DJC62 caused severe defects in growth and development, which affected almost all organs. Furthermore, it could be shown that the double mutant is more sensitive to osmotic stress and treatment with abscisic acid, but surprisingly exhibited enhanced tolerance to drought. Taken together, these findings indicate that DJC31 and DJC62 might act as important regulators of chaperone-dependent signaling pathways involved in plant development and stress responses.

Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis

The Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.