Proteasome inhibition in oxidative stress neurotoxicity: implications for heat shock proteins

Recent studies have demonstrated that inhibition of the proteasome, an enzyme responsible for the majority of intracellular proteolysis, may contribute to the toxicity associated with oxidative stress. In the present study we demonstrate that exposure to oxidative injury (paraquat, H(2)O(2), FeSO(4)) induces a rapid increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential, inhibition of proteasome activity, and induction of cell death in neural SH-SY5Y cells. Application of proteasome inhibitors (MG115, epoxomycin) mimicked the effects of oxidative stressors on mitochondrial membrane potential and cell viability, and increased vulnerability to oxidative injury. Neural SH-SY5Y cells stably transfected with human HDJ-1, a member of the heat shock protein family, were more resistant to the cytotoxicity associated with oxidative stressors. Cells expressing increased levels of HDJ-1 displayed similar degrees of ROS formation following oxidative stressors, but demonstrated a greater preservation of mitochondrial function and proteasomal activity following oxidative injury. Cells transfected with HDJ-1 were also more resistant to the toxicity associated with proteasome inhibitor application. These data support a possible role for proteasome inhibition in the toxicity of oxidative stress, and suggest heat shock proteins may confer resistance to oxidative stress, by preserving proteasome function and attenuating the toxicity of proteasome inhibition.     

集胞藻PCC6803中S2P同源蛋白基因sll0862缺失突变株对热胁迫与氧化胁迫的响应

原核生物中S2P参与应答外界环境刺激,然而行光合作用的蓝细菌-集胞藻PCC6803的S2P同源蛋白功能未知。【目的】考察集胞藻PCC6803中S2P同源蛋白sll0862是否参与外界环境刺激的应答。【方法】监测在高温和氧化胁迫的条件下sll0862基因缺失突变株与野生株在生长速率或存活率上的差异,利用水样调制叶绿素荧光仪(water-PAM,脉冲-振幅-调制叶绿素荧光仪)测量在高温和氧化胁迫的条件下突变株与野生株叶绿素荧光参数的差异,来考察其光合作用差异。【结果】sll0862突变株与野生株在正常的培养环境中生长速率并无差异,但是将sll0862突变株与野生株在48℃加热处理半小时后,sll0862突变株的存活率明显低于野生株。当初始OD730值为0.1的藻液中添加终浓度为1 mmol/L双氧水的时候,sll0862突变株的生长速率比野生株明显低,而且氧化胁迫条件下突变株与野生株的调制叶绿素荧光有差异。【结论】集胞藻PCC6803中sll0862基因的缺失导致突变体对高温与氧化胁迫响应出现缺陷,提示有功能的sll0862参与响应热和氧化胁迫。研究结果为进一步阐述S2P同源蛋白sll0862在集胞藻PCC6803中的功能奠定基础。     

Heat shock proteins and effects of heat shock in plants

Soybean seedlings when exposed to a heat shock respond in a manner very similar to that exhibited by cultured cells, and reported earlier [2]. Maximum synthesis of heat shock proteins (HSPs) occurs at 40C. The heat shock response is maintained for a relatively short time under continuous high temperature. After 2.5 hr at 40 C the synthesis of HSPs decreases reaching a very low level by 6 hr. The HSPs synthesized by cultured cells and seedlings are identical and there is a large degree of similarity in HSPs synthesized between the taxonomically widely separated species, soybean and corn. Storage protein synthesis in the developing soybean embryo is not inhibited but is actually stimulated during a heat shock, unlike most other non-HSPs, whose synthesis is greatly reduced. Seedlings respond differently to a gradual increase in temperature than they do a sudden heat shock. There is an upward shift of several degrees in the temperature at which maximum protein synthesis occurs and before it begins to be inhibited. In addition, there appears to be a protection of normal protein synthesis from heat shock inhibition when the temperature increase is gradual. An additional function of the heat shock phenomenon might be the protection of seedlings from death caused by extreme heat stress. The heat shock response appears to have relevance to plants in the field.     

Vitamin C mitigates heat damage by reducing oxidative stress,inducing HSP expression in TM4 Sertoli cells

Heat stress is a major stressor that can lead to male reproductive dysfunction. Sertoli cells play a crucial role in spermatogenesis by providing germ cells with structural and nutritional support, and contributing to blood–testis barrier formation. Vitamin C (Vc) is an antioxidant capable of neutralizing reactive oxygen species and preventing lipid peroxidation widely used because it is inexpensive and highly accessible. In the present study, we investigated the protective effect of Vc on TM4 cells following heat stress. Pretreatment with Vc could effectively inhibit apoptosis (p < 0.01), lipid peroxidation, and lactate dehydrogenase (LDH) activity. However, a significant increase in the malondialdehyde (MDA) level and LDH activity (p < 0.01) was observed in TM4 cells without Vc‐pretreatment, in conjunction with vacuole degeneration and karyopyknosis. In addition, both the messenger RNA and protein levels of CryAB, Hsp27, Hsp70, and Hsp110 substantially increased in the 3 and 12 hr recovery groups (p < 0.01). Vc also prevented microtubule aggregation following heat stress. These results suggest that pretreatment with Vc‐protected TM4 cells against heat stress by reducing the level of oxidative stress and inducing heat shock protein expression.     

