Identification of the small heat shock protein, HSP21, of shrimp Penaeus monodon and the gene expression of HSP21 is inactivated after white spot syndrome virus (WSSV) infection

The white spot syndrome virus (WSSV) is the causative agent of a severe disease in cultivated shrimp. The virus causes high mortality and leads to heavy stress on shrimps. In response to a variety of stresses, living organisms express particular sets of genes such as HSPs. In this study, a HSP21 gene, categorized into the small heat shock protein (smHSP) family, of shrimp Penaeus monodon was identified by annotating the EST databases established from WSSV-infected and WSSV-uninfected shrimp. The shrimp HSP21 gene was 555 bp in length. The thermal aggregation assay showed that the HSP21 had chaperone activity. The result of real-time PCR indicated that HSP21 was constitutive and inducible and was highly expressed in almost all organs such as the epithelium, gill, stomach, midgut, lymphoid organ, hepatopancreas, nervous tissue and heart, but less expressed in haemolymph. However, HSP21 gene showed down-regulation after WSSV infection. It suggests that gene regulation of HSP21 was seriously affected by WSSV.     

Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp, <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Heat shock proteins (Hsps) are a class of highly conserved proteins produced in virtually all living organisms from bacteria to humans. Hsp60 and Hsp10, the most important mitochondrial chaperones, participate in environmental stress responses. In this study, the full-length complementary DNAs (cDNAs) of Hsp60 (PmHsp60) and Hsp10 (PmHsp10) were cloned from Penaeus monodon. Sequence analysis showed that PmHsp60 and PmHsp10 encoded polypeptides of 578 and 102 amino acids, respectively. The expression profiles of PmHsp60 and PmHsp10 were detected in the gills and hepatopancreas of the shrimps under pH challenge, osmotic stress, and heavy metal exposure, and results suggested that PmHsp60 and PmHsp10 were involved in the responses to these stimuli. ATPase and chaperone activity assay indicated that PmHsp60 could slow down protein denaturation and that Hsp60/Hsp10 may be combined to produce a chaperone complex with effective chaperone and ATPase activities. Overall, this study provides useful information to help further understand the functional mechanisms of the environmental stress responses of Hsp60 and Hsp10 in shrimp.     

Molecular identification and expression of heat shock cognate 70 (HSC70) in the Pacific white shrimp Litopenaeus vannamei

Heat shock protein 70s (HSP70s) are fundamental chaperone proteins that are indispensable to most living organisms. In order to investigate the function of HSP70 and heat shock response in shrimp, a heat shock cognate (HSC70) gene of the white shrimp (Litopenaeus vannamei), containing a 1959-bp open reading frame, was cloned and characterized. The amino acid sequence, 71.5 kDa of molecular weight, shares 80-99.6% homology with 12 diverse species' HSP70s and HSC70s. In fact, some segments of the eukaryotic HSC70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp HSC70. Moreover, multi-tissue RT-PCR was performed to assay the basal expressions of HSC70 in the heart, gill, hepatopancreas, stomach, gut, and muscle. The results demonstrate that the basal expressions of HSC70 in theses organs are similar to that of beta-actin. Furthermore, quantitative real-time experiments showed that HSC70 was up-regulated in hepatopancreas (4.6-fold), stomach (5.9-fold), gut (2.6-fold), and muscle (3.5-fold) but not in the heart (1.7-fold) and gill (1.6-fold) after 2 h of heat shock. Nevertheless, the HSC70 was found to be highly expressed in the heart and gill following 6 h of heat shock. This suggests that HSC70 in white shrimp possess both short-term and long-term responses to heat shock stress, indicating this HSC70 may be a heat-dependent HSC70 member. Finally, we constructed an expression vector to generate HSC70 in Escherichia coli BL21, which displayed immune cross-reactivity with mouse HSP70 antibody. In conclusion, the identification and expression of white shrimp HSC70 gene present useful data for studying the molecular mechanism of heat shock response and the effect of heat shock proteins in shrimps' cytoprotection.     

Stress-induced interaction between p38 MAPK and HSP70

p38 MAPK, one of the four MAPK subfamilies in mammalian cells, is activated by environmental stresses and pro-inflammatory cytokines, playing fundamental roles in many biological processes. Despite all that is known on the structure and functions of p38, many questions still exist. The coupling of activation and nuclear translocation represents an important aspect of p38 signaling. In our effort in exploring the potential chaperone for p38 translocation, we performed an endogenous pull-down assay and identified HSP70 as a potential interacting protein of p38. We confirmed the interaction between p38 and HSP70 in vitro and in vivo, and identified their interaction domains. We also showed stress-induced nuclear co-localization of these two proteins. Our preliminary result indicated that HSP70 was related to the phosphorylation of MK2, a specific nuclear downstream target of p38, suggesting HSP70 is a potential chaperone for the nuclear translocation of p38.     

