Recruitment of Ahsa1 to Hsp90 is regulated by a conserved peptide that inhibits ATPase stimulation.

Hsp90 is a molecular chaperone that acts on its clients through an ATP-dependent and conformationally dynamic functional cycle. The cochaperone Accelerator of Hsp90 ATPase, or Ahsa1, is the most potent stimulator of Hsp90 ATPase activity. Ahsa1 stimulates the rate of Hsp90 ATPase activity through a conserved motif, NxNNWHW. Metazoan Ahsa1, but not yeast, possesses an additional 20 amino acid peptide preceding the NxNNWHW motif that we have called the intrinsic chaperone domain (ICD). The ICD of Ahsa1 diminishes Hsp90 ATPase stimulation by interfering with the function of the NxNNWHW motif. Furthermore, the NxNNWHW modulates Hsp90's apparent affinity to Ahsa1 and ATP. Lastly, the ICD controls the regulated recruitment of Hsp90 in cells and its deletion results in the loss of interaction with Hsp90 and the glucocorticoid receptor. This work provides clues to how Ahsa1 conserved regions modulate Hsp90 kinetics and how they may be coupled to client folding status.

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 role of cellular proteostasis in antitumor immunity.

Immune checkpoint blockade therapy is perhaps the most important development in cancer treatment in recent memory. It is based on decades of investigation into the biology of immune cells and the role of the immune system in controlling cancer growth. While the molecular circuitry that governs the immune system in general-and antitumor immunity in particular-is intensely studied, far less attention has been paid to the role of cellular stress in this process. Proteostasis, intimately linked to cell stress responses, refers to the dynamic regulation of the cellular proteome and is maintained through a complex network of systems that govern the synthesis, folding, and degradation of proteins in the cell. Disruption of these systems can result in the loss of protein function, altered protein function, the formation of toxic aggregates, or pathologies associated with cell stress. However, the importance of proteostasis extends beyond its role in maintaining proper protein function; proteostasis governs how tolerant cells may be to mutations in protein-coding genes and the overall half-life of proteins. Such gene expression changes may be associated with human diseases including neurodegenerative diseases, metabolic disease, and cancer and manifest at the protein level against the backdrop of the proteostasis network in any given cellular environment. In this review, we focus on the role of proteostasis in regulating immune responses against cancer as well the role of proteostasis in determining immunogenicity of cancer cells.

Structural basis for cargo regulation of COPII coat assembly.

Using cryo-electron microscopy, we have solved the structure of an icosidodecahedral COPII coat involved in cargo export from the endoplasmic reticulum (ER) coassembled from purified cargo adaptor Sec23-24 and Sec13-31 lattice-forming complexes. The coat structure shows a tetrameric assembly of the Sec23-24 adaptor layer that is well positioned beneath the vertices and edges of the Sec13-31 lattice. Fitting the known crystal structures of the COPII proteins into the density map reveals a flexible hinge region stemming from interactions between WD40 beta-propeller domains present in Sec13 and Sec31 at the vertices. The structure shows that the hinge region can direct geometric cage expansion to accommodate a wide range of bulky cargo, including procollagen and chylomicrons, that is sensitive to adaptor function in inherited disease. The COPII coat structure leads us to propose a mechanism by which cargo drives cage assembly and membrane curvature for budding from the ER.

Aha-type co-chaperones: the alpha or the omega of the Hsp90 ATPase cycle?

Heat shock protein 90 (Hsp90) is a dimeric molecular chaperone that plays an essential role in cellular homeostasis. It functions in the context of a structurally dynamic ATP-dependent cycle to promote conformational changes in its clientele to aid stability, maturation, and activation. The client activation cycle is tightly regulated by a cohort of co-chaperone proteins that display specific binding preferences for certain conformations of Hsp90, guiding Hsp90 through its functional ATPase cycle. Aha-type co-chaperones are well-known to robustly stimulate the ATPase activity of Hsp90 but other roles in regulating the functional cycle are being revealed. In this review, we summarize the work done on the Aha-type co-chaperones since the 1990s and highlight recent discoveries with respect to the complexity of Hsp90 cycle regulation.