Effect of heat stress on development in vitro and in vivo and on synthesis of heat shock proteins in porcine embryos

The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42°–45.5°C and for 10–180 min) was examined. Synthesis of 70 kDa hsp (hsp 70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42°C-10 min (236.6 ± 71.4; P < 0.05) and 43°C-30 min (276.8 ± 89.4; P < 0.005) compared to control (173.9 ± 53.9). The 42°C-180 min group (158.0 ± 27.1 μm) had a greater increase in diameter after 24 hr in culture following heat stress compared to control (82.5 ± 47.3 μm), while heat stress with 43°C for ≧60 min, 44°–44.5°C for ≧30 min, or 45°-45.5°C for ≧10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42°C-180 min, 43°C-10 min, 43°C-30 min, 44°C-10 min, or 45°C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42°C for 180 min, 43°C for 30 min, 44°C for 10 min, and 45°C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute and temporary rise in temperature. However, no increase of hsp70 and hsp90 was observed in the heat-stressed porcine embryos, while hsp70 was detected in the nonheat-stressed porcine embryos. The precise mechanism of the thermotolerance was unclear. © 1996 Wiley-Liss, Inc.     

Molecular genetics of heat tolerance and heat shock proteins in cereals

Heat stress is common in most cereal-growing areas of the world. In this paper, we summarize the current knowledge on the molecular and genetic basis of thermotolerance in vegetative and reproductive tissues of cereals. Significance of heat stress response and expression of heat shock proteins (HSPs) in thermotolerance of cereal yield and quality is discussed. Major avenues for increasing thermotolerance in cereals via conventional breeding or genetic modification are outlined.     

Selenium and vitamin E ameliorate lead acetate-induced hepatotoxicity in rats via suppression of oxidative stress,mRNA of heat shock proteins,and NF-kB production

BackgroundLead exposure results in a terrible rise in heat shock protein levels.ObjectiveThis research was conducted to look at the effects of lead poisoning on heat shock response, oxidative stress, and inflammatory markers in albino rats, as well as the power of selenium and vitamin E to resist lead toxic effects.MethodsEight groups of albino rats are used. Each group contained six rats where the first group represented the negative control, and the other groups were treated with olive oil, vitamin E, selenium, lead, (vitamin E + lead), (selenium + lead), and (vitamin E + selenium + lead). All the treatments lasted for 28 days. Then, the mRNA expression of interested heat shock proteins (HSP90, HSP70, and HSP60) was assessed. For oxidative stress disruption, we investigated nitric oxide (NO) and malondialdehyde (MDA) content, and enzymatic and non-enzymatic antioxidants activity respectively in rat livers.Resultsour results revealed the synergetic protective effect of the combination of two antioxidants (vitamin E and selenium) against lead poising. This was clear in regulating HSPs expression, inflammatory markers, glucose, lipid profile, liver functions, and antioxidant enzymes more than the treatment with one antioxidant.ConclusionPb is a toxic material that can induce HSPs and inflammatory markers expression. Selenium and vitamin E can give excellent effects in ameliorating Pb toxicity when used together.     

Adaptation to heat of cardiomyoblasts in culture protects them against heat shock: role of nitric oxide and heat shock proteins

Dosed adaptation to environmental factors is an efficient non-drug means for increasing the resistance of organs or the body as a whole. We demonstrated earlier that nitric oxide (NO) plays an important role in adaptive defense of the organism, in particular due to activation of heat shock protein (HSP) synthesis. A key question remained open—to what extent the formation of adaptive defense depends on central mechanisms and to what extent on the intracellular mechanisms immediately responding to the adapting factor, and whether the NO-dependent activation of HSP synthesis plays a role in adaptation of isolated cells. In the present study we looked into the possibility of producing a protective effect of adaptation to heat in cell culture. A 6-day adaptation to heat limited to 17% the decrease in metabolic activity induced by heat shock in H9c2 cardiomyoblasts. The development of adaptation was associated with increased NO production. Treatment of cells with the inhibitor of NO synthase L-NNA (100 M) prevented the development of adaptive protection. Adaptation of cell culture enhanced synthesis of HSP70 but not HSP27. Blockade of HSP70 synthesis with quercetin (50 M) left unchanged the protective effect of adaptation. Inhibition of NO synthesis restricted the adaptation-induced HSP70 synthesis. Therefore, the formation of adaptation at the cell level may result from a direct action of an environmental factor without participation of neurohumoral factors. Such adaptation involves NO-dependent mechanisms divorced from the activation of HSP70 synthesis.     

