SGT, a Hsp90beta binding partner, is accumulated in the nucleus during cell apoptosis

In this study, we reported that small glutamine-rich TPR-containing protein (SGT) interacted with not only Hsp90alpha but also Hsp90beta. Confocal analysis showed that treatment of cells with Hsp90-specific inhibitor geldanamycin (GA) disrupted the interaction of SGT with Hsp90beta and this contributed to the increase of nuclear localization of SGT in HeLa cells. The increased nuclear localization of SGT was further confirmed by the Western blotting in GA-treated HeLa cells and H1299 cells. In our previous study, SGT was found to be a new pro-apoptotic factor, so we wondered whether the sub-cellular localization of SGT was related with cell apoptosis. By confocal analysis we found that the nuclear import of SGT was significantly increased in STS-induced apoptotic HeLa cells, which implied that the sub-cellular localization of SGT was closely associated with Hsp90beta and apoptosis.     

Heat Shock Inhibition of CDK5 Increases NOXA Levels through miR-23a Repression

Hyperthermia is a proteotoxic stress that is lethal when exposure is extreme but also cytoprotective in that sublethal exposure leads to the synthesis of heat shock proteins, including HSP70, which are able to inhibit stress-induced apoptosis. CDK5 is an atypical cyclin-dependent kinase family member that regulates many cellular functions including motility and survival. Here we show that exposure of a human lymphoid cell line to hyperthermia causes CDK5 insolubilization and loss of tyrosine-15 phosphorylation, both of which were prevented in cells overexpressing HSP70. Inhibition of CDK5 activity with roscovitine-sensitized cells to heat induced apoptosis indicating a protective role for CDK5 in inhibiting heat-induced apoptosis. Both roscovitine and heat shock treatment caused increased accumulation of NOXA a pro-apoptotic BH3-only member of the BCL2 family. The increased abundance of NOXA by CDK5 inhibition was not a result of changes in NOXA protein turnover. Instead, CDK5 inhibition increased NOXA mRNA and protein levels by decreasing the expression of miR-23a, whereas overexpressing the CDK5 activator p35 attenuated both of these effects on NOXA and miR-23a expression. Lastly, overexpression of miR-23a prevented apoptosis under conditions in which CDK5 activity was inhibited. These results demonstrate that CDK5 activity provides resistance to heat-induced apoptosis through the expression of miR-23a and subsequent suppression of NOXA synthesis. Additionally, they indicate that hyperthermia induces apoptosis through the insolubilization and inhibition of CDK5 activity.     

Structural and functional characterization of human SGT and its interaction with Vpu of the human immunodeficiency virus type 1

The small glutamine-rich tetratricopeptide repeat protein (SGT) belongs to a family of cochaperones that interacts with both Hsp70 and Hsp90 via the so-called TPR domain. Here, we present the crystal structure of the TPR domain of human SGT (SGT-TPR), which shows that it contains typical features found in the structures of other TPR domains. Previous studies show that full-length SGT can bind to both Vpu and Gag of human immunodeficiency virus type 1 (HIV-1) and the overexpression of SGT in cells reduces the efficiency of HIV-1 particle release. We show that SGT-TPR can bind Vpu and reduce the amount of HIV-1 p24, which is the viral capsid, secreted from cells transfected with the HIV-1 proviral construct, albeit at a lower efficiency than full-length SGT. This indicates that the TPR domain of SGT is sufficient for the inhibition of HIV-1 particle release but the N- and/or C-terminus also have some contributions. The SGT binding site in Vpu was also identified by using peptide array and confirmed by GST pull-down assay.     

