Hsp90 Activity Research Articles

Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Corneal Endothelial Cell Proliferation

We have performed clinical research on cell-based therapy for corneal endothelial decompensation since 2013. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for promoting proliferation of human corneal endothelial cells (HCECs). HCECs were cultured in media supplemented with various low-molecular-weight compounds to screen for the effect of those compounds on cell proliferation. Activation of substrates of p38 MAPK and cell cycle regulatory proteins were evaluated by western blotting. Corneal endothelial wounds were created in a rabbit model, and p38 MAPK was applied in eye drop form, followed by evaluation of cell proliferation in the corneal endothelium by Ki67-immunostaining. HCECs cultured with SB203580 exhibited hexagonal morphology and similar size and morphology, whereas control HCECs cultured without inhibitor exhibited monolayer morphology and varied in size and morphology. Flow cytometry demonstrated that cell proliferation was significantly increased by SB203580. Western blotting showed activation of ATF2 and HSP27 (substrates of p38 MAPK), and upregulation of cyclin D and downregulation of p27 were induced by inhibiting p38 MAPK. In the rabbit model, promotion of wound healing of the corneal endothelium was associated with significant upregulation of Ki67-positive proliferating cells following topical administration of SB203580 when compared with untreated endothelium (50.9% and 36.1%, respectively). Activation of p38 MAPK signaling due to culture stress might suppress the proliferation of HCECs, whereas a p38 MAPK inhibitor can counteract this activation and enable efficient in vitro HCEC expansion.

Stimulation of the ATPase activity of Hsp90 by zerumbone modification of its cysteine residues destabilizes its clients and causes cytotoxicity.

Hsp90 is an ATP-dependent molecular chaperone that assists folding and conformational maturation/maintenance of many proteins. It is a potential cancer drug target because it chaperones oncoproteins. A prokaryotic homolog of Hsp90 (HtpG) is essential for thermo-tolerance in some bacteria and virulence of zoonotic pathogens. To identify a new class of small molecules which target prokaryotic and eukaryotic Hsp90s, we studied the effects of a naturally occurring cyclic sesquiterpene, zerumbone, which inhibits proliferation of a wide variety of tumor cells, on the activity of Hsp90. Zerumbone enhanced the ATPase activity of cyanobacterial Hsp90 (Hsp90SE), yeast Hsp90, and human Hsp90α. It also enhanced the catalytic efficiency of Hsp90SE by greatly increasing kcat Mass analysis showed that zerumbone binds to cysteine side chains of Hsp90SE covalently. Mutational studies identified 3 cysteine residues (one per each domain of Hsp90SE) that are involved in the enhancement, suggesting the presence of allosteric sites in the middle and C-terminal domains of Hsp90SE Treatment of cyanobacterial cells with zerumbone caused them to become very temperature-sensitive, a phenotype reminiscent of cyanobacterial Hsp90 mutants, and also decreased the cellular level of linker polypeptides that are clients for Hsp90SE Zerumbone showed cellular toxicity on cancer-derived mammalian cells by inducing apoptosis. In addition, zerumbone inhibited the binding of Hsp90/Cdc37 to client kinases. Altogether, we conclude that modification of cysteine residues of Hsp90 by zerumbone enhances its ATPase activity and inhibits physiological Hsp90 function. The activation of Hsp90 may provide new strategies to inhibit its chaperone function in cells.

Heat shock protein 70 and anti-heat shock protein 70 antibodies in patients with chronic glomerulonephritis.