Mobile phones, heat shock proteins and cancer

There are several reports which indicate that electromagnetic radiation (such as from mobile phones) at non-thermal levels may elicit a biological effect in target cells or tissues. Whether or not these biological effects lead to adverse health effects, including cancer, is unclear. To date there is limited scientific evidence of health issues, and no mechanism by which mobile phone radiation could influence cancer development. In this paper, we develop a theoretical mechanism by which radiofrequency radiation from mobile phones could induce cancer, via the chronic activation of the heat shock response. Upregulation of heat shock proteins (Hsps) is a normal defence response to a cellular stress. However, chronic expression of Hsps is known to induce or promote oncogenesis, metastasis and/or resistance to anticancer drugs. We propose that repeated exposure to mobile phone radiation acts as a repetitive stress leading to continuous expression of Hsps in exposed cells and tissues, which in turn affects their normal regulation, and cancer results. This hypothesis provides the possibility of a direct association between mobile phone use and cancer, and thus provides an important focus for future experimentation.     

AtHsfA2 modulates expression of stress responsive genes and enhances tolerance to heat and oxidative stress in Arabidopsis

In nature, plants are subject to changes of tempera-ture. Thus, like other organisms, plants have evolved strategies for preventing damage caused by rapid changes in temperature and for repairing what damage is unavoidable. Heat stress responses have been well documented in a wide range of organisms. In all spe-cies studied, the heat shock (HS) response is charac-terized by a rapid production and a transient accumu-lation of specific families of proteins known as heat shock proteins (Hsps) th…     

Induction of stress response and differential expression of 70 kDa stress proteins by sodium fluoride in hela and rat brain tumor 9l cells

We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr-phosphatases but not to Tyr-phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali-resistant 65-kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat-shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat-shock protein70 (the glucose-regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well-characterized regulatory elements revealed the activation of the heat-shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF-κB, AP-2, and CRE-like elements were detected in NaF-treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed. J. Cell. Biochem. 69:221–231, 1998. © 1998 Wiley-Liss, Inc.     

Agonistic encounters and cellular angst: social interactions induce heat shock proteins in juvenile salmonid fish

Juvenile salmonid fish readily form dominance hierarchies when faced with limited resources. While these social interactions may result in profound behavioural and physiological stress, it is unknown if this social stress is evident at the level of the cellular stress response—specifically, the induction of stress or heat shock proteins (Hsps). Thus, the goal of our study was to determine if Hsps are induced during hierarchy formation in juvenile rainbow trout (Oncorhynchus mykiss). To this end, we measured levels of three Hsps, Hsp70, Hsc (heat shock cognate)70 and Hsp90 in the white muscle, liver and brain of trout that had been interacting for 36 h, 72 h or 6 days. Our data indicate that Hsps are induced in both dominant and subordinate fish in a time- and tissue-specific manner. In further mechanistic experiments on fasted and cortisol-treated fish, we demonstrated that high plasma cortisol does not affect Hsp induction in trout white muscle or liver, but both conditions may be part of the mechanism for Hsp induction with social stress in the brain. We conclude that the behavioural and physiological stress experienced by juvenile rainbow trout in dominance hierarchies can be extended to the induction of Hsps.     

Guidelines for the nomenclature of the human heat shock proteins

The expanding number of members in the various human heat shock protein (HSP) families and the inconsistencies in their nomenclature have often led to confusion. Here, we propose new guidelines for the nomenclature of the human HSP families, HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), and HSPB (small HSP) as well as for the human chaperonin families HSPD/E (HSP60/HSP10) and CCT (TRiC). The nomenclature is based largely on the more consistent nomenclature assigned by the HUGO Gene Nomenclature Committee and used in the National Center of Biotechnology Information Entrez Gene database for the heat shock genes. In addition to this nomenclature, we provide a list of the human Entrez Gene IDs and the corresponding Entrez Gene IDs for the mouse orthologs.     