The Hsp90 Chaperone: 1H and 19F Dynamic Nuclear Magnetic Resonance Spectroscopy Reveals a Perfect Enzyme.

Hsp90 is a crucial chaperone whose ATPase activity is fundamental for stabilizing and activating a diverse array of client proteins. Binding and hydrolysis of ATP by dimeric Hsp90 drive a conformational cycle characterized by fluctuations between a compact, N- and C-terminally dimerized catalytically competent closed state and a less compact open state that is largely C-terminally dimerized. We used 19F and 1H dynamic nuclear magnetic resonance (NMR) spectroscopy to study the opening and closing kinetics of Hsp90 and to determine the kcat for ATP hydrolysis. We derived a set of coupled ordinary differential equations describing the rate laws for the Hsp90 kinetic cycle and used these to analyze the NMR data. We found that the kinetics of closing and opening for the chaperone are slow and that the lower limit for kcat of ATP hydrolysis is ∼1 s-1. Our results show that the chemical step is optimized and that Hsp90 is indeed a "perfect" enzyme.

A novel method for site-specific chemical SUMOylation: SUMOylation of Hsp90 modulates co-chaperone binding in vitro.

SUMO is covalently attached to lysine side chains in target proteins by the action of a cascade of E1, E2, and E3 ligases. Unlike ubiquitin, SUMO does not target proteins for degradation but rather plays a regulatory role in activating target proteins or directing them to multiprotein complexes. Isolating SUMOylated proteins from native sources is challenging because of the low stoichiometry of SUMOylation that occurs for any given target protein in cells. Here we report a novel strategy to couple SUMO to the site of a target lysine for the purpose of in vitro study. Introduction of a single cysteine after the C terminal diglycine motif and a cysteine in place of a target lysine in a substrate protein allows for efficient and specific crosslinking of SUMO using a homo-bifunctional maleimide crosslinker. We demonstrate that SUMO can be crosslinked in this manner to amino acid position 178 in the dimeric molecular chaperone, Hsp90. Chemically SUMOylated Hsp90 has very similar ATPase activity compared to unmodified Hsp90 but displays preferential co-chaperone binding in vivo. Our novel strategy can easily be applied to other SUMOylated or ubiquitinated target protein in vitro.

The activity of the Drosophila Vestigial protein is modified by Scalloped-dependent phosphorylation.

The Drosophila vestigial gene is required for proliferation and differentiation of the adult wing and for differentiation of larval and adult muscle identity. Vestigial is part of a multi-protein transcription factor complex, which includes Scalloped, a TEAD-class DNA binding protein. Binding Scalloped is necessary for translocation of Vestigial into the nucleus. We show that Vestigial is extensively post-translationally modified and at least one of these modifications is required for proper function during development. We have shown that there is p38-dependent phosphorylation of Serine 215 in the carboxyl-terminal region of Vestigial. Phosphorylation of Serine 215 occurs in the nucleus and requires the presence of Scalloped. Comparison of a phosphomimetic and non-phosphorylatable mutant forms of Vestigial shows differences in the ability to rescue the wing and muscle phenotypes associated with a null vestigial allele.

Regulation of RNA interference by Hsp90 is an evolutionarily conserved process.

RNAi is a highly conserved mechanism in almost every eukaryote with a few exceptions including the model organism Saccharomyces cerevisiae. A recent study showed that the introduction of the two core components of canonical RNAi systems, Argonaute and Dicer, from another budding yeast, Saccharomyces castellii, restores RNAi in S. cerevisiae. We report here that a functional RNAi system can be reconstituted in yeast with the introduction of only S. castellii Dicer and human Argonaute2. Interestingly, whether or not TRBP2 was present, human Dicer was unable to restore RNAi with either S. castellii or human Argonaute. Contrary to previous reports, we find that human Dicer, TRBP2 and Argonaute2 are not sufficient to reconstitute RNAi in yeast when bona fide RNAi precursors are co-expressed. We and others have previously reported that Hsp90 regulates conformational changes in human and Drosophila Argonautes required to accommodate the loading of dsRNA duplexes. Here we show that the activities of both human and S. castellii Argonaute are subject to Hsp90 regulation in S. cerevisiae. In summary, our results suggest that regulation of the RNAi machinery by Hsp90 may have evolved at the same time as ancestral RNAi.