We evaluated the heat shock system 70 (HSP70) in patients with chronic glomerulonephritis (CGN). Seventy-six patients with CGN patients were included in our study. Ten patients with mild proteinuria (median 0.48 [0.16-0.78] g/24h) and ten healthy subjects served as positive and negative controls, respectively. Urinary levels of HSP70, interleukin-10, and serum levels of anti-HSP70 were measured by ELISA. The immunohistochemical peroxidase method was used to study the expression of HSP70 and Foxp3+ in kidney biopsies. TregFoxP3+ cells in the interstitium were determined morphometrically. Median urinary HSP70 levels in patients with nephrotic syndrome (NS) [6.57 (4.49-8.33) pg/mg] and subnephrotic range proteinuria [5.7 (4.12-6.9) pg/mg] were higher (p < 0.05) than in positive [3.7 (2.5-4.82) pg/mg] and negative [3.78 (2.89-4.84) pg/mg] controls. HSP70 expression index in tubular cells positively correlated with urinary HSP70 (Rs = 0.948, р < 0.05) and proteinuria (Rs = 0.362, p < 0.05). The number of TregFoxp3+ cells in the kidney interstitium and interleukin-10 excretion were lower in patients with NS. Anti-HSP70 antibody serum levels in patients with NS [21.1 (17.47-29.72) pg/ml] and subnephrotic range proteinuria [24.9 (18.86-30.92) pg/ml] were significantly higher than in positive [17.8 (12.95-23.03) pg/ml] and negative [18.9 (13.5-23.9) pg/ml] controls. In patients with CGN, increasing proteinuria was associated with higher HSP70 renal tissue and urinary levels. However, activation of HSP70 in patients with nephrotic syndrome did not lead to an increase in tissue levels of TregFoxp3+ cells or to the release of IL-10.

PO-504 The impact of KRAS mutations on the cytotoxic effects of afatinib and allitinib in non-small cell lung cancer cell line

IntroductionEGFR alterations (overexpression and mutations) are frequent events in non-small cell lung cancer (NSCLC). In the last years, second-generation EGFR-targeted therapies, such as afatinib such as allitinib were designed, having a potent irreversible inhibitor action of EGFR and other ErbB family members. Besides the EGFR mutation status, there are no predictive biomarkers of response to this new generation of inhibitors.On this context, the aim of our study was to compare the cytotoxic effects of two irreversible anti-EGFR inhibitors in a panel of NSCLC cell line and assess the impact of KRAS mutations in the response to these agents.Material and methodsTotal of 15 NSCLC cell lines were used. Cytotoxicity was assessed by (MTS). According to GI50 score, cell lines were classified into three groups: higly sensitive (HS), moderate sensitive (MS) and resistant (R). Mutational status of EGFR, KRAS, BRAF and PIK3CA was determined by direct sequencing. Sensitive H292 cell line was transfected with KRAS mutations (p.G12D and p.G12S), then profile of MAPK phospho-protein was assessed by RPPA. Subsequently, cytotoxicity, colony formation, migration and invasion were measured. In vivo chorioallantoic membrane assay (CAM) was used to evaluate the impact of KRAS mutations on tumour proliferation.Results and discussionsWe found 2 cell lines with EGFR and 5 with KRAS mutations. GI50 score show that 2 cell lines treated with afatinib and 7 treated with allitinib exhibited a HS phenotype. We observed IC50 values of 0.95±0.17 µM for H292 wild-type, a six times increase (6.56±0.23 µM) in the H292-KRAS-G12D and an eight times increase (8.47±0.15 µM) in the H292-KRAS-G12S cells. Protein array of KRAS mutant cell lines exhibited an AKT, CREB, HSP27, JNK and TOR activation. In addition, colony formation, migration and invasion potential were increased. Cam assay revealed increased tumour perimeter in KRAS mutant cell lines.ConclusionWe demonstrated that allitinb is most potent anti-EGFR agent. Importantly KRAS mutations increase aggressive behaviour and can constitute a potential predictive biomarker of therapy response. Activation of intracellular protein, revelled by array represent a potential target for therapies combination. In additional, KRAS mutant cell lines increased the proliferative potential measured by CAM in vivo model.

