热激蛋白90与热激应答

热激蛋白90(heat shock protein 90,HSP90)作为机体重要的分子伴侣之一,主要是维持机体内环境的稳态.在机体遭受内外界刺激时,体内氧化-抗氧化平衡失调诱发机体热激应答,诱导HSP90高表达来抵御刺激对机体造成的损伤.     

热休克蛋白90 抑制剂的活性初步评价

目的:初步评价经计算机模拟筛选的Fs系列化合物对A549细胞的增殖抑制作用与对热休克蛋白90活性的抑制。方法:首先采用SRB(磺基罗丹明B)法观察26个化合物对A549肿瘤细胞增殖抑制活性,再进一步应用Western Blot方法对此类化合物在蛋白水平上对热休克蛋白90活性的抑制进行评价。以热休克蛋白70的表达增加作为热休克蛋白90活性是否被抑制的参考指标。结果:在Fs系列化合物中Fs-1、Fs-8、Fs-24对A549细胞增殖有明显抑制,且能明显上调Hsp70蛋白的表达,但同时Hsp90蛋白的表达量并不受影响。其余化合物在两种方法中显示均无明显抑制作用。结论:在经计算机模拟筛选出的26种Fs系列化合物中Fs-1、Fs-8、Fs-24可抑制A549细胞的增殖,可能是通过对Hsp90活性的抑制而发挥作用,经筛选其余Fs系列化合物抑制A549细胞增殖与抑制Hsp90活性作用均不显著。Fs-1、Fs-8、Fs-24类化合物作用的初步探索将为研制Hsp90靶向抑制剂类药物开辟新途径。     

Cell-Impermeable Peptide Fragments of Survivin and Hsp70/Hsp90-Organizing Protein Inhibit the Hsp90-Dependent Migration and Invasion of Tumor Cells

Biophysics - Membrane-associated and secreted extracellular heat shock proteins 90 (Hsp90) have important roles in the migration, invasion, and metastasis of tumor cells and are proposed as...     

Heat Shock Protein 90 regulates the stability of c-Jun in HEK293 Cells

The 90-kDa heat shock protein (HSP90) normally functions as a molecular chaperone participating in folding and stabilizing newly synthesized proteins, and refolding denatured proteins. The HSP90 inhibitor geldanamycin (GA) occupies the ATP/ADP binding pocket of HSP90 so inhibits its chaperone activity and causes subsequent degradation of HSP90 client proteins by proteasomes. Here we show that GA reduces the level of endogenous c-Jun in human embryonic kidney 293 (HEK293) cells in a time and dose dependent manner, and that this decrease can be reversed by transfection of HSP90 plasmids. Transfection of HSP90 plasmids in the absence of GA increases the level of endogenous c-Jun protein, but has no obvious affect on c-Jun mRNA levels. We also showed that HSP90 prolongs the half-life of c-Jun by stabilizing the protein; the proteasome inhibitor N-benzoyloxy-carbonyl (Z)-Leu-Leu-leucinal (MG132) blocks the degradation of c-Jun promoted by GA. Transfection of HSP90 plasmids did not obviously alter phosphorylation of c-Jun, and a Jun-2 luciferase activity assay indicated that over-expression of HSP90 elevated the total protein activity of c-Jun in HEK293 cells. All our evidence indicates that HSP90 stabilizes c-Jun protein, and so increases the total activity of c-Jun in HEK293 cells.     

Identification of Mixed Lineage Leukemia 1(MLL1) Protein as a Coactivator of Heat Shock Factor 1(HSF1) Protein in Response to Heat Shock Protein 90 (HSP90) Inhibition

Heat shock protein 90 (HSP90) inhibition inhibits cancer cell proliferation through depleting client oncoproteins and shutting down multiple oncogenic pathways. Therefore, it is an attractive strategy for targeting human cancers. Several HSP90 inhibitors, including AUY922 and STA9090, show promising effects in clinical trials. However, the efficacy of HSP90 inhibitors may be limited by heat shock factor 1 (HSF1)-mediated feedback mechanisms. Here, we identify, through an siRNA screen, that the histone H3 lysine 4 methyltransferase MLL1 functions as a coactivator of HSF1 in response to HSP90 inhibition. MLL1 is recruited to the promoters of HSF1 target genes and regulates their expression in response to HSP90 inhibition. In addition, a striking combination effect is observed when MLL1 depletion is combined with HSP90 inhibition in various human cancer cell lines and tumor models. Thus, targeting MLL1 may block a HSF1-mediated feedback mechanism induced by HSP90 inhibition and provide a new avenue to enhance HSP90 inhibitor activity in human cancers.     