Hsp90 inhibition leads to an increase in surface expression of multiple immunological receptors in cancer cells.

Heat shock protein 90 (Hsp90) is a molecular chaperone important for maintaining protein homeostasis (proteostasis) in the cell. Hsp90 inhibitors are being explored as cancer therapeutics because of their ability to disrupt proteostasis. Inhibiting Hsp90 increases surface density of the immunological receptor Major Histocompatibility Complex 1 (MHC1). Here we show that this increase occurs across multiple cancer cell lines and with both cytosol-specific and pan-Hsp90 inhibitors. We demonstrate that Hsp90 inhibition also alters surface expression of both IFNGR and PD-L1, two additional immunological receptors that play a significant role in anti-tumour or anti-immune activity in the tumour microenvironment. Hsp90 also negatively regulates IFN-γ activity in cancer cells, suggesting it has a unique role in mediating the immune system's response to cancer. Our data suggests a strong link between Hsp90 activity and the pathways that govern anti-tumour immunity. This highlights the potential for the use of an Hsp90 inhibitor in combination with another currently available cancer treatment, immune checkpoint blockade therapy, which works to prevent immune evasion of cancer cells. Combination checkpoint inhibitor therapy and the use of an Hsp90 inhibitor may potentiate the therapeutic benefits of both treatments and improve prognosis for cancer patients.

Second Virtual International Symposium on Cellular and Organismal Stress Responses, September 8-9, 2022.

The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8-9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI.

Fluorine- and rhenium-containing geldanamycin derivatives as leads for the development of molecular probes for imaging Hsp90.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone responsible for protein quality control in cells. Hsp90 has been shown to be overexpressed in many human cancers. This has prompted extensive research on Hsp90 inhibitors as novel anticancer agents and, more recently, the development of molecular probes for imaging Hsp90 expression in vivo. This work describes the development of various fluorine-containing and rhenium-containing geldanamycin derivatives as leads for the development of corresponding (18)F-labeled and (99m)Tc-labeled PET and SPECT probes for molecular imaging of Hsp90 expression. All compounds were evaluated in an in vitro ATPase activity assay using Hsp90 isoform Hsp82p. Fluorobenzoylated geldanamycin derivative 5 displayed comparable inhibitory potency like parent compound geldanamycin.

Structure of the Sec13/31 COPII coat cage.

Endomembranes of eukaryotic cells are dynamic structures that are in continuous communication through the activity of specialized cellular machineries, such as the coat protein complex II (COPII), which mediates cargo export from the endoplasmic reticulum (ER). COPII consists of the Sar1 GTPase, Sec23 and Sec24 (Sec23/24), where Sec23 is a Sar1-specific GTPase-activating protein and Sec24 functions in cargo selection, and Sec13 and Sec31 (Sec13/31), which has a structural role. Whereas recent results have shown that Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles, we now show that Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24. We present a three-dimensional reconstruction of these Sec13/31 cages at 30 A resolution using cryo-electron microscopy and single particle analysis. These results reveal a novel cuboctahedron geometry with the potential to form a flexible lattice and to generate a diverse range of containers. Our data are consistent with a model for COPII coat complex assembly in which Sec23/24 has a non-structural role as a multivalent ligand localizing the self-assembly of Sec13/31 to form a cage lattice driving ER cargo export.

A Small Molecule Inhibitor of Pex3-Pex19 Interaction Disrupts Glycosome Biogenesis and Causes Lethality in Trypanosoma brucei.