Abstract LB-085: A new role for LKB1 to regulate Heat Shock Protein 90 activity

Abstract Approximately 30% of human non-small cell lung cancer (NSCLC) patients harbor a somatic KRAS mutation resulting, in aberrant activation of downstream signaling pathways that control cell proliferation, cell growth, and cell survival. Importantly, alleles of LKB1, a serine/threonine kinase that functions as a tumor suppressor, are somatically inactivated in ~30% of NSCLCs within KRAS-mutant NSCLC. The loss of LKB1 gives rise to aggressive, highly metastatic, and highly drug resistant tumors. We have previously demonstrated that the inactivation of the tumor suppressor lkb1 rendered mutant kras murine NSCLC resistant to targeted agents including BET bromodomain and kinase inhibitors. However, the mechanism by which LKB1 inactivation attenuates the efficacy of the therapies remains elusive. Consequently, investigation of the molecular-basis of drug resistance to formulate effective therapies for this KRAS-mutated and LKB1-inactivated (KRAS/LKB1) subset of patients is warranted. Using lung adenocarcinoma cells that lack LKB1 expression, we have established isogenic cells that express vector control, wild-type LKB1, or kinase-dead LKB1. Our preliminary data using these isogenic cell lines suggest the previously unrealized function of LKB1 to activate HSP90 to stabilize client signaling proteins. Furthermore, we have performed p23 immunoprecipitation (IP) followed by Western blot for HSP90 to assess the HSP90 ATPase activity. In the cells ectopically expressing wild-type LKB1, p23 co-immunoprecipitated with HSP90 showing that HSP90 is in active conformation. In contrast, few HSP90 co-immunoprecipitated with p23 in the cells that express the kinase-dead version of LKB1 or vector control, demonstrating that little HSP90 is in active conformation without LKB1 kinase activity. Using NetworKIN, a web-based bioinformatic analysis, we find putative LKB1 phosphorylation motifs on HSP90. These results suggest that LKB1 phosphorylates and activates HSP90. Consequently, the inactivation of LKB1 contributes to the destabilization of key HSP90 client signaling proteins and inadvertently promotes compensatory mechanisms to stabilize the select signaling molecules. Using our LKB1-isogenic cell lines, we demonstrated that LKB1 activates HSP90 to stabilize RAF1, resulting in MAPK pathway activation. In contrast, LKB1 inactivation led to reduced HSP90 activity, which reduced MAPK activity and elevated IGF-1R-mediated phosphatidylinositol 3-kinase (PI3-K) activity in vivo and in vitro models of mutant KRAS NSCLC. The aberrant activation of PI3-K pathway augments survival by the stabilization of the anti-apoptotic molecule, MCL1. The use of HSP90 inhibitors does not fully inhibit MAPK or PI3-K pathways due to the compensatory mechanisms chaperoning the IGF-1R axis. Importantly, we confirmed that canonical LKB1-AMPK-mTOR pathway does not fully control PI3K or MAPK signaling. Taken together, our data supports the presence of an HSP90-independent mechanism to stabilize kinases, including IGF-1R, to make KRAS/LKB1 NSCLC resistant to targeted therapies. Citation Format: Jeffrey H. Becker, Michael P. Kahle, Margaret Soucheray, Ines Pulido, Fatima Al-Shahrour, Manuel Sanchez del Pino, Kwok-Kin Wong, Julian Carretero, Takeshi Shimamura. A new role for LKB1 to regulate Heat Shock Protein 90 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-085.

Abstract 2697: Alterations in the HSP70 interactome are induced by overexpression of MUC1 in pancreatic cancer

Abstract Heat Shock Protein 70 (HSP70) is an evolutionarily well-conserved molecular chaperone involved in several cellular processes such as folding of proteins, modulating protein-protein interactions and transport of proteins across membranes. HSP70 works synergistically with a series of chaperones, co-chaperones, adaptors, and folding enzymes known as the HSP70 “interactome”. Previous work has demonstrated that HSP70 interactome changes can be promoted by overexpression of oncoproteins. An important “client” of HSP70 is the oncoprotein MUC1. MUC1 is overexpressed and promotes drug resistance in pancreatic cancer. We applied quantitative affinity-purification mass spectrometry (AP-MS) to characterize the HSP70 interactome differences between PC3/NEO and PC3/MUC1 cell lines. 439 HSP70-interacting proteins were identified and quantified via LC-MS/MS. Interestingly while 53% of HSP70 interactions remained constant upon MUC1 overexpression, 29% significantly increased and 18% decreased. Many of the altered proteins were key co-chaperones that regulate and maintain HSP70 activity. High levels of MUC1 promoted HSP70 association with 15 co-chaperones (HSPA5, DNAJB1, STIP1, TXNDC5, HSPB1, CCT2, TCP1, P4HB, CCT6A, CCT8, PDIA3 and HSP90B). Conversely, MUC1 overexpression triggered the loss of HSP70 interaction with only 2 co-chaperones (PPIB and DNAJA2). We are currently validating these altered interactions using an array of biochemical techniques. Our hope is that by understanding these HSP70 interactome changes, we may be able to uncover novel ways to inhibit MUC1 function in pancreatic cancer. Citation Format: . Nitika, Donald J. Wolfgeher, Pinku Mukherjee, Andrew W. Truman. Alterations in the HSP70 interactome are induced by overexpression of MUC1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2697.