Triggers of the HSP70 stress response: environmental responses and laboratory manipulation in an Antarctic marine invertebrate (Nacella concinna)

The Antarctic limpet, Nacella concinna, exhibits the classical heat shock response, with up-regulation of duplicated forms of the inducible heat shock protein 70 (HSP70) gene in response to experimental manipulation of seawater temperatures. However, this response only occurs in the laboratory at temperatures well in excess of any experienced in the field. Subsequent environmental sampling of inter-tidal animals also showed up-regulation of these genes, but at temperature thresholds much lower than those required to elicit a response in the laboratory. It was hypothesised that this was a reflection of the complexity of the stresses encountered in the inter-tidal region. Here, we describe a further series of experiments comprising both laboratory manipulation and environmental sampling of N. concinna. We investigate the expression of HSP70 gene family members (HSP70A, HSP70B, GRP78 and HSC70) in response to a further suite of environmental stressors: seasonal and experimental cold, freshwater, desiccation, chronic heat and periodic emersion. Lowered temperatures (−1.9°C and −1.6°C), generally produced a down-regulation of all HSP70 family members, with some up-regulation of HSC70 when emerging from the winter period and increasing sea temperatures. There was no significant response to freshwater immersion. In response to acute and chronic heat treatments plus simulated tidal cycles, the data showed a clear pattern. HSP70A showed a strong but very short-term response to heat whilst the duplicated HSP70B also showed heat to be a trigger, but had a more sustained response to complex stresses. GRP78 expression indicates that it was acting as a generalised stress response under the experimental conditions described here. HSC70 was the major chaperone invoked in response to long-term stresses of varying types. These results provide intriguing clues not only to the complexity of HSP70 gene expression in response to environmental change but also insights into the stress response of a non-model species.     

Impact of seasonal variation on HSP70 expression quantitated in stressed fish hepatocytes

Heat shock protein 70 (HSP70) is an effective molecular chaperone, playing a role in cell protection from damage in response to stress stimuli. Here, we report the impact of environmental stress on hepatocyte HSP70 expression in Mugil cephalus living in either a contaminated (Ennore) or uncontaminated (Kovalam) estuary over the course of two seasons. Oxidative and nitrative stress was determined along with quantification of HSP70 by enzyme-linked immunosorbent assay (ELISA) after electroelution from polyacrylamide gels. Fish from Ennore showed significantly higher levels of oxidative and nitrative stress and HSP70 expression than fish from Kovalam. Also, there was significant seasonal variation in all oxidative, nitrative stress marker levels and HSP70 expression which peaked during summer. These results provide direct evidence that HSP70 overexpression in fish hepatocytes under stress may aid cell survival by protecting against oxidative and nitrative stress-induced changes. In addition, seasonal variation may have a significant impact on HSP70 expression.     

Identification of a novel inducible cytosolic Hsp70 gene in Chinese shrimp <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> and comparison of its expression with the cognate Hsc70 under different stresses

The heat shock protein 70 (Hsp70) family is widely expressed in eukaryotic cells as the major chaperone protein. In this study, the full-length complementary DNA (cDNA) of a novel inducible cytosolic Hsp70 family member (FcHsp70) was cloned from Fenneropenaeus chinensis. FcHsp70 full-length cDNA consists of 2,511 bp with a 1,890-bp open reading frame encoding 629 amino acids. Three Hsp70 protein family signatures, IDLGTTYS, IIDLGGGTFDVSIL, and IVLVGGSTRIPKVQK, were found in the predicted FcHsp70 amino acid sequence. Phylogenetic analysis showed that FcHsp70 was categorized together with the inducible HSP70s reported in other crustaceans. Compared to the previously identified cognate Hsp70 (FcHsc70) in F. chinensis, the expression of FcHsp70 showed quite different expression profiles when the shrimp were subjected to different stresses including heat shock and heavy metal treatments. Under heat shock treatment, the expression of FcHsp70 showed much higher up-regulation than FcHsc70. Copper treatment also induced higher up-regulation of FcHsp70 than FcHsc70. Cadmium treatment did not induce the expression of FcHsp70, but caused down-regulation of FcHsc70. The different expression profiles of FcHsp70 and FcHsc70 in shrimp may indicate their different reactions to different stresses. Therefore, Hsp70 or Hsc70 could be developed as a biomarker to indicate different stresses in shrimp.     

