Production of heat shock proteins, cytokines, and nitric oxide in toxic stress

Expression of heat shock proteins Hsp27, Hsp90, and Hsp70 and production of tumor necrosis factors (TNF-alpha, TNF-beta), interferon-gamma (IFN-gamma), interleukin-2, -3, -6, and nitric oxide (NO) were studied under conditions of acute and chronic intoxication of animals with lipopolysaccharides. Injection of endotoxin increased expression of heat shock proteins Hsp70 and Hsp90-alpha in mouse cells. Acute toxic stress also provoked a sharp increase in the production of TNF-alpha, TNF-beta, and NO in mouse cells. The production of other cytokines (interleukins and IFN-gamma) was changed insignificantly. In the model of chronic toxic stress, changes in the production of Hsp70, Hsp90, TNF, and NO were followed during 11 days after the beginning of the toxin injections. The expression of Hsp70 and Hsp90 in acute stress was significantly higher than at the final stage of the chronic exposure. The changes in the TNF and NO productions, on one hand, and the production of heat shock proteins, on the other hand, were synchronous. The findings indicate that repeated injections of increasing endotoxin doses result in a decreased ability of the body cells to respond to stress by overproduction of heat shock proteins, TNF, and NO.     

The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

The heat shock response (HSR) is a highly conserved molecular response to various types of stresses, including heat shock, during which heat-shock proteins (Hsps) are produced to prevent and repair damages in labile proteins and membranes. In cells, protein unfolding in the cytoplasm is thought to directly enable the activation of the heat shock factor 1 (HSF-1), however, recent work supports the activation of the HSR via an increase in the fluidity of specific membrane domains, leading to activation of heat-shock genes. Our findings support the existence of a plasma membrane-dependent mechanism of HSF-1 activation in animal cells, which is initiated by a membrane-associated transient receptor potential vanilloid receptor (TRPV). We found in various non-cancerous and cancerous mammalian epithelial cells that the TRPV1 agonists, capsaicin and resiniferatoxin (RTX), upregulated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70 and Hsp90 respectively, while the TRPV1 antagonists, capsazepine and AMG-9810, attenuated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70, Hsp90, respectively. Capsaicin was also shown to activate HSF-1. These findings suggest that heat-sensing and signaling in mammalian cells is dependent on TRPV channels in the plasma membrane. Thus, TRPV channels may be important drug targets to inhibit or restore the cellular stress response in diseases with defective cellular proteins, such as cancer, inflammation and aging.     

A natural extracellular factor that induces Hsp72, inhibits apoptosis, and restores stress resistance in aged human cells

Experiments with cultured cells showed that most cellular stress resistance components are specialized for certain types of damage. For example, superoxide dismutase protects from oxidative damage; DNA repair enzymes guard against mutagens and other DNA-damaging agents. On the other hand, the major inducible heat shock protein Hsp72 protects cells from a large variety of stresses and thus represents a generalized repair/stress resistance component. Hsp72 not only refolds damaged proteins but also interferes with programmed cell death signaling pathways, thus providing cells with time to repair the damage, hence its universality as a stress protector. In the present study we demonstrate the occurrence in murine and human ascites fluids (AF) of a natural nontoxic extracellular factor (ascites Hsp72-inducing factor, AHIF) capable of activating Hsp72 expression in different types of cells via a pathway distinct from the heat shock response pathway. AHIF is unique in that it is the first physiological factor capable of inducing synthesis of Hsp72 not only in young cells but, remarkably, also in aged human cells that largely have lost the ability to express Hsp72 in response to stresses, a manifestation at the cellular level of a progressive impairment in the ability to adapt to environmental changes which characterizes aging. Pretreatment of aged human cells with AF triggers Hsp72 expression at levels seen in young stressed cells and protects cells from a variety of otherwise lethal stressful treatments such as heat shock, TNF, UV irradiation, etoposide, and menadione. Activation of Hsp72 expression is essential for antiapoptotic action of AHIF because specific inhibition of Hsp72 expression by antisense RNA abolishes the cytoprotective effect of AF. In view of an important link between stress resistance and longevity in different organisms, the abilities of AHIF make it a unique candidate for the role of a systemic regulator of the aging process. While a cell-autonomous stress response diminishes with aging, aged cells retain the ability to respond to an extracellular factor which induces the expression of Hsp72. This finding opens up exciting possibilities for using AF factor to restore stress resistance to old cells and organisms and the possibility of interfering with the aging process. The ability to induce stress resistance in young cells and to restore it in aged cells could serve as a basis for developing effective antiapoptotic therapies.     