CDK11p58 protein kinase activity is associated with Bcl-2 down-regulation in pro-apoptosis pathway

CDK11^p58, a G2/M-specific protein kinase, has been shown to be associated with apoptosis in many cell lines, with largely unknown mechanisms. Our previous study proved that CDK11^p58-enhanced cycloheximide (CHX)-induced apoptosis in SMMC-7721 hepatocarcinoma cells. Here we report for the first time that ectopic expression of CDK11^p58 down-regulates Bcl-2 expression and its Ser70, Ser87 phosphorylation in CHX-induced apoptosis in SMMC-7721 cells. Overexpression of Bcl-2 counteracts the pro-apoptotic activity of CDK11^p58. Furthermore, we confirm that the kinase activity of CDK11^p58 is essential to the down-regulation of Bcl-2 as well as apoptosis. Taken together, these results demonstrate that CDK11^p58 down-regulates Bcl-2 in pro-apoptosis pathway depending on its kinase activity, which elicits survival signal in hepatocarcinoma cells.     

Small glutamine-rich tetratricopeptide repeat-containing protein is composed of three structural units with distinct functions

Previously, we identified the human small glutamine-rich tetratricopeptide repeat-containing protein (SGT) as a co-chaperone. The tetratricopeptide repeat (TPR) domain in SGT is responsible for interacting with Hsc70. In this study, we demonstrated that the TPR domain of SGT also interacted with Hsp90. Moreover, we investigated the functional significance of regions of SGT outside the TPR domain. Evidently, the N-terminal domain of SGT is necessary and sufficient for its self-association; and, SGT may be a dimer elongated in shape. The C-terminal glutamine-rich region has the capacity to interact with short peptide segments composed of consecutive non-polar amino acids. The C-terminal fragment of SGT indeed plays a role in the association of SGT with in vitro translated rat type 1 glucose transporter, an integral membrane protein folded in a non-physiological state. Moreover, in the presence of SGT, the degradation of the transporter in reticulocyte lysates is inhibited. Taking together, SGT can be separated into three structural units with distinct functions.     

热休克反应中小鼠肾HSP70和乳酸脱氢酶同工酶表达变化的研究

用免疫组织化学和聚丙烯酰胺凝胶同工酶电泳方法研究了小鼠肾在热休克(46℃,30分钟)恢复期(4h和12h)HSP70的表达和乳酸脱氢酶(LDH)同工酶的变化。结果表明:(1)HSP70主要定位于肾小管上皮细胞胞质中,细胞核内未见表达;(2)HSP70免疫阳性反应在肾髓质较肾皮质强,肾小管较肾小球强;(3)热休克诱导小鼠肾LDH同工酶活性增强。提示:LDH同工酶可能对细胞热耐受性的建立有重要作用。     

Chitooligosaccharides induce apoptosis of human hepatocellular carcinoma cells via up-regulation of Bax

Chitooligosaccharides (COS) have been shown to regulate various cellular and biological functions. However, the effect of COS on apoptosis of hepatocellular carcinoma cells remains unclear. In this study, the activity and mechanism of COS against human hepatocellular carcinoma cells (SMMC-7721 cells) were investigated in vitro. The experiments showed that COS notably induced the apoptosis of SMMC-7721 cells and increased the cleavage of poly(ADP-ribose) polymerase. It presented a dose-dependent manner, and the apoptotic rate amounted to about 38% after treatment with 0.8 mg/ml COS for 72 h. The mRNA and protein levels of Bax were up-regulated by COS. These results demonstrated that COS induced apoptosis of SMMC-7721 cells. The possible mechanism is that COS up-regulate pro-apoptotic protein Bax, and trigger the cells a start-up of the apoptosis program.     

The effect of dietary lipid manipulation on murine splenic lymphocytes apoptosis and heat shock protein over expression

In this study, we kept BALB/c mice on a hyperlipidic diet for 120 days and then assessed the predisposition to apoptosis and the appearance of heat shock protein (Hsp) on splenic lymphocytes. By immunoblot analysis, bands corresponding to Hsp 60 and Hsp 70 in cells from mice kept on a saturated fatty acid diet showed a greater expression already after 1 month while two other bands, which correspond to Hsp 25 and Hsp 27, were slightly present after 1 month of treatment. In cells from mice kept on a diet rich in unsaturated fatty acid, there was a marked expression of Hsp 25 and Hsp 27 after only 30 days of treatment, which was maintained constant for up to 4 months; while for bands corresponding to Hsp 60 and Hsp 70, a significant minor signal was only detectable after 2-4 months from the beginning of the treatment. Splenic lymphocytes from animals kept on a lipidic diet containing saturated fatty acids were more susceptible to death by apoptosis, while cells of animals treated with unsaturated fatty acid were shown to be more resistant.     