Abstract 2325: Protein homeostasis adaptation to Hsp70 inhibition in cancer

Abstract Cancer cells experience acute stress conditions due to their increased proliferation rate and synthesis of misfolded proteins. However, by modulating the mechanisms that control protein-folding, cancer cells avoid cell death. Thus, it is perhaps not surprising that molecular chaperones, like Hsp70, are upregulated in cancer cells compared to their normal counterparts. These data suggest that these chaperones are potential targets for cancer therapy. Previous work in our lab demonstrated the dependence of patient-driven rhabdomyosarcoma cell survival on cytoplasmic Hsp70 activity, thanks to the use of a specific Hsp70 inhibitor, MAL3-101. In particular, we discovered that treatment of on RMS13 cell line with an Hsp70 inhibitor, known as MAL3-101, activates the PERK driven unfolded protein response that results in CHOP-dependent cell death (Sabinis et al., 2016). Nevertheless, the mechanism underlying how Hsp70 inhibition triggered apoptosis was poorly understood. By taking advantage of a MAL3-101-resistant cell line (RMS13-R), we have now determined which compensatory mechanism alters MAL3-101-driven cell death. We found that both endoplasmic reticulum-associated degradation (ERAD) and autophagy are upregulated in RMS13-R cells, underlying the increased demand on two protein degradation pathways upon inhibition of Hsp70. Specifically, autophagy related genes were upregulated, and increased conversion of LC3BI to LC3BII and accumulation of LC3BII was detected in RMS13-R cells. Further experiments demonstrated that autophagy was further induced by MAL3-101 treatment in RMS13-R cells, as evidenced by an increase in the messages and proteins corresponding to key autophagy components. Finally, we discovered that only autophagy inhibition—but not inhibition of ERAD—re-sensitized RMS13-R cells to Hsp70 inhibition, which was apparent from an induction of apoptotic markers and cell death. These data highlight a pro-survival role for autophagy induction upon exposure to an Hsp70 inhibitor in cancer, and provide a link between Hsp70, proteasomal degradation, the unfolded protein response, and autophagy in rhabdomyosarcoma. Citation Format: Sara Sannino, Christopher J. Guerriero, Amit J. Sabnis, Trever G. Bivona, Jeffrey L. Brodsky. Protein homeostasis adaptation to Hsp70 inhibition in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2325.

Herpes Simplex Virus 1 Inhibits TANK-Binding Kinase 1 through Formation of the Us11-Hsp90 Complex.

The Us11 protein of herpes simplex virus 1 (HSV-1) is an accessory factor with multiple functions. In virus-infected cells, it inhibits double-stranded RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase, RIG-I, and MDA-5. However, its precise role is incompletely defined. By screening a human cDNA library, we showed that the Us11 protein targets heat shock protein 90 (Hsp90), which inactivates TANK binding kinase 1 (TBK1) and antiviral immunity. When ectopically expressed, HSV-1 Us11 precludes TBK1 from access to Hsp90 and interferon (IFN) promoter activation. Consistently, the Us11 protein, upon HSV infection, suppresses the expression of beta interferon (IFN-β), RANTES, and interferon-stimulated genes. This is mirrored by a blockade in the phosphorylation of interferon regulatory factor 3. Mechanistically, the Us11 protein associates with endogenous Hsp90 to disrupt the Hsp90-TBK1 complex. Furthermore, Us11 induces destabilization of TBK1 through a proteasome-dependent pathway. Accordingly, Us11 expression facilitates HSV growth. In contrast, TBK1 expression restricts viral replication. These results suggest that control of TBK1 by Us11 promotes HSV-1 infection.IMPORTANCE TANK binding kinase 1 plays a key role in antiviral immunity. Although multiple factors are thought to participate in this process, the picture is obscure in herpes simplex virus infection. We demonstrated that the Us11 protein of HSV-1 forms a complex with heat shock protein 90, which inactivates TANK binding kinase 1 and IFN induction. As a result, expression of the Us11 protein promotes HSV replication. These experimental data provide a new insight into the molecular network of virus-host interactions.

Cytosolic Hsp70 and Hsp40 chaperones enable the biogenesis of mitochondrial β-barrel proteins.