C. elegans STI-1, the Homolog of Sti1/Hop, Is Involved in Aging and Stress Response

Environmental and physiological stresses such as heat shock, oxidative stress, heavy metals, and pathogenic conditions induce cellular stress response. This response is often mediated by heat shock proteins that function as molecular chaperones. A stress-inducible cochaperone, Sti1/Hop (Hsp organizer protein), functions as an adaptor protein that simultaneously binds with Hsp70 and Hsp90 to transfer client proteins from Hsp70 to Hsp90. However, the biological role of STI-1 in vivo is poorly understood in metazoans. Here, we report the characterization of the Caenorhabditis elegans homolog of Sti1/Hop, which is approximately 56% identical with human STI-1. C. elegans STI-1 (CeSTI-1) is expressed in the pharynx, intestine, nervous system, and muscle from larvae to adults. Analysis of proteins immunoprecipitated with anti-STI-1 antibody by mass spectrometry revealed that CeSTI-1 can bind with both Hsp70 and Hsp90 homologs like its mammalian counterpart. sti-1 expression is elevated by heat stress, and an sti-1(jh125) null mutant shows decreased fertility under heat stress conditions. These mutants also show abnormally high lethality in extreme heat and may be functioning with DAF-16 in thermotolerance. In addition, sti-1(jh125) mutants have a shortened life span. Our results confirm that CeSTI-1 is a cochaperone protein that may maintain homeostatic functions during episodes of stress and can regulate longevity in nematodes.     

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp 70 and Hsp60 in response to heat and ethanol

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.     

番茄热激蛋白90的全基因组鉴定及分析

热激蛋白90(Heat shock protein 90,Hsp90)是植物应对不良环境胁迫产生的一类特定的抗逆蛋白。文章以番茄(Solanum lycopersicum L.)基因组数据为平台,借助生物信息学方法对Hsp90基因家族进行鉴定与分析。结果表明,番茄至少含有7个Hsp90基因,不均匀分布在6条染色体上,氨基酸序列长度为267~794aa,内含子数目为2~19;共线性分析发现两对基因(Hsp90-1和Hsp90-3,Hsp90-5和Hsp90-7)以片段重复形式存在。MEME(Multiple Em for Motif Elicitation)分析显示,番茄Hsp90基因编码的氨基酸序列具有多个保守基序;聚类分析揭示番茄、水稻(Oryza sativa L.)和拟南芥(Arabidopsis thaliana L.)Hsp90基因可以分为5组,存在3对直系同源基因和4对旁系同源基因;基于RNA-seq数据库表达分析发现,3个基因(Hsp90-5、Hsp90-6和Hsp90-7)在营养器官和生殖器官中表达量较高,4个基因(Hsp90-1、Hsp90-2、Hsp90-3和Hsp90-4)除在番茄转色后10 d的果实中表达量较高外,其余组织中表达量均较低;对Hsp90基因启动子序列进行分析,发现了多个参与植物对逆境胁迫的顺式作用元件,如HSE、CCAAT-box。此外,qRT-PCR检测结果表明,在叶片热胁迫条件下,番茄Hsp90基因的表达量均存在增强趋势,表明这些基因参与了番茄叶片应对高温胁迫的反应。研究结果为鉴定番茄Hsp90基因的功能和进化起源奠定了基础。     

Reduced thermotolerance in aged cells results from a loss of an hsp72- mediated control of JNK signaling pathway