The Extracellular Protein of Luteococcus japonicus subsp. casei Reactivates Cells Inactivated by UV Irradiation or Heat Shock

The culture liquid of Luteococcus japonicus subsp. casei was found to be able to reactivate cells of this bacterium inactivated by UV irradiation or heat shock. The antistress activity of the culture liquid was due to the presence of an extracellular exometabolite of a protein nature with a molecular mass of more than 10 kDa. When the bacterium was grown in a nutrient broth or glucose-containing mineral medium, the antistress protein was secreted by cells in the logarithmic growth phase. The reactivating effect of the antistress protein was inversely proportional to the survival rate of stressed cells.     

植物热激蛋白90的结构和功能

文章从结构和功能两个方面介绍植物热激蛋白90的研究进展。     

HeLa细胞热休克蛋白的代谢动力学

 HeLa细胞经45℃,15min应激后,可诱导一组分子量为100,85,73,70,54kD的热休克蛋白,其中分子量为73/70kD的HSP产量最高。在产生HSP的同时,正常蛋白质合成受到抑制,并在随后数小时内恢复。HSP73/70在应激后能迅速诱导产生,应激后4—6小时为其合成高峰,10小时后明显减少,24小时恢复正常。其分解遵循指数规律,半衰期为49.9小时。     

The 90-kDa Heat Shock Protein Hsp90 Protects Tubulin against Thermal Denaturation

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.     

Tumor Cell Migration and Invasion Are Enhanced by Depletion of Rap1 GTPase-activating Protein (Rap1GAP)

The functional significance of the widespread down-regulation of Rap1 GTPase-activating protein (Rap1GAP), a negative regulator of Rap activity, in human tumors is unknown. Here we show that human colon cancer cells depleted of Rap1GAP are endowed with more aggressive migratory and invasive properties. Silencing Rap1GAP enhanced the migration of confluent and single cells. In the latter, migration distance, velocity, and directionality were increased. Enhanced migration was a consequence of increased endogenous Rap activity as silencing Rap expression selectively abolished the migration of Rap1GAP-depleted cells. ROCK-mediated cell contractility was suppressed in Rap1GAP-depleted cells, which exhibited a spindle-shaped morphology and abundant membrane protrusions. Tumor cells can switch between Rho/ROCK-mediated contractility-based migration and Rac1-mediated mesenchymal motility. Strikingly, the migration of Rap1GAP-depleted, but not control cells required Rac1 activity, suggesting that loss of Rap1GAP alters migratory mechanisms. Inhibition of Rac1 activity restored membrane blebbing and increased ROCK activity in Rap1GAP-depleted cells, suggesting that Rac1 contributes to the suppression of contractility. Collectively, these findings identify Rap1GAP as a critical regulator of aggressive tumor cell behavior and suggest that the level of Rap1GAP expression influences the migratory mechanisms that are operative in tumor cells.     

The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein

Background

Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear.

Results

In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells.

Conclusions

Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD.     

Expression of Heat Shock Protein 90 (hsp90) in Neural and Nonneural Tissues of the Control and Hyperthermic Rabbit

Tissue-specific differences were apparent in the constitutive level of hsp90 in various body tissues of the unstressed rabbit. Western blotting with monoclonal antibody 29A revealed very low levels in muscle and highest levels in neural regions (cerebellum, cerebral hemispheres, and retina) and in testes and thymus. Intermediate levels were apparent in other tissues such as liver, kidney, heart, and small intestine. Following hyperthermia, induction of hsp90 was not detected with 1-D Western blotting in tissues which demonstrated high constitutive levels; however, elevations were noted in tissues which showed lower constitutive amounts of the protein, such as kidney, heart, and muscle. Immunocytochemical studies revealed that hsp90 is preferentially localized to neuronal cell populations in the rabbit brain and that this pattern does not alter following hyperthermic conditions which result in glial induction of hsp70. In kidney, where constitutive levels of hsp90 are lower than in brain, an induction of hsp90 was noted in renal tubules following hyperthermia.     

Stimulation of Cortical Myosin Phosphorylation by p114RhoGEF Drives Cell Migration and Tumor Cell Invasion

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.     

