Heat Shock Protein Degradation Research Articles

USP14 governs CYP2E1 to promote nonalcoholic fatty liver disease through deubiquitination and stabilization of HSP90AA1

Nonalcoholic fatty liver disease (NAFLD) begins with excessive triglyceride accumulation in the liver, and overly severe hepatic steatosis progresses to nonalcoholic steatohepatitis (NASH), which is characterized by lipid peroxidation, inflammation, and fibrosis. Ubiquitin-specific proteinase 14 (USP14) regulates inflammation, hepatocellular carcinoma and viral infection, but the effect of USP14 on NAFLD is unknown. The aim of this study was to reveal the role of USP14 in the progression of NAFLD and its underlying mechanism. We demonstrated that hepatic USP14 expression was significantly increased in NAFLD in both humans and mice. Hepatic USP14 overexpression exacerbated diet-induced hepatic steatosis, inflammation and fibrosis in mice, in contrast to the results of hepatic USP14 knockdown. Furthermore, palmitic/oleic acid-induced lipid peroxidation and inflammation in hepatocytes were markedly increased by USP14 overexpression but decreased by USP14 knockdown. Notably, in vivo or in vitro data show that USP14 promotes NAFLD progression in a cytochrome p4502E1 (CYP2E1)-dependent manner, which exacerbates hepatocyte oxidative stress, impairs the mitochondrial respiratory chain and inflammation by promoting CYP2E1 protein levels. Mechanistically, we demonstrated by immunoprecipitation and ubiquitination analysis that USP14 inhibits the degradation of heat shock protein 90 alpha family class A member 1 (HSP90AA1) by decreasing its lysine 48-linkage ubiquitination. Meanwhile, upregulation of HAP90AA1 protein promotes CYP2E1 protein accumulation. Collectively, our data indicate that an unknown USP14-HSP90AA1-CYP2E1 axis contributes to NAFLD progression, and we propose that inhibition of USP14 may be an effective strategy for NASH treatment.

Effects of Postmortem Aging on Small Heat Shock Protein Degradation of 3 Bovine Muscles

Effects of Postmortem Aging on Small Heat Shock Protein Degradation of 3 Bovine Muscles

Sulforaphane inhibits pancreatic cancer through disrupting Hsp90–p50Cdc37 complex and direct interactions with amino acids residues of Hsp90

Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl) butane)], an isothiocyanate derived from cruciferous vegetables, has been shown to possess potent chemopreventive activity. We analyzed the effect of sulforaphane on the proliferation of pancreatic cancer cells. Sulforaphane inhibited pancreatic cancer cell growth in vitro with IC50s of around 10–15 μM and induced apoptosis. In pancreatic cancer xenograft mouse model, administration of sulforaphane showed remarkable inhibition of tumor growth without apparent toxicity noticed. We found that sulforaphane induced the degradation of heat shock protein 90 (Hsp90) client proteins and blocked the interaction of Hsp90 with its cochaperone p50Cdc37 in pancreatic cancer cells. Using nuclear magnetic resonance spectroscopy (NMR) with an isoleucine-specific labeling strategy, we overcame the protein size limit of conventional NMR and studied the interaction of sulforaphane with full-length Hsp90 dimer (170 kDa) in solution. NMR revealed multiple chemical shifts in sheet 2 and the adjacent loop in Hsp90 N-terminal domain after incubation of Hsp90 with sulforaphane. Liquid chromatography coupled to mass spectrometry further mapped a short peptide in this region that was tagged with sulforaphane. These data suggest a new mechanism of sulforaphane that disrupts protein–protein interaction in Hsp90 complex for its chemopreventive activity.

Nephrotoxin-Induced Renal Cell Injury Involving Biochemical Alterations and Its Prevention With Antioxidant