Aged organisms exhibit a greatly decreased ability to induce the major heat shock protein, Hsp72, in response to stresses, a phenomenon that can also be observed in cell cultures (Heydari AR, Takahashi R, Gutsmann A, You S and Richardson A (1994) Hsp70 and aging. Experientia 50: 1092–1098). Hsp72 was shown to protect cells from a variety of stresses. The protective function of Hsp72 has been commonly ascribed to its chaperoning ability. However, recently we showed that Hsp72 protects cells from heat shock by suppression of a stress-kinase JNK, an essential component of the heat-induced apoptotic pathway (Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S, Shifrin VI and Sherman MY (1997) Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 272: 18033–18037). Here we demonstrate that because of the diminished inducibility of Hsp72 in aged cells, Hsp72-mediated control of JNK signaling pathway is compromised. This results in increased rate of apoptotic cell death following heat shock. We show that forced expression of Hsp72 in aged cells from an adenovirus-based vector completely suppresses activation of JNK by heat shock and consequently protects from heat-induced apoptosis. We also demonstrate for the first time that it is possible to restore endogenous expression of Hsp72 in aged cells. This can be achieved by treatment with the proteasome inhibitor MG132. Induction of Hsp72 in aged cells under these conditions leads to suppression of JNK activation by a heat shock and restoration of thermotolerance manifested in a lower rate of apoptosis.     

Induced hsp70 is in small, cytoplasmic complexes in a cell culture model of renal ischemia: a comparative study with heat shock

A number of clinical conditions are known to result in the induction of heat shock proteins, but detailed studies on stress response have focused mostly on heat shock as a model. We have analyzed the induction and intracellular distribution of heat shock proteins in a reversible adenosine triphosphate (ATP) depletion model of renal ischemia. Two Hsp70 homologues, Hsp70 in the cytoplasm and BiP in the endoplasmic reticulum (ER) lumen, were found significantly induced during the recovery phase of ATP depletion. Other members of the heat shock protein family, such as Hsp90, constitutive Hsc70, and a related protein Hop60, were not induced. The induction of stress proteins on ATP depletion differed from that after heat shock in the kinds of proteins elaborated, their induction kinetics, and their intracellular distributions. Biochemical fractionation and indirect immunofluorescence experiments indicated that Hsp70 was predominantly cytoplasmic in the recovery phase of ischemia-like stress. Velocity sedimentation on sucrose gradients showed that induced Hsp70 sedimented as small, soluble complexes, ranging in size from 4S20,w to 8S20,w. The results suggest a role for induced Hsp70 that may be different from one of protecting aggregated proteins as under heat shock and emphasize the need for their characterization in other clinical conditions that result in stress response.     

Distribution,phosphorylation, and activities of Hsp25 in heat-stressed H9c2 myoblasts: a functional link to cytoprotection

The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) association with F-actin bundles in the cytoplasm. The intranuclear granulation of Hsp25 and its association with F-actin were sensitive to drugs affecting Hsp25 phosphorylation (cantharidin, sodium orthovanadate, SB203580, SB202190). Isoform analysis of Hsp25 translocated to the nucleus-free cytoskeletal fraction revealed only mono- and biphosphorylated Hsp25 and no unphosphorylated Hsp25. Transfected luciferase with initial localization in the nucleosol became colocalized with the Hsp25-containing granules after a heat shock treatment that denatured the enzyme in the cells. The association of Hsp25 with actin filaments after a mild heat stress conferred protection from subsequent F-actin-damaging treatments with cytochalasins (D and B) or severe heat stress. We hypothesize that (1) the binding of heat-denatured nucleosolic proteins to the Hsp25 contained in specific granular structures may serve for the subsequent chaperoning or degradation of the bound proteins, and (2) the actin cytoskeleton is stabilized by the direct targeting of phosphorylated Hsp25 to microfilament bundles.     

棉花粉蚧热休克蛋白基因的鉴定

热休克蛋白(heat shock proteins,Hsps)是生物体或细胞受到热胁迫后新合成的一类遗传上高度保守的蛋白,在昆虫应对外界环境因子胁迫时起着重要作用。为了系统研究棉花粉蚧Phenacoccus solenopsis Hsp基因家族,对棉花粉蚧转录组基因注释信息进行分析、获得目标序列,并应用NCBI上Blast X等软件进行比对、共鉴定出24条热激蛋白(Hsp)基因,包括3个Hsp90、8个Hsp70、2个Hsp60和11个s Hsp(small heat shock protein,s Hsp)基因。对棉花粉蚧与模式昆虫家蚕Bombyx mori、黑腹果蝇Drosophila melanogaster、赤拟谷盗Tribolium castaneum系统进化关系分析显示,昆虫的小分子量热休克蛋白s Hsp具有很强的种属特异性,Hsp70家族的保守性比s Hsp强。棉花粉蚧热激蛋白基因的鉴定为深入研究该虫Hsp与生长发育、抗逆境的相互关系奠定了基础。     