Proteomics-based analysis of a counter-oxidative stress system in Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative anaerobic pathogen associated with chronic periodontitis. Although anaerobic, P. gingivalis exhibits a high degree of aerotolerance, which enables it to survive within periodontal pockets. The aim of the present study was to examine the effect of oxidative stress on protein expression in P. gingivalis to obtain a better understanding of the mechanism underlying its aerotolerance. To accomplish this, P. gingivalis cells were grown under conditions of hemin limitation (0.01 microg/mL) to avoid the oxygen protective effect of hemin on oxidative stress. The proteins were then extracted from cultures either left untreated or subjected to oxidative stress and separated by 2-DE. The resultant protein expression profiles were examined by image scanning, and those found to differ depending on the presence or absence of aeration were subjected to MALDI-MS and then analyzed using the ORF database of P. gingivalis W83 from The Institute of Genomic Research. Oxidative stress was found to affect the expression of numerous proteins in P. gingivalis cells. In particular, the levels of HtpG, GroEL, DnaK, AhpC, TPR domain protein, and trigger factor were substantially increased.     

Methionine protects against hyperthermia-induced cell injury in cultured bovine mammary epithelial cells

The aim of this study was to investigate the effects of methionine on cell proliferation, antioxidant activity, apoptosis, the expression levels of related genes (HSF-1, HSP70, Bax and Bcl-2) and the expression levels of protein (HSP70) in mammary epithelial cells, after heat treatment. Methionine (60 mg/L) increased the viability and attenuated morphological damage in hyperthermia-treated bovine mammary epithelial cells (BMECs). Additionally, methionine significantly reduced lactate dehydrogenase leakage, malondialdehyde formation, nitric oxide, and nitric oxide synthase activity. Superoxide dismutase, catalase, and glutathione peroxidase enzymatic activity was increased significantly in the presence of methionine. Bovine mammary epithelial cells also exhibited a certain amount of HSP70 reserve after methionine pretreatment for 24 h, and the expression level of the HSP70 gene and protein further increased with incubation at 42 °C for 30 min. Compared to the control, the expression of HSF-1 mRNA increased, and there was a significantly reduced expression of Bax/Bcl-2 mRNA and a reduced activity of caspase-3 against heat stress. Methionine also increased survival and decreased early apoptosis of hyperthermia-treated BMECs. Thus, methionine has cytoprotective effects on hyperthermia-induced damage in BMECs.     

Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress

We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H₂O₂-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H₂O₂-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.     

Expression of exercise-induced HSP70 in long-distance runner''s leukocytes

This study was designed to investigate the expression of heat shock protein 70 (HSP70), after acute moderate intensity exercise, in human peripheral blood leukocytes of trained runners and untrained controls. Ten male long-distance trained runners (TR) and untrained sedentary control subjects (SED) ran for 1 h at 70% of heart rate reserve (HRR). Basal HSP70 expression in TR was usually lower than that in SED, but basal HSP70 gene expression in TR was usually higher than that in SED. Although expression rates of exercise-induced HSP70 in both groups were similar, levels of HSP70 in SED were significantly higher than in TR. Significant increases in leukocytes, neutrophils, and lymphocytes after exercise were observed in both groups, but there were some differences between groups. We conclude that 1 h treadmill running at 70% HRR intensity is a sufficient stimulus to leukocytosis, neutrocytosis, lymphocytosis, and HSP70 proteins and gene expression in leukocytes. Adaptation to training was observed in TR.     