Aging effects on the habitual expression of HSP70 mRNA in the hippocampus of rats

Heat shock proteins are induced by stressful stimuli and have been shown to protect cells and organs from such stresses both in vitro and in vivo. This study examined the regulation of HSP70 mRNA expression and detected the effect of aging on RNA expression in hippocampus of rats. The stress models were built by using forced-swimming in 25 degrees C and 4 degrees C water, respectively. Two groups of male rats, 2-month-old and 16-month-old, respectively, were randomly divided into three subgroups: acute stress (AS) model, chronic habituation stress (CHS) model and chronic dishabituation stress (CDS) model. Observation of exploratory behavior in an open-field (OF) test indicated stress levels. The expression of HSP70 mRNA in hippocampus was measured by RT-PCR after 0, 30, 60, 180, and 360 min of stress, respectively. Results showed that the number of quadrant crossing in both aged CHS and young CHS groups decreased gradually with the process of stress, reflecting an adaptation to the stress condition. Repeated swimming in warm water resulted in habitual expression of HSP70 mRNA in both young and aged CHS group, indicating an adaptation to the stress. The RNA expression of young CHS group was significantly stronger than that of the aged CHS group at 30, 60, 180, and 360 min after stress (P < 0.05). Meanwhile, in an intensive stress level in which the rats swam in 4 degrees C water, a high expression level of HSP70 mRNA was achieved in CDS groups, producing a dishabituation that proved the habitual expression from the other side. These results showed that senescence dramatically affected both exploratory behavior and HSP70 mRNA expression in rats' hippocampus. The results also suggested that chronic stress could lead to the habituational expression of HSP70 mRNA, but high intensive stress could reverse the habituational state and lead to the dishabituational expression. Moreover, the duration of stimuli is one of the important factors that affect the level of HSP70 mRNA expression.     

Induction of heat-shock (stress) protein gene expression by selected natural and anthropogenic disturbances in the octocoral Dendronephthya klunzingeri

Previously it was found that the expression of selected heat-shock proteins is upregulated in corals after exposure to elevated temperature. We published that HSPs are suitable markers in sponges to monitor the degree of environmental stress on these animals. In the present study the heat-shock proteins (HSPs) with a molecular weight of 90 kDa have been selected to prove their potential usefulness as biomarkers under controlled laboratory conditions and in the field. The studies have been performed with the octocoral Dendronephthya klunzingeri4.5-fold higher steady-state level of the respective mRNA. Also animals taken from stressed locations in the field showed an increased expression. The amount of HSP90 protein in D. klunzingeri was found to be strongly increased under thermal stress, or exposure to polychlorinated biphenyl (congener 118), but not after treatment with cadmium. Field studies revealed that samples taken from a nonstressed area have a low level of HSP90, but those collected from locations at which the corals are under physical stress (sedimentation through landfilling) show a high expression of HSP90. It is concluded that the chaperone HSP90 might become a suitable biomarker to monitor environmental stress on corals.     

Nitroreductase-based therapy of prostate cancer, enhanced by raising expression of heat shock protein 70, acts through increased anti-tumour immunity

Gene-directed enzyme-prodrug therapy (GDEPT) using nitroreductase (NTR), with efficient adenoviral delivery, and CB1954 (CB), is an effective means of directly killing tumours. However, an immune-mediated bystander effect remains an important product of GDEPT since it is often critical to the elimination of untransduced tumour cells both locally and at distal metastatic sites through generation of tumour-specific immunity without the need for tumour antigen identification or the generation of a personalised vaccine. The mode of induced tumour cell death is thought to contribute to the immunisation process, together with the induction and release of stress proteins. Here, RM-9 murine prostate tumour cells were efficiently killed by adenovirally delivered NTR/CB treatment both in vitro and in vivo, and bystander effects were observed. Cells appeared to die by pathways that suggest necrosis more than that of classical apoptosis. NTR/CB-induced expression of a range of stress proteins was determined by proteomic analysis, revealing chiefly heat shock protein (HSP)25 and HSP70 upregulation, whilst immune responses in vivo were weak. In an attempt to enhance the anti-tumour effect, an adenoviral vector was constructed that co-expressed NTR and HSP70, the latter being a known immune stimulator and chaperone of antigen. This combination elicited significantly enhanced protection over NTR alone for both the treated tumour and a subsequent re-challenge. Protection was CD4⁺ and CD8⁺ T cell-dependent and was associated with tumour-specific CTL, IFNγ and IL-5 responses. The use of such a cytotoxic and immunomodulatory gene combination in cancer therapy warrants further pursuit.     