Stress-induced inhibition of the NF-kappaB signaling pathway results from the insolubilization of the IkappaB kinase complex following its dissociation from heat shock protein 90

Activation of the stress response attenuates proinflammatory responses by suppressing cytokine-stimulated activation of the NF-kappaB signaling pathway. In this study, we show that the activation of the cellular stress response, either by heat shock treatment or after exposure to sodium arsenite, leads to a transient inhibition of IkappaBalpha phosphorylation. Inhibition of IkappaBalpha phosphorylation after stress was associated with the detergent insolubilization of the upstream kinases, IkappaB kinase alpha (IKKalpha) and IkappaB kinase beta, components involved in IkappaBalpha phosphorylation. Pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of stress-induced protein denaturation, reduced the stress-dependent detergent insolubility of the IKK complex and restored the cytokine-stimulated phosphorylation of IkappaB. The stress-dependent insolubility of the IKK complex appeared reversible; as the cells recovered from the heat shock treatment, the IKK complex reappeared within the soluble fraction of cells and was again capable of mediating the phosphorylation of IkappaBalpha in response to added cytokines. Treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) function, also resulted in IKK detergent insolubility and proteasome-mediated degradation of the IKK complex. Furthermore, while IKKalpha coprecipitated with Hsp90 in control cells, coprecipitation of the two proteins was greatly reduced in those cells early after stress or following exposure to geldanamycin. Stress-induced transient insolubilization of the IkappaB kinase complex following its dissociation from Hsp90 represents a novel mechanism by which the activation of the stress response inhibits the NF-kappaB signaling pathway in response to proinflammatory stimuli.     

Characterization of a small heat shock protein, Mx Hsp16.6, of Myxococcus xanthus

A number of heat shock proteins in Myxococcus xanthus were previously identified by two-dimensional (2D) gel electrophoresis. One of these protein was termed Mx Hsp16.6, and the gene encoding Mx Hsp16.6 was isolated. Mx Hsp16.6 consists of 147 amino acid residues and has an estimated molecular weight of 16,642, in accordance with the apparent molecular mass in the 2D gel. An alpha-crystallin domain, typically conserved in small heat shock proteins, was found in Mx Hsp16.6. Mx Hsp16.6 was not detected during normal vegetative growth but was immediately induced after heat shock. Expression of the hsp16.6 gene was not induced by other stresses, such as starvation, oxidation, and high osmolarity. Mx Hsp16.6 was mostly localized in particles formed after heat shock and precipitated by low-speed centrifugation. Furthermore, Mx Hsp16.6 was detected in highly electron-dense particles in heat-shocked cells by immunoelectron microscopy, suggesting that it forms large complexes with heat-denatured proteins. An insertion mutation in the hsp16.6 gene resulted in lower viability during heat shock and lower acquired thermotolerance. Therefore, it is likely that Mx Hsp16.6 plays critical roles in the heat shock response in M. xanthus.     

Heat shock proteins 90 kDa: diversity, structure, functions

The review presents data on diversity, structure, functions and gene expression of the high conserved family of heat shock proteins 90 kDa (Hsp90). They are specialized molecular chaperones that fulfill the folding, maintenance of structural integrity and conformational regulation of a subset of proteins involved in important cellular processes, such as transduction of signals, cell cycle control etc. A composition and functioning of the Hsp90 chaperone machine are considered. Hsp90s play a significant role in growth and development of organisms carrying out conformational regulation of many regulatory proteins and protecting cells under stress. The review summarizes the results of investigations of different organisms, mainly animals and yeasts, with emphasis on the facts on Hsp90s in plants.     

Heat shock responses for understanding diseases of protein denaturation

Extracellular stresses induce heat shock response and render cells resistant to lethal stresses. Heat shock response involves induction of heat shock proteins (Hsps). Recently the roles of Hsps in neurodegenerative diseases and cancer are attracting increasing attention and have accelerated the study of heat shock response mechanism. This review focuses on the stress sensing steps, molecules involved in Hsps production, diseases related to Hsp malfunctions, and the potential of proteomics as a tool for understanding the complex signaling pathways relevant to these events.