Hsp90 Induces Acsl4-dependent Glioma Ferroptosis via Dephosphorylate Ser637 at Drp1

Background: Ferroptosis is a newly identified form of regulated cell death (RCD) characterized by the iron-dependent lipid reactive oxygen species (ROS) accumulation, but its exact mechanism in gliomas remains elusive. Acyl–coenzyme A (CoA) synthetase long-chain family member 4 (Acsl4), a pivotal enzyme in the regulation of lipid biosynthesis, has been found to benefit the initiation of ferroptosis, but its role in gliomas likewise needs clarification. Erastin, widely investigated as an inducer of ferroptosis, was recently found to regulate lipid peroxidation by regulating Acsl4 other than glutathione peroxidase 4 (GPX4) in ferroptosis. Methods: Relationship between Hsp90, Drp1 and Acsl4 was determined by Co-immunoprecipitation/ Mass spectrometry and western blot assay. The impact of Hsp90 and Drp1 on Acsl4-dependent ferroptosis was examined by lipid peroxidation indicators in patient-derived PL1 and PG7 cells. The morphological changes of mitochondria are observed by confocal-fluorescence microscopy and transmission electron microscope. Therapeutic efficacy of Erastin-induced ferroptosis in vivo was examined in xenograft mouse models.Results: In this study, we demonstrated that heat shock protein 90 (Hsp90) and dynamin-related protein 1 (Drp1) actively regulated Acsl4 expression in erastin-induced ferroptosis in gliomas. Hsp90 overexpression and calcineurin (CN)–mediated Drp1 dephosphorylation at serine 637 (Ser637) promoted ferroptosis by altering mitochondrial morphology and increasing Acsl4-mediated lipid peroxidation. Importantly, the Hsp90–Acsl4 pathway mediated Acsl4-dependent ferroptosis, amplifying the anticancer activity of erastin in vitro and in vivo. Conclusions: Our study not only uncovered an important role of Hsp90–Drp1–Acsl4 pathway in erastin-induced ferroptosis but also reveals an efficient mechanism of Acsl4 as a potential therapeutic target to ferroptosis-mediated glioma therapy.

Heat Shock Protein 90 as Therapeutic Target for CVDs and Heart Ageing

Cardiovascular diseases (CVDs) are the leading cause of death globally, representing approximately 32% of all deaths worldwide. Molecular chaperones are involved in heart protection against stresses and age-mediated accumulation of toxic misfolded proteins by regulation of the protein synthesis/degradation balance and refolding of misfolded proteins, thus supporting the high metabolic demand of the heart cells. Heat shock protein 90 (HSP90) is one of the main cardioprotective chaperones, represented by cytosolic HSP90a and HSP90b, mitochondrial TRAP1 and ER-localised Grp94 isoforms. Currently, the main way to study the functional role of HSPs is the application of HSP inhibitors, which could have a different way of action. In this review, we discussed the recently investigated role of HSP90 proteins in cardioprotection, atherosclerosis, CVDs development and the involvements of HSP90 clients in the activation of different molecular pathways and signalling mechanisms, related to heart ageing.

HSP90 Inhibitor 17-AAG Attenuates Nucleus Pulposus Inflammation and Catabolism Induced by M1-Polarized Macrophages

Overactivated inflammation and catabolism induced by proinflammatory macrophages are involved in the pathological processes of intervertebral disc (IVD) degeneration (IVDD). Our previous study suggested the protective role of inhibiting heat shock protein 90 (HSP90) in IVDD, while the underlying mechanisms need advanced research. The current study investigated the effects of HSP90 inhibitor 17-AAG on nucleus pulposus (NP) inflammation and catabolism induced by M1-polarized macrophages. Immunohistochemical staining of degenerated human IVD samples showed massive infiltration of macrophages, especially M1 phenotype, as well as elevated levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP)13. The conditioned medium (CM) of inflamed NP cells (NPCs) enhanced M1 polarization of macrophages, while the CM of M1 macrophages but not M2 macrophages promoted the expression of inflammatory factors and matrix proteases in NPCs. Additionally, we found that 17-AAG could represent anti-inflammatory and anti-catabolic effects by modulating both macrophages and NPCs. On the one hand, 17-AAG attenuated the pro-inflammatory activity of M1 macrophages via inhibiting nuclear factor-κB (NF-κB) pathway and mitogen-activated protein kinase (MAPK) pathways. On the other hand, 17-AAG dampened M1-CM-induced inflammation and catabolism in NPCs by upregulating HSP70 and suppressing the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway. Moreover, both in vitro IVD culture models and murine disc puncture models supported that 17-AAG treatment decreased the levels of inflammatory factors and matrix proteases in IVD tissues. In conclusion, HSP90 inhibitor 17-AAG attenuates NP inflammation and catabolism induced by M1 macrophages, suggesting 17-AAG as a promising candidate for IVDD treatment.