Bacterial Lipopolysaccharide Augments Febrile-Range Hyperthermia-Induced Heat Shock Protein 70 Expression and Extracellular Release in Human THP1 Cells

Sepsis, a devastating and often lethal complication of severe infection, is characterized by fever and dysregulated inflammation. While infections activate the inflammatory response in part through Toll-like receptors (TLRs), fever can partially activate the heat shock response with generation of heat shock proteins (HSPs). Since extracellular HSPs, especially HSP70 (eHSP70), are proinflammatory TLR agonists, we investigated how exposure to the TLR4 agonist, bacterial lipopolysaccharide (LPS) and febrile range hyperthermia (FRH; 39.5°C) modify HSP70 expression and extracellular release. Using differentiated THP1 cells, we found that concurrent exposure to FRH and LPS as well as TLR2 and TLR3 agonists synergized to activate expression of inducible HSP72 (HSPA1A) mRNA and protein via a p38 MAP kinase-requiring mechanism. Treatment with LPS for 6 h stimulated eHSP70 release; levels of eHSP70 released at 39.5°C were higher than at 37°C roughly paralleling the increase in intracellular HSP72 in the 39.5°C cells. By contrast, 6 h exposure to FRH in the absence of LPS failed to promote eHSP70 release. Release of eHSP70 by LPS-treated THP1 cells was inhibited by glibenclamide, but not brefeldin, indicating that eHSP70 secretion occurred via a non-classical protein secretory mechanism. Analysis of eHSP70 levels in exosomes and exosome-depleted culture supernatants from LPS-treated THP1 cells using ELISA demonstrated similar eHSP70 levels in unfractionated and exosome-depleted culture supernatants, indicating that LPS-stimulated eHSP70 release did not occur via the exosome pathway. Immunoblot analysis of the exosome fraction of culture supernatants from these cells showed constitutive HSC70 (HSPA8) to be the predominant HSP70 family member present in exosomes. In summary, we have shown that LPS stimulates macrophages to secrete inducible HSP72 via a non-classical non-exosomal pathway while synergizing with FRH exposure to increase both intracellular and secreted levels of inducible HSP72. The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.     

Activity of the Heat Shock Protein 90 Inhibitor Ganetespib in Melanoma

Heat shock protein 90 (HSP90) is involved in the regulation of diverse biological processes such as cell signaling, proliferation and survival, and has been recently recognized as a potential target for cancer therapy. Ganetespib is a potent ATP competitive inhibitor of HSP90. Ganetespib downregulated the expression of multiple signal transducing molecules including EGFR, IGF-1R, c-Met, Akt, B-RAF and C-RAF, resulting in pronounced decrease in phosphorylation of Akt and Erk1/2 in a panel of five cutaneous melanoma cell lines including those harboring B-RAF and N-RAS mutations. Ganetespib exhibited potent antiproliferative activity on all five of these cell lines, with IC50 values between 37.5 and 84 nM. Importantly, Ganetespib is active on B-RAF mutated melanoma cells that have acquired resistance to B-RAF inhibition. Ganetespib induced apoptosis and cell cycle arrest at G1 and/or G2/M phase. Ganetespib induced cell cycle arrest was accompanied by altered expression of cyclin-dependent kinase inhibitor (CDKI) p21^Cip1 and p27^Kip1, cyclins B1, D1 and E, and/or cyclin-dependent kinases 1, 2 and 4. HSP90 is functionally important for melanoma cells and HSP90 inhibitors such as ganetespib could potentially be effective therapeutics for melanoma with various genetic mutations and acquired resistance to B-RAF inhibition.     

Oxidative Stress and Heat Shock Protein Induction in Human Cells

Agents which induce heat shock protein synthesis in cultured monolayers of Hela cells such as hyperthermia, ethanol and sodium arsenite can also cause increases in the levels of lipid peroxidation as determined by the formation of TBA-products. The heat induced increases may be diminished by addition to the medium of mannitol or EGTA. These compounds are known to depress heat shock protein synthesis.

Following hyperthermia there is also a decrease in protein synthesis. In vitro studies indicate possible damage to ribosomes, and since the heat induced loss of protein synthetic capacity can be increased by superoxide dismutase inhibitors, and prevented by mannitol, such effects may be linked to the increases observed in lipid peroxidation. It is suggested that a connection exists between lipid peroxidation and heat shock protein gene activation.