三角帆蚌热休克蛋白60基因克隆及其表达分析

利用3'RACE技术, 对高通量转录组测序所得三角帆蚌HSP60基因（hcHSP60）长片段（2629 bp）进行了3'末端克隆, 经拼接得到hcHSP60 cDNA全长序列。采用多种分子生物学软件对hcHSP60 cDNA全长序列进行了特征分析, 并采用RT-PCR技术检测了其组织分布及经冷热应激后的表达变化。结果显示, hcHSP60 cDNA全长为2807 bp, 其中开放阅读框为1707 bp, 编码568个氨基酸, 预测分子量大小为61.04 ku, pH 7.0时的理论等电点为5.63。序列中不存在信号肽与跨膜结构。氨基酸序列保守性分析表明, hcHSP60氨基酸序列与光滑双脐螺HSP60同源性最高（达82%）, 而与牙鲆、白云金丝鱼HSP60的同源性最低（为75%）。RT-PCR检测结果表明, hcHSP60在肝胰腺中的表达水平最高, 而在血液中的表达水平最低。30℃与35℃水温处理三角帆蚌4h后, 各组织中hcHSP60表达水平明显上升, 表明hcHSP60可能在三角帆蚌耐热应激反应中起着重要作用。       

HSP70 inhibition by the small-molecule 2-phenylethynesulfonamide impairs protein clearance pathways in tumor cells

The evolutionarily conserved stress-inducible HSP70 molecular chaperone plays a central role in maintaining protein quality control in response to various forms of stress. Constitutively elevated HSP70 expression is a characteristic of many tumor cells and contributes to their survival. We recently identified the small-molecule 2-phenylethyenesulfonamide (PES) as a novel HSP70 inhibitor. Here, we present evidence that PES-mediated inhibition of HSP70 family proteins in tumor cells results in an impairment of the two major protein degradation systems, namely, the autophagy-lysosome system and the proteasome pathway. HSP70 family proteins work closely with the HSP90 molecular chaperone to maintain the stability and activities of their many client proteins, and PES causes a disruption in the HSP70/HSP90 chaperone system. As a consequence, many cellular proteins, including known HSP70/HSP90 substrates, accumulate in detergent-insoluble cell fractions, indicative of aggregation and functional inactivation. Overall, PES simultaneously disrupts several cancer critical survival pathways, supporting the idea of targeting HSP70 as a potential approach for cancer therapeutics.     

Autophagy-related genes serve as heat shock protein 90 co-chaperones in disease resistance against cassava bacterial blight

Heat shock protein 90 (HSP90) is involved in plant growth and various stress responses via regulating protein homeostasis. Autophagy keeps cellular homeostasis by recycling the components of cellular cytoplasmic constituents. Although they have similar effects on cellular protein homeostasis, the direct association between HSP90 and autophagy signaling remains unclear in plants, especially in tropical crops. In this study, the correlation between HSP90 and autophagy signaling was systematically analyzed by protein–protein interaction in cassava, one of the most important economy fruit in tropic. In addition, their effects on plant disease response and underlying mechanisms in cassava were investigated by functional genomics and genetic phenotype assay. The potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex interacts with MeATGs and subsequently triggers autophagy signaling, conferring improved disease resistance to cassava bacterial blight (CBB). On the contrary, HSP90 inhibitor and autophagy inhibitor decreased disease resistance against CBB in cassava, and autophagy may be involved in the potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex-mediated multiple immune responses. This study highlights the precise modulation of autophagy signaling by potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex in autophagy-mediated disease resistance to CBB.     

Human heat shock cognate protein (HSC70/HSPA8) interacts with negatively charged phospholipids by a different mechanism than other HSP70s and brings HSP90 into membranes

Heat shock proteins (HSP) are critical elements for the preservation of cellular homeostasis by participating in an array of biological processes. In addition, HSP play an important role in cellular protection from various environmental stresses. HSP are part of a large family of different molecular mass polypeptides, displaying various expression patterns, subcellular localizations, and diversity functions. An unexpected observation was the detection of HSP on the cell surface. Subsequent studies have demonstrated that HSP have the ability to interact and penetrate lipid bilayers by a process initiated by the recognition of phospholipid heads, followed by conformational changes, membrane insertion, and oligomerization. In the present study, we described the interaction of HSPA8 (HSC70), the constitutive cytosolic member of the HSP70 family, with lipid membranes. HSPA8 showed high selectivity for negatively charged phospholipids, such as phosphatidylserine and cardiolipin, and low affinity for phosphatidylcholine. Membrane insertion was mediated by a spontaneous process driven by increases in entropy and diminished by the presence of ADP or ATP. Finally, HSPA8 was capable of driving into the lipid bilayer HSP90 that does not display any lipid biding capacity by itself. This observation suggests that HSPA8 may act as a membrane chaperone.