Plumbagin induces autophagy and apoptosis of SMMC-7721 cells in vitro and in vivo

Plumbagin (PL), an active naphthoquinone compound, has been demonstrated to be a potential anticancer agent. However, the underlying anticancer mechanism is not fully understood. In this study, the human hepatocellular carcinoma (HCC) SMMC-7721 cell line was studied in an in vitro model. The cell proliferation was inhibited by PL in a dose- and time-dependent manner. Electron microscopy, acridine orange staining, and immunofluorescence were used to evaluate autophagosome formation and LC3 protein expression in PL-treated SMMC-7721 cells. Real-time polymerase chain reaction and Western blot showed that PL treatment suppressed the expression of apoptosis and autophagy factors (LC3, Beclin1, Atg7, and Atg5), which are associated with tumor apoptosis and autophagy in SMMC-7721 cells. In the study of in vitro tumor nude mouse models, PL can inhibit tumor growth. Cell apoptosis and autophagy of the transplanted tumors were evaluated by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining, and Western blot. In addition, in the in vivo studies of HCC cells, we found that pretreatment with the autophagy inhibitor 3-methyladenine blocked the formation of apoptosis induced by PL. In contrast, administration of the apoptosis inhibitor Z-VAD did not affect PL-induced autophagy. Taken together, our findings strongly suggest that PL is a promising drug with significant antitumor activity in HCC.     

乌司他丁对大鼠脑缺血再灌注损伤后的保护作用及其机制研究

目的:探究乌司他丁在脑缺血再灌注损伤中的脑保护作用机制。方法:原代分离培养雄性SD大鼠脑皮质细胞,部分细胞经siRNA沉默HSP70基因。细胞先以无糖培养基在低氧条件下培养,12 h后复糖复氧模拟体外缺血再灌注损伤,并实施乌司他丁预处理干预,流式细胞术检测各组细胞的凋亡率,western-blotting检测Bcl-2,Bax,HSP70,JNK和p-JNK蛋白的表达。结果:与对照组比较,模型组脑组织细胞凋亡率明显增多(P0.05)、Bcl-2和Bax的表达量均有上调,Bcl-2/Bax的比值显著降低(P0.01)、HSP70的表达无显著变化;与模型组比较,乌司他丁处理组脑组织细胞凋亡率明显降低(P0.05)、Bax的表达量显著下调(P0.05),Bcl-2/Bax的比值显著上调(P0.05),HSP70的表达显著上调(P0.05),JNK的表达无显著变化、p-JNK则显著下调(P0.05)。HSP70沉默后乌司他丁的脑保护作用消失,对以上蛋白的表达无显著影响。结论:乌司他丁可能是通过上调HSP70表达进而抑制JNK信号转导通路对缺血再灌注引起的脑损伤起保护作用。     

热休克蛋白70在心血管疾病中的保护作用

热休克蛋白(heat shock protein70,HSP70)是HSP家族中重要成员,在生物细胞中含量最高,可诱导性最强,具有保护细胞免受刺激损伤,促进受损细胞修复及抗炎、抗凋亡、耐受缺血/缺氧损伤等多种生物学功能。许多研究发现在心肌组织中HSP70表达升高可减轻心肌细胞损伤程度,利于损伤心肌细胞的恢复,在预防和延缓心血管疾病中起到重要作用。因此,热休克蛋白70诱导剂在心血管疾病的防治中具有潜在的临床价值。本文主要对HSP70在心血管疾病中的保护作用进行综述。     

Two specific inhibitors of the phosphatidylinositol 3-kinase LY294002 and wortmannin up-regulate β1,4-galactosyltransferase I and thus sensitize SMMC-7721 human hepatocarcinoma cells to cycloheximide-induced apoptosis