The first multi-omic interaction network in the human pathogen Candida glabrata provides novel insights into the virulence regulator, Hsp90

Candida glabrata-caused candidiasis is growing but treatments remain limited by paucity of drug targets, intrinsic azole resistance and increasing resistance to other drug classes. Drug resistance is one of numerous virulence traits regulated by the chaperone, heat shock protein 90 (Hsp90) in Candida albicans via its interactions with 5% of the genome. Hsp90 also regulates key drug resistance mechanisms in C. glabrata, but little else was known about Hsp90 in this organism. Therefore, CgHsp90 interactions were elucidated by genetic and proteomic methods. A genetic network was produced by a chemical-genetic, synthetic-sick screen on a gene-deletion library covering 16% of the genome; whilst quantitative proteomics was undertaken by tandem mass tagging on wild-type cells. Both experiments were undertaken at 30°C, 37°C and 39°C and Hsp90 was perturbed using sub-lethal concentrations of Hsp90 inhibitor. Efforts to identify Hsp90 interactors at these host-infection associated temperatures produced a genetic network of 68 genes and a protein network of 2298 proteins. Of these, 4 genes and 261 proteins interacted with Hsp90 at all three temperatures, indicating a core Hsp90 interaction network. Intriguingly, both networks had enrichment for the “antibiotic biosynthesis” GO term. Two genes, BCY1 and MCM16, were found to interact with Hsp90 at multiple temperatures in both networks. These data indicate the divergence of Hsp90 networks between C. glabrata and its close relatives and offer important targets for further research. Presented here is the first multi-omic interaction network in C. glabrata, focused on the virulence and drug resistance regulator, Hsp90.

The Potential of Hsp90 in Targeting Pathological Pathways in Cardiac Diseases

Heat shock protein 90 (Hsp90) is a molecular chaperone that interacts with up to 10% of the proteome. The extensive involvement in protein folding and regulation of protein stability within cells makes Hsp90 an attractive therapeutic target to correct multiple dysfunctions. Many of the clients of Hsp90 are found in pathways known to be pathogenic in the heart, ranging from transforming growth factor β (TGF-β) and mitogen activated kinase (MAPK) signaling to tumor necrosis factor α (TNFα), Gs and Gq g-protein coupled receptor (GPCR) and calcium (Ca2+) signaling. These pathways can therefore be targeted through modulation of Hsp90 activity. The activity of Hsp90 can be targeted through small-molecule inhibition. Small-molecule inhibitors of Hsp90 have been found to be cardiotoxic in some cases however. In this regard, specific targeting of Hsp90 by modulation of post-translational modifications (PTMs) emerges as an attractive strategy. In this review, we aim to address how Hsp90 functions, where Hsp90 interacts within pathological pathways, and current knowledge of small molecules and PTMs known to modulate Hsp90 activity and their potential as therapeutics in cardiac diseases.

The Disassociation of the A20/HSP90 Complex via Downregulation of HSP90 Restores the Effect of A20 Enhancing the Sensitivity of Hepatocellular Carcinoma Cells to Molecular Targeted Agents

NF-κB (nuclear factor κB) is a regulator of hepatocellular cancer (HCC)-related inflammation and enhances HCC cells’ resistance to antitumor therapies by promoting cell survival and anti-apoptosis processes. In the present work, we demonstrate that A20, a dominant-negative regulator of NF-κB, forms a complex with HSP90 (heat-shock protein 90) and causes the disassociation of the A20/HSP90 complex via downregulation of HSP90. This process restores the antitumor activation of A20. In clinical specimens, the expression level of A20 did not relate with the outcome in patients receiving sorafenib; however, high levels of HSP90 were associated with poor outcomes in these patients. A20 interacted with and formed complexes with HSP90. Knockdown of HSP90 and treatment with an HSP90 inhibitor disassociated the A20/HSP90 complex. Overexpression of A20 alone did not affect HCC cells. Downregulation of HSP90 combined with A20 overexpression restored the effect of A20. Overexpression of A20 repressed the expression of pro-survival and anti-apoptosis-related factors and enhanced HCC cells’ sensitivity to sorafenib. These results suggest that interactions with HSP90 could be potential mechanisms of A20 inactivation and disassociation of the A20/HSP90 complex and could serve as a novel strategy for HCC treatment.