Effects of heat shock on in vitro development and intracellular oxidative state of bovine preimplantation embryos

We investigated the effects of heat shock on developmental competence of bovine embryos and intracellular oxidative state. After in vitro fertilization, embryos were exposed to heat shock at 41 degrees C for 6 hr on days 0, 2, 4, and 6, respectively. On day 2, cleavage rate was not significantly different in all groups. However, the percentage of embryos developing to blastocyst stage after exposure to heat shock on day 0 (18.8 +/- 4.3%) and day 2 (23.6 +/- 3.7%) were significantly decreased compared with control (37.5 +/- 4.0%), day 4 (40.0 +/- 7.4%), and day 6 (38.1 +/- 2.0%). In addition, the total cell number of blastocysts was significantly decreased by heat shock on day 0 (107.5 +/- 6.6) and day 2 (112.8 +/- 5.7) compared with the control (143.2 +/- 9.4). To evaluate intracellular oxidative state by heat shock, embryos exposed to heat shock on days 0, 2, 4, and 6 were incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCHFDA) and fluorescence of oxidized DCHFDA by reactive oxygen species (ROS) was detected under fluorescent microscope. The intensity of fluorescence was significantly increased when embryos were exposed to heat shock on days 0 and 2. However, heat shock on day 4 and day 6 did not increase the fluorescence intensity. These results indicate that (1) heat shock to earlier stage embryos causes a decrease in development to blastocysts and cell proliferation and (2) the decrease in development by heat shock could be involved in an increase of intracellular oxidative stress. Mol. Reprod. Dev. 67: 77-82, 2004.     

Small heat shock proteins and adaptation of various Drosophila species to hyperthermia

The dynamics and the level of accumulation of small heat shock proteins (sHSP group 21–27) after a heat exposure were studied in three Drosophila species differing in thermotolerance. The southern species Drosophila virilis, having the highest thermotolerance, surpassed thermosensitive D. lummei and D. melanogaster in the level of sHSPs throughout the temperature range tested. The results suggest an important role of sHSPs in the molecular mechanisms of adaptation to adverse environmental conditions, particularly to hyperthermia.     

Studies on heat shock proteins in sea urchin development

Work on stress proteins in sea urchin embryos carried out over the last 20 years is reviewed and the following major results are described. Entire sea urchin embryos, if subjected to a rise in temperature at any postblastular stage undergo a wave of heat shock protein (hsp) synthesis and survive. If subjected to the same rise between fertilization and blastula formation, they are not yet able to synthesize hsp and die. Four clones coding for the major hsp, hsp70, have been isolated and sequenced; evidence for the existence of a heat shock factor has been provided, and a mechanism for the developmental regulation of hsp synthesis discussed. Intraembryonic and intracellular hsp location has been described; and a mechanism for achievement of thermotolerance proposed. A chaperonine role for a constitutive mitochondrial hsp56 has been suggested, as well as a role for the constitutive hsp70 in cell division. Heat shock, if preceded by 12-O-tetradecanoylphorbol-12-acetate (TPA) treatment causes apoptosis.     

果蝇热激蛋白的研究进展

热休克蛋白(heat shock proteins,HSPs)是生物体受到应激刺激时诱导产生的一组保守性蛋白,普遍存在于各种生物体中。近年来,果蝇Drosophila作为生命科学与人类疾病研究的重要模式生物,其热激蛋白的研究取得了许多新的进展。文章对果蝇热激蛋白的类别、热激蛋白基因的表达调控机制、热激蛋白的分子伴侣功能、调节细胞存亡和影响发育及寿命等相关生物学功能进行综述,并对热激蛋白在神经退行性疾病治疗中的应用前景作展望。     

Respiratory activity of yeast Yarrowia lipolytica under oxidative stress and heat shock

Heat shock (45°C) and the effect of oxidants (H₂O₂) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37°C) or treatment with a nonlethal dose of oxidants (0.5 mM H₂O₂) for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.     

Differential effect of oxidative stress on the apoptosis of early and late passage human diploid fibroblasts: implication of heat shock protein 60

Since an attenuated response to stress is a characteristic of senescence, a cellular senescence model was used to examine the mechanism of resistance against oxidative stress using human diploid fibroblasts (HDF). With increasing passage, the HDF showed increased production of reactive oxygen species (ROS). Late passage HDF were resistant to the lethal effects of oxidative stress, showing less cleavage of pro-caspase-3 and PARP than those of early ones. Since heat shock proteins (Hsps) are not only cytoprotective but also interfere with the apoptotic cascade, the expression patterns of Hsps during cellular senescence were next examined. Oxidative stress induced a decrease in the mitochondrial Hsp60 levels with a concomitant increase in the cytosolic Hsp60 levels in the early passage HDF, but not in late ones. To show that the resistance to oxidative stress is a specific effect of Hsp60, the levels of Hsp60 were knocked down by siRNA. As expected the Hsp60 knock-down cells were more resistant to oxidative stress. These findings show that Hsp60 is a key player in the resistance mechanism against oxidative stress and aging.