Mitochondrial β-barrel proteins are encoded in the nucleus, translated by cytosolic ribosomes, and then imported into the organelle. Recently, a detailed understanding of the intramitochondrial import pathway of β-barrel proteins was obtained. In contrast, it is still completely unclear how newly synthesized β-barrel proteins reach the mitochondrial surface in an import-competent conformation. In this study, we show that cytosolic Hsp70 chaperones and their Hsp40 cochaperones Ydj1 and Sis1 interact with newly synthesized β-barrel proteins. These interactions are highly relevant for proper biogenesis, as inhibiting the activity of the cytosolic Hsp70, preventing its docking to the mitochondrial receptor Tom70, or depleting both Ydj1 and Sis1 resulted in a significant reduction in the import of such substrates into mitochondria. Further experiments demonstrate that the interactions between β-barrel proteins and Hsp70 chaperones and their importance are conserved also in mammalian cells. Collectively, this study outlines a novel mechanism in the early events of the biogenesis of mitochondrial outer membrane β-barrel proteins.

HSC70 is a chaperone for wild-type and mutant cardiac myosin binding protein C.

Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover.

Heptamer Peptide Disassembles Native Amyloid in Human Plasma Through Heat Shock Protein 70.

Proteostasis, which includes the repair and disposal of misfolded proteins, depends, in part, on the activity of heat shock proteins (HSPs), a well-known class of chaperone molecules. When this process fails, abnormally folded proteins may accumulate in cells, tissues, and blood. These species are a hallmark of protein aggregation diseases, but also amass during aging, often in the absence of an identified clinical disorder. We report that a neuroprotective cyclic heptapeptide, CHEC-7, which has been applied systemically as a therapeutic in animal neurodegeneration models, disrupts such aggregates and inhibits amyloidogenesis when added in nanomolar concentrations to human plasma. This effect includes aggregates of amyloid beta (Aβ1-40, 1-42), prominent features of Alzheimer's disease pathology. The activity of endogenous HSP70, a recently discovered target of the peptide, is required as demonstrated by both antibody blocking and application of pifithrin-μ, an HSP70 inhibitor. CHEC-7 is the first high-affinity compound to stimulate HSP70's disaggregase activity and therefore enable this endogenous mechanism in a human systemic environment, increasing the likelihood of a convenient therapy for protein aggregate disease, including age-related failures of protein repair.

Andrographolide induces degradation of mutant p53 via activation of Hsp70

The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein(Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.

Low-intensity pulsed ultrasound stimulation facilitates in vitro osteogenic differentiation of human adipose-derived stem cells via up-regulation of heat shock protein (HSP)70, HSP90, and bone morphogenetic protein (BMP) signaling pathway.

Low-intensity pulsed ultrasound (LIPUS) has positive effects on osteogenic differentiation. However, the effect of LIPUS on osteogenic differentiation of human adipose-derived stem cells (hASCs) is unclear. In the present study, we investigated whether LIPUS could promote the proliferation and osteogenic differentiation of hASCs. hASCs were isolated and osteogenically induced with LIPUS stimulation at 20 and 30 mW cm−2 for 30 min day−1. Cell proliferation and osteogenic differentiation potential of hASCs were respectively analyzed by cell counting kit-8 assay, Alizarin Red S staining, real-time polymerase chain reaction, and Western blotting. The results indicated that LIPUS stimulation did not significantly affect the proliferation of hASCs, but significantly increased their alkaline phosphatase activity on day 6 of culture and markedly promoted the formation of mineralized nodules on day 21 of culture. The mRNA expression levels of runt-related transcription factor, osteopontin, and osteocalcin were significantly up-regulated by LIPUS stimulation. LIPUS stimulation did not affect the expression of heat shock protein (HSP) 27, HSP40, bone morphogenetic protein (BMP)-6 and BMP-9, but significantly up-regulated the protein levels of HSP70, HSP90, BMP-2, and BMP-7 in the hASCs. Further studies found that LIPUS increased the mRNA levels of Smad 1 and Smad 5, elevated the phosphorylation of Smad 1/5, and suppressed the expression of BMP antagonist Noggin. These findings indicated that LIPUS stimulation enhanced osteogenic differentiation of hASCs possibly through the up-regulation of HSP70 and HSP90 expression and activation of BMP signaling pathway. Therefore, LIPUS might have the potential to promote the repair of bone defect.

Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide

Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104 M−1 s−1, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.

Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides

Proteostasis is critical to maintain organismal viability, a process counteracted by aging-dependent protein aggregation. Chaperones of the heat shock protein (HSP) family help control proteostasis by reducing the burden of unfolded proteins. They also oversee the formation of protein aggregates. Here, we explore how AMPylation, a posttranslational protein modification that has emerged as a powerful modulator of HSP70 activity, influences the dynamics of protein aggregation. We find that adjustments of cellular AMPylation levels in Caenorhabditis elegans directly affect aggregation properties and associated toxicity of amyloid-β (Aβ), of a polyglutamine (polyQ)-extended polypeptide, and of α-synuclein (α-syn). Expression of a constitutively active C. elegans AMPylase FIC-1(E274G) under its own promoter expedites aggregation of Aβ and α-syn, and drastically reduces their toxicity. A deficiency in AMPylation decreases the cellular tolerance for aggregation-prone polyQ proteins and alters their aggregation behavior. Overexpression of FIC-1(E274G) interferes with cell survival and larval development, underscoring the need for tight control of AMPylase activity in vivo. We thus define a link between HSP70 AMPylation and the dynamics of protein aggregation in neurodegenerative disease models. Our results are consistent with a cytoprotective, rather than a cytotoxic, role for such protein aggregates.

A Rationally Designed Hsp70 Variant Rescues the Aggregation-Associated Toxicity of Human IAPP in Cultured Pancreatic Islet β-Cells.

Molecular chaperones are key components of the protein homeostasis system against protein misfolding and aggregation. It has been recently shown that these molecules can be rationally modified to have an enhanced activity against specific amyloidogenic substrates. The resulting molecular chaperone variants can be effective inhibitors of protein aggregation in vitro, thus suggesting that they may provide novel opportunities in biomedical and biotechnological applications. Before such opportunities can be exploited, however, their effects on cell viability should be better characterised. Here, we employ a rational design method to specifically enhance the activity of the 70-kDa heat shock protein (Hsp70) against the aggregation of the human islet amyloid polypeptide (hIAPP, also known as amylin). We then show that the Hsp70 variant that we designed (grafted heat shock protein 70 kDa-human islet amyloid polypeptide, GHsp70-hIAPP) is significantly more effective than the wild type in recovering the viability of cultured pancreatic islet β-cells RIN-m5F upon hIAPP aggregation. These results indicate that a full recovery of the toxic effects of hIAPP aggregates on cultured pancreatic cells can be achieved by increasing the specificity and activity of Hsp70 towards hIAPP, thus providing evidence that the strategy presented here provides a possible route for rationally tailoring molecular chaperones for enhancing their effects in a target-dependent manner.

Intra-molecular pathways of allosteric control in Hsp70s.

The 70 kDa heat-shock protein (Hsp70) is undoubtedly the most versatile of all molecular chaperones. Hsp70 is involved in numerous cellular protein folding processes, accompanying proteins throughout their lifespan from de novo folding at the ribosome to degradation at the proteasome, surveilling protein stability and functionality. Several properties of this ATP-dependent chaperone constitute the molecular basis for this versatility. With its substrate binding domain (SBD), Hsp70 transiently interacts with a short degenerative linear sequence motif found practically in all proteins and, in addition, with more folded protein conformers. Binding to polypeptides is tightly regulated by ATP binding and hydrolysis in the nucleotide binding domain, which is coupled to the SBD by an intricate allosteric mechanism. Hsp70 is regulated by a host of J-cochaperones, which act as targeting factors by regulating the ATPase activity of Hsp70 in synergism with the substrates themselves, and by several families of nucleotide exchange factors. In this review, I focus on the allosteric mechanism, which allows Hsp70s to interact with substrates with ultrahigh affinity through a non-equilibrium mode of action and summarize what mutagenesis and structural studies have taught us about the pathways and mechanics of interdomain communication.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

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

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

The Molecular Chaperone HSP90 Promotes Notch Signaling in the Germline of Caenorhabditis elegans.