Dietary Flavone Baicalein Combinate with Genipin Attenuates Inflammation Stimulated by Lipopolysaccharide in RAW264.7 Cells or Pseudomonas aeruginosa in Mice via Regulating the Expression and Phosphorylation of AKT

Mounting evidence has shown that single-targeted therapy might be inadequate to achieve satisfactory effects. Thus, drug combinations are gaining attention as they can regulate multiple targets to obtain more beneficial effects. Heat shock protein 90 (HSP90) is a molecular chaperone that assists the protein assembly and folding of client proteins and maintains their stability. Interfering with the interaction between HSP90 and its client proteins by inhibiting the latter’s activity may offer a new approach toward combination therapy. The HSP90 client protein AKT plays an important role in the inflammatory response syndrome caused by infections. In this study, the dietary flavone baicalein was identified as a novel inhibitor of HSP90 that targeted the N-terminal ATP binding pocket of HSP90 and hindered the chaperone cycle, resulting in AKT degradation. Combining baicalein with genipin, which was extracted from Gardenia jasminoides, could inhibit the pleckstrin homology domain of AKT, significantly increasing the anti-inflammatory effects both in vitro and in vivo. This synergistic effect was attributed to the reduction in AKT expression and phosphorylation. Thus, elucidating the mechanism underlying this effect will provide a new avenue for the clinical application and development of synergistic anti-inflammatory drugs.

SGT1 SGS-domain mutations impair interactions with the barley MLA6 immune receptor in association with loss of NLR protein

The Mla (Mildew resistance locus a) of barley (Hordeum vulgare L.) is an effective model for cereal immunity against fungal pathogens. Like many resistance proteins, variants of the MLA coiled-coil nucleotide-binding leucine-rich-repeat (CC-NLR) receptor require the HRS complex to function, which includes HSP90 (Heat Shock Protein 90), RAR1 (Required for Mla12 Resistance 1), and SGT1 (Suppressor of G-two allele of Skp1). However, functional analysis of Sgt1 has been particularly difficult as deletions are often lethal. Recently, we identified rar3 (Required for Mla6 resistance 3), an in-frame Sgt1ΔKL308-309 mutation in the SGS domain that alters resistance conferred by MLA, but without lethality. Here we use autoactive MLA6 and heterologous yeast-two-hybrid strains with stably integrated HvRar1 and HvHsp90, to determine that this mutation weakens, but doesn’t entirely disrupt, the interaction between SGT1 and MLA. This causes a concomitant reduction in MLA6 protein accumulation below the apparent threshold required for effective resistance. The ΔKL308-309 deletion had a lesser effect on intramolecular interactions than alanine or arginine substitutions, and MLA variants that display diminished interactions with SGT1 appear to be disproportionately affected by the SGT1ΔKL308-309 mutation. We hypothesize that those dimeric plant CC-NLRs that appear unaffected by Sgt1 silencing are those with the strongest intermolecular interactions with it. Combining our data with recent work in CC-NLRs, we propose a cyclical model of the MLA-HRS resistosome interactions.

Heterogeneous Responses and Isoform Compensation Dim Therapeutic Window of Hsp90 ATP-Binding Inhibitors in Cancer

The rare capacity for heat shock protein-90 (Hsp90) chaperones to support almost the entire cellular signaling networks was viewed as a potential breakthrough point to combat tumor resistance to single oncogene-based therapeutics. Over two decades, several generations of Hsp90 ATP-binding inhibitors have entered numerous cancer clinical trials, but few have advanced to FDA approval for treatment of human cancers. Herein, we report that Hsp90 expression dramatically vary especially among different types of non-cancer cells and organs. The highly variable levels of Hsp90 from as low as 1.7% to as high as 9% of their total cellular proteins were responsible for either an extreme sensitivity or an extreme resistance to a classical Hsp90 ATP-binding inhibitor. Among randomly selected cancer cell lines, the same client proteins for regulation of cell growth exhibited unexpectedly heterogenous reactions in response to Hsp90 ATP-binding inhibitor, inconsistent with the current understanding. Finally, a minimum amount (<10%) of Hsp90β was still required for client protein stability and cell survival even in the presence of full Hsp90α. These new findings of Hsp90 expression in host and isoform compensation in tumor cells could complicate biomarker selection, toxicity readout and clinical efficacy of Hsp90-ATP-binding inhibitors in cancer clinical trials.