Previous study indicated that β1,4-galactosyltransferase I (β1,4GT1) was up-regulated by cycloheximide (CHX) and thus enhanced apoptosis induced by CHX in SMMC-7721 cells. In this study, we reported that constitutively active Akt protein (myr-Akt) inhibited CHX-induced apoptosis in SMMC-7721 cells through down-regulating β1,4GT1. However, the two PI3K inhibitors LY294002 and wortmannin treatment up-regulated β1,4GT1 through enhancing Sp1 protein expression and consequently increased CHX-induced SMMC-7721 cells apoptosis. Besides, our results suggested that β1,4GT1 and cell surface galactose residues synthesized by elevated β1,4GT1 played an important role in SMMC-7721 cells apoptosis treated with CHX and PI3K inhibitor together. Moreover, we found that CHX accentuated β1,4GT1 through down-regulating Akt expression to mediate SMMC-7721 cells apoptosis. Taken together, PI3K inhibitors LY294002 and wortmannin up-regulated β1,4GT1 and enhanced CHX-induced apoptosis in SMMC-7721 cells, which suggested that PI3K inhibitors might have therapeutic potential when combined with CHX in the treatment of hepatoma.     

Intracellular delivery of HSP70 using HIV-1 Tat protein transduction domain

Heat shock protein 70 (HSP70) is an intracellular stress protein that confers cytoprotection to a variety of cellular stressors. Several lines of evidence have suggested that augmentation of the heat shock response by increasing the expression of HSP70 represents a potential therapeutic strategy for the treatment of critically ill patients. The Tat protein of human immunodeficiency virus 1 (HIV-1) has been used previously to deliver functional cargo proteins intracellularly when added exogenously to cultured cells. We generated a Tat-HSP70 fusion protein using recombinant methods and treated HSF -/- cells with either Tat-HSP70 or recombinant HSP70 prior to exposure to hyperoxia or lethal heat shock. We showed that biologically active, exogenous HSP70 can be delivered into cells using the HIV-1 Tat protein, and that the Tat-mediated delivery of HSP70 confers cytoprotection against thermal stress and hyperoxia and may represent a novel approach to augmenting intracellular HSP70 levels.     

Regulation of heat shock-induced apoptosis by sensitive to apoptosis gene protein

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species scavenger, but its antioxidant properties have not been completely defined. In this report, we demonstrate that modulation of SAG expression in U937 cells regulates heat shock-induced apoptosis. When we examined the protective role of SAG against heat shock-induced apoptosis with U937 cells transfected with the cDNA for SAG, a clear inverse relationship was observed between the amount of SAG expressed in target cells and their susceptibility to apoptosis. We also observed a significant decrease in the endogenous production of reactive oxygen species and oxidative DNA damage in SAG-overexpressed cells compared to control cells on exposure to heat shock. In addition, transfection of PC3 cells with SAG small interfering RNA markedly decreased the expression of SAG, enhancing the susceptibility of heat shock-induced apoptosis. Taken together, these results indicate that SAG may play an important role in regulating the apoptosis induced by heat shock presumably through maintaining the cellular redox status.     

扁蒴藤素对人鼻咽癌HNE2 细胞增殖的抑制作用研究

目的：探讨扁蒴藤素对人鼻咽癌HNE2 细胞增殖的抑制作用,明确HSP70 在肿瘤发展过程中的抑制作用。方法：噻唑蓝法 （MTT）检测扁蒴藤素对HNE2 细胞生长抑制作用,流式细胞术检测扁蒴藤素诱导HNE2 细胞凋亡,免疫印迹法检测Caspase、 PARP、酪氨酸激酶、AKT 及Bcl-2 家族蛋白表达。结果：扁蒴藤素抑制HNE2 细胞的生长,促使Caspase 9,Caspase 3和PARP 蛋 白被切割,上调Bim 等促凋亡蛋白的表达,减少Bcl-xL等抗凋亡蛋白的表达,下调EGFR 等受体酪氨酸激酶, 抑制AKT 磷酸化, 上调热休克蛋白70（HSP70）的表达。用热休克反应抑制剂KNK437 抑制HSP70 的表达可以增强扁蒴藤素促进细胞凋亡的能力。 结论：扁蒴藤素通过下调受体酪氨酸激酶,激活caspase 介导的凋亡通路抑制鼻咽癌HNE2 细胞的增殖,抑制HSP70 的表达可增 强其抗肿瘤作用。