In a genetic screen to identify genes that promote GLP-1/Notch signaling in Caenorhabditis elegans germline stem cells, we found a single mutation, om40, defining a gene called ego-3. ego-3(om40) causes several defects in the soma and the germline, including paralysis during larval development, sterility, delayed proliferation of germline stem cells, and ectopic germline stem cell proliferation. Whole genome sequencing identified om40 as an allele of hsp-90, previously known as daf-21, which encodes the C. elegans ortholog of the cytosolic form of HSP90. This protein is a molecular chaperone with a central position in the protein homeostasis network, which is responsible for proper folding, structural maintenance, and degradation of proteins. In addition to its essential role in cellular function, HSP90 plays an important role in stem cell maintenance and renewal. Complementation analysis using a deletion allele of hsp-90 confirmed that ego-3 is the same gene. hsp-90(om40) is an I→N conservative missense mutation of a highly conserved residue in the middle domain of HSP-90. RNA interference-mediated knockdown of hsp-90 expression partially phenocopied hsp-90(om40), confirming the loss-of-function nature of hsp-90(om40). Furthermore, reduced HSP-90 activity enhanced the effect of reduced function of both the GLP-1 receptor and the downstream LAG-1 transcription factor. Taken together, our results provide the first experimental evidence of an essential role for HSP90 in Notch signaling in development.

Phosphoproteomics analyses reveal that phosphorylation of heat shock protein 70 (hsp70) by protein kinase B (Akt1) on ser631 controls activity of mitochondrial superoxide‐2 (SOD2).

BackgroundSOD2 is a nuclear encoded gene whose product must be imported into the mitochondria for activation. Previously, we showed that hsp70 chaperones SOD2 to the mitochondria. The chaperone activity of hsp70 is negatively regulated by the C‐terminus of hsp70‐interacting protein (CHIP). However, the mechanism that controls SOD2 activity proportional to activating signals to maintain mitochondrial redox balance is unknown.MethodsTo identify the signaling protein that regulates activity of hsp70, HEK‐293T cells were transfected with GST‐tagged‐hsp70. The GST epitope was immuno‐precipitated and the differential band was sent for mass spectrometry and Akt1 was identified. To examine the contributions of Akt1 to SOD2 induction in response to stimuli, we used PI3K inhibitor, Ly294002 treatment as well as Akt1‐siRNA knockdown methods. Pulmonary artery endothelial cells (PAECs) were transfected with Akt1‐siRNA and we re‐expressed myristoylated Akt1 or dominant negative Akt1. We then quantified SOD2 protein and activity in isolated mitochondria using Western blots and colorimetric assays. To identify the amino acid residue (s) in hsp70 that are phosphorylated by Akt1, we incubated recombinant Akt1 proteins with purified hsp70 in an in‐vitro kinase reaction and the band containing phosphorylated hsp70 was sent for phosphoproteomics analysis and site identification using LC‐MS/MS. We characterized the newly identified residue by point mutation and assessed the effect of mutations on SOD2 localization to the mitochondria by hsp70 using immuno‐fluorescent staining. To examined the effect of hsp70 phosphorylation on SOD2 activity in response to stimuli, we knockeddown Akt1 using siRNA and generated global akt1 null mice and measured SOD2 import, and activity and rates of superoxide and hydrogen peroxide levels in cells and lung lysates following stimulation using LC‐MS/MS following mitoSOX/mito‐B stainings. We determined the effect of hsp70 phosphorylation on the interaction between CHIP and hsp70, and the functional relevance of CHIP interaction on SOD2 function using CHIP overexpression, ubiquitin assays and immunoprecipitation methods.ResultsInduction of Akt1 activity enhanced SOD2 activity in PAECs within minutes following PDGF stimulation, reaching a peak at 20 minutes and is then downregulated. Conversely, inhibition of Akt1 activity robustly decreased SOD2 protein and activity in isolated mitochondria and increased mitochondrial O2− formation compared to controls. Re‐expression of myr‐Akt1 restored SOD2 activity whereas DN‐Akt1 failed to improve SOD2 activity. Mechanistically, we demonstrated that Akt1 binds to and phosphorylates hsp70 at ser‐631. Substitution of ser for Ala at position 631 in hsp70 blocked the PDGF mediated induction of SOD2 accumulation in the mitochondria. Phosphorylated hsp70 inhibited the chaperone interaction with CHIP, and conversely, dephosphorylated hsp70 preferentially recruited CHIP and facilitated SOD2 degradation. Treatment with mito‐TEMPO induced PP2A/2C protein expression, inactivated Akt1 and dephosphorylated hsp70‐ser‐631, leading to the eventual termination of SOD2 import.ConclusionAkt1 integrates activation and inhibition signals, links them to hsp70 to regulates SOD2 activity.Support or Funding InformationSupported by NIHKO8 (AJA), Children Research Institute (CRI) and Department of Pediatrics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.