Trypanosomatid parasites, including Trypanosoma and Leishmania, are infectious zoonotic agents for a number of severe diseases such as African sleeping sickness and American trypanosomiasis (Chagas disease) that affect millions of people, mostly in the emergent world. The glycosome is a specialized member of the peroxisome family of organelles found in trypanosomatids. These organelles compartmentalize essential enzymes of the glycolytic pathway, making them a prime target for drugs that can kill these organisms by interfering with either their biochemical functions or their formation. Glycosome biogenesis, like peroxisome biogenesis, is controlled by a group of proteins called peroxins (Pex). Pex3 is an early acting peroxin that docks Pex19, the receptor for peroxisomal membrane proteins, to initiate biogenesis of peroxisomes from the endoplasmic reticulum. Identification of Pex3 as the essential master regulator of glycosome biogenesis has implications in developing small molecule inhibitors that can impede Pex3-Pex19 interaction. Low amino acid sequence conservation between trypanosomatid Pex3 and human Pex3 (HsPex3) would aid in the identification of small molecule inhibitors that selectively interfere with the trypanosomatid Pex3-Pex19 interaction. We tested a library of pharmacologically active compounds in a modified yeast two-hybrid assay and identified a compound that preferentially inhibited the interaction of Trypanosoma brucei Pex3 and Pex19 versus HsPex3 and Pex19. Addition of this compound to either the insect or bloodstream form of T. brucei disrupted glycosome biogenesis, leading to mislocalization of glycosomal enzymes to the cytosol and lethality for the parasite. Our results show that preferential disruption of trypanosomal Pex3 function by small molecule inhibitors could help in the accelerated development of drugs for the treatment of trypanosomiases.

Enhanced Home Care Interventions for Community Residing Adults Compared With Usual Care on Health and Cost-effectiveness Outcomes: A Systematic Review.

ABSTRACT: The need for home care services is expanding around the world with increased attention to the resources required to produce them. To assist decision making, there is a need to assess the cost-effectiveness of alternative programs within home care. Electronic searches were performed in five databases (before February 2020) identifying 3292 potentially relevant studies that assessed new or enhanced home care interventions compared with usual care for adults with an accompanying economic evaluation. From these, 133 articles were selected for full-text screening; 17 met the inclusion criteria and were analyzed. Six main areas of research were identified including the following: alternative nursing care (n = 4), interdisciplinary care coordination (n = 4), fall prevention (n = 4), telemedicine/remote monitoring (n = 2), restorative/reablement care (n = 2), and one multifactorial undernutrition intervention study. Risk of bias was found to be high/weak (n = 7) or have some concerns/moderate (n = 6) rating, in addition to inconsistent reporting of important information required for economic evaluations. Both health and cost outcomes had mixed results. Cost-effective interventions were found in two areas including alternative nursing care and reablement/restorative care. Clinicians and decision makers are encouraged to carefully evaluate the quality of the studies because of issues with risk of bias and incomplete reporting of economic outcomes.

Structural design of cage and coat scaffolds that direct membrane traffic.

Trafficking within the exocytic and endocytic pathways of eukaryotic cells involves the generation of caged transport carriers that mediate communication between compartments through vesicle budding and fusion. Structural studies of vesicle cage structures using X-ray crystallography and cryo-electron microscopy approaches reveal new insight into cargo-dependent coat assembly mechanisms. Clathrin and coat protein complex II (COPII) use conserved primary element alpha-solenoid and WD40 structural motifs found in self-assembling cage scaffolds to generate unique geometries that sort cargo and produce vesicles. These studies emphasize molecular and structural principles that reflect the properties of self-assembling nanomachines to regulate cargo capacity in trafficking pathways.

The ER chaperone calnexin controls mitochondrial positioning and respiration.

Chaperones in the endoplasmic reticulum (ER) control the flux of Ca2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.

The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation.

Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.

An Hsp90 co-chaperone protein in yeast is functionally replaced by site-specific posttranslational modification in humans.

Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone. To properly chaperone its clientele, Hsp90 proceeds through an ATP-dependent conformational cycle influenced by posttranslational modifications (PTMs) and assisted by a number of co-chaperone proteins. Although Hsp90 conformational changes in solution have been well-studied, regulation of these complex dynamics in cells remains unclear. Phosphorylation of human Hsp90α at the highly conserved tyrosine 627 has previously been reported to reduce client interaction and Aha1 binding. Here we report that these effects are due to a long-range conformational impact inhibiting Hsp90α N-domain dimerization and involving a region of the middle domain/carboxy-terminal domain interface previously suggested to be a substrate binding site. Although Y627 is not phosphorylated in yeast, we demonstrate that the non-conserved yeast co-chaperone, Hch1, similarly affects yeast Hsp90 (Hsp82) conformation and function, raising the possibility that appearance of this PTM in higher eukaryotes represents an evolutionary substitution for HCH1.