BackgroundAlthough nephrotoxic agents or nephrotoxins are known to induce acute renal cell injury, their cytotoxic action is not fully elucidated. It is thus crucial to explore such a cytotoxic mechanism and the increasing volume of reports indicated a significant involvement of oxidative stress. To test this possibility, we investigated if a nephrotoxin would exert oxidative stress, leading to renal cell injury accompanied by certain biochemical alterations. We also examined if specific antioxidant might help prevent such oxidative cell injury. These studies may then help establish a prophylactic or preventive modality for renal cell injury induced by nephrotoxins.MethodsAs glycerol has been commonly used for studying acute renal failure in animals, whether it would induce cellular injury was tested in renal proximal tubular OK cells in vitro. Cells were exposed to the varying concentrations of glycerol and cell number/viability was determined in 24 hours. Severity of oxidative stress was assessed by lipid peroxidation assay. Possible effects of glycerol on biochemical parameters were also examined on glyoxalase I activity and heat shock protein 90 using spectrophotometric (enzymatic) assay and Western blot analysis.ResultsGlycerol (2.5%) was highly cytotoxic to OK cells, inducing 95% cell death in 24 hours. Lipid peroxidation assay indicated that nearly 3-fold greater oxidative stress was exerted by this glycerol. Concurrently, glyoxalase I activity was drastically lost by 75% and heat shock protein 90 was partially degraded following glycerol exposure. However, N-acetylcysteine, a potent glutathione-based antioxidant, was capable of almost completely preventing the glycerol-mediated adverse outcomes, such as cell death, glyoxalase I inactivation, and heat shock protein 90 degradation.ConclusionsGlycerol is cytotoxic, capable of inducing specific biochemical alterations such as inactivation of glyoxalase I and degradation of heat shock protein 90, which may reflect a breakdown of the cellular detoxification and defense systems, leading ultimately to OK cell death. Nevertheless, as N-acetylcysteine can provide full cytoprotection against such glycerol toxicity, it could be considered a prophylactic modality for nephrotoxin-induced oxidative renal cell injury and death.KeywordsGlycerol; Glyoxalase I; Heat shock protein; N-acetylcysteine; Renal cell injury

Degradation of HIF-1alpha under Hypoxia Combined with Induction of Hsp90 Polyubiquitination in Cancer Cells by Hypericin: a Unique Cancer Therapy

The perihydroxylated perylene quinone hypericin has been reported to possess potent anti-metastatic and antiangiogenic activities, generated by targeting diverse crossroads of cancer-promoting processes via unique mechanisms. Hypericin is the only known exogenous reagent that can induce forced poly-ubiquitination and accelerated degradation of heat shock protein 90 (Hsp90) in cancer cells. Hsp90 client proteins are thereby destabilized and rapidly degraded. Hsp70 client proteins may potentially be also affected via preventing formation of hsp90-hsp70 intermediate complexes. We show here that hypericin also induces enhanced degradation of hypoxia-inducible factor 1α (HIF-1α) in two human tumor cell lines, U87-MG glioblastoma and RCC-C2VHL−/− renal cell carcinoma and in the non-malignant ARPE19 retinal pigment epithelial cell line. The hypericin-accelerated turnover of HIF-1α, the regulatory precursor of the HIF-1 transcription factor which promotes hypoxic stress and angiogenic responses, overcomes the physiologic HIF-1α protein stabilization which occurs in hypoxic cells. The hypericin effect also eliminates the high HIF-1α levels expressed constitutively in the von-Hippel Lindau protein (pVHL)-deficient RCC-C2VHL−/− renal cell carcinoma cell line. Unlike the normal ubiquitin-proteasome pathway-dependent turnover of HIF-α proteins which occurs in normoxia, the hypericin-induced HIF-1α catabolism can occur independently of cellular oxygen levels or pVHL-promoted ubiquitin ligation of HIF-1α. It is mediated by lysosomal cathepsin-B enzymes with cathepsin-B activity being optimized in the cells through hypericin-mediated reduction in intracellular pH. Our findings suggest that hypericin may potentially be useful in preventing growth of tumors in which HIF-1α plays pivotal roles, and in pVHL ablated tumor cells such as renal cell carcinoma through elimination of elevated HIF-1α contents in these cells, scaling down the excessive angiogenesis which characterizes these tumors.

The Heatshockprotein-90 Inhibitor IPI-504 Potently Overcomes Tyrosinekinase Inhibitor-Induced Activation of Cell Protection Mechanisms and Degrades HSP-Client Proteins KIT and FLT3 In Acute Leukemia Models

AbstractAbstract 3982Activating mutations of FLT3 and KIT class III receptor tyrosine kinases (TK) are associated with acute leukemias and systemic mastocytosis. Despite the encouraging therapeutic potential of TK inhibitors (TKI), monotherapy often is not sufficient to obtain lasting responses. Here we show that TKI therapy may lead to cellular protection mechanisms including stabilization/repair of DNA, as well as protection of the function of heat shock proteins. We further demonstrate that degradation of heat shock protein 90 (HSP90) client proteins, FLT3 and KIT, by the HSP90 inhibitor IPI-504, leads to antitumor effects in acute leukemia and mastocytosis models; including additive to synergistic antiproliferative and proapoptotic activity after TKI treatment. To explore the immediate downstream signaling effects of mutant FLT3 and KIT inhibition in acute leukemia and mastocytosis models, we performed unbiased phoshoproteomic analyses of cells before and after TKI treatment. Phosphopeptides that displayed a significant difference in phosphorylation before and after FLT3/KIT inhibition with Imatinib or Sunitinib at IC90 were immunoaffinity purified, and identified by tandem mass spectrometry. Degradation of FLT3 and KIT after HSP90 inhibition was studied by immunoblotting. Antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504 alone, or in combination with TKI treatment, were studied in different leukemia and mastocytosis cell models, and in an isogenic BaF/3 model harboring different clinically relevant KIT and FLT3 mutations. Phoshoproteomics revealed a significant upregulation in phosphorylation of DNA and protein function stabilizing proteins, including HSP members in mutant KIT and FLT3 cell lines. Both FLT3 and KIT turned out to be client proteins of HSP90, and IPI-504 was able to degrade FLT3 and KIT protein, resulting in a strong potentiation of the antiproliferative and proapoptotic effects achieved by TK inhibitors alone. Of interest, IPI-504 displayed antitumor activity even as monotherapy in some leukemia and mastocytosis cell line models. Our data suggest that TK inhibition of FLT3 or KIT causes initiation of cell protective mechanisms, including activation of HSPs, which can therapeutically be targeted by HSP90 inhibitors such as IPI-504. These results provide a rationale to combine TKI with HSP inhibitors to optimize TKI therapy in patients with hematopoietic malignancies. Disclosures:Normant:Infinity pharmaceuticals: Employment.

First Prize (Tie): Oxidative Renal Cell Injury Induced by Calcium Oxalate Crystal and Renoprotection with Antioxidants: A Possible Role of Oxidative Stress in Nephrolithiasis

Calcium oxalate (CaOx) is one of the key elements for kidney stone formation, but the exact mechanism needs to be defined. CaOx has been shown to cause renal cell injury through oxidative stress, leading to potential crystal deposition in the kidneys. We thus investigated if CaOx crystal would induce such renal cell injury in vitro and also explored how it would be carried out. Renal tubular epithelial LLC-PK(1) cells were employed, and CaOx monohydrate (COM) was used as CaOx crystal in this study. Cytotoxic effects of COM were assessed on cell viability and biochemical parameters, while protective effect of antioxidants against COM was also examined. COM demonstrated its cytotoxicity on LLC-PK(1) cells, exhibiting a approximately 35% cell viability reduction with 500 microg/mL COM in 6 hours. This was presumably attributed to oxidative stress, indicated by lipid peroxidation assay, and N-acetylcysteine (NAC), a potent antioxidant, indeed neutralized such COM cytotoxicity. Although COM also induced inactivation of glutathione-dependent enzymes and partial degradation of heat shock protein 90, these adverse effects were completely prevented with NAC. Moreover, such reduced cell viability with COM was rather associated with apoptosis, evidenced by DNA analysis. COM is cytotoxic to LLC-PK(1) cells through oxidative stress, leading to the cell viability reduction, adverse effects on biochemical parameters, and, consequently, apoptosis. However, NAC effectively averted such severe cytotoxic effects, sustaining the renal cell integrity. Thus, NAC may provide full renoprotection against COM assault, preventing renal cell injury and ultimate stone formation.

Taxotere-Induced Inhibition of Human Endothelial Cell Migration Is a Result of Heat Shock Protein 90 Degradation

In addition to effects on tumor cell proliferation and apoptosis, microtubule-binding agents are potent inhibitors of angiogenesis. The cancer chemotherapeutic drug Taxotere (docetaxel) inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) migration in vitro at concentrations substantially lower than required to cause cell cycle arrest or apoptosis. Here, we show that Taxotere caused the ubiquitination and subsequent proteasomal degradation of heat shock protein 90 (Hsp90) in HUVEC. This prevented signaling from the focal adhesions and VEGF receptors and inhibited integrin activation. Taxotere prevented the VEGF-induced phosphorylation of focal adhesion kinase, Akt, and endothelial nitric oxide synthase (eNOS), all of which are Hsp90 client proteins. Taxotere completely blocked the VEGF-induced increase in eNOS activity, and the addition of a NO donor reversed the inhibitory effect of Taxotere on VEGF-induced migration. A similar reversal occurred with a proteasomal inhibitor of Hsp90 degradation. Furthermore, overexpression of Hsp90 rescued HUVEC from the inhibition of VEGF-induced migration by Taxotere. Previous studies have suggested that tubulin is also a client protein of Hsp90, and immunocytochemical analysis showed that Taxotere caused the dissociation of Hsp90 from tubulin. We suggest that uncomplexed Hsp90 is more susceptible to ubiquitination and subsequent proteasomal degradation than the bound form. Although inhibitors of Hsp90 are currently under clinical investigation as antitumor agents, this seems to be the first account of a drug that reduces Hsp90 function by enhancing its proteasomal degradation. Further, the loss of Hsp90 and the inactivation of Hsp90 client proteins are previously undescribed actions of Taxotere that may contribute to its antiangiogenic activity.