Molecular cloning, mRNA expression, and characterization of heat shock protein 10 gene from humphead snapper Lutjanus sanguineus

Heat shock protein 10 (HSP10) gene of humphead snapper (Lutjanus sanguineus), designated as ByHSP10, was cloned by rapid amplification of cDNA ends (RACE) techniques with the primers designed from the known EST sequence identified from the subtracted cDNA library of the head kidney of humphead snapper. Sequence analysis showed the full length cDNA of ByHSP10 was 529 bp, containing a 5′ terminal untranslated region (UTR) of 51 bp, a 3′ terminal UTR of 181 bp, and an open reading frame (ORF) of 297 bp encoding a polypeptide of 99 amino acids. Based on the deduced amino acid sequence, the theoretical molecular mass of ByHSP10 was calculated to be 10.92 kDa with an isoelectric point of 9.46. Moreover, chaperonins hsp10/cpn10 signature was found in the amino acids sequence of ByHSP10 by PredictProtein. BLAST analysis revealed that the amino acids of ByHSP10 had the highest homology of 88% compared with other HSP10s. Fluorescent real-time quantitative RT-PCR was used to examine the expression of ByHSP10 gene in eight kinds of tissues of humphead snapper after the challenge with Vibrio harveyi. There was a clear time-dependent expression pattern of ByHSP10 in head kidney, spleen and thymus after bacteria challenge. The expression of mRNA reached the maximum level at the time point of 9 h, 6 h and 24 h, respectively and then returned to control level in 36 h. The up-regulated mRNA expression of ByHSP10 in humphead snapper after bacteria challenge indicated that the HSP10 gene was inducible and might be involved in immune response. A phylogenetic tree was constructed based on the ORF nucleotide sequences of HSP10 for 30 species. The relatonships among them were generally in agreement with the traditional taxonomy which suggested that HSP10 genes could aid in the system classification research.     

二化螟热休克蛋白70基因的克隆及热胁迫下的表达分析

热休克蛋白70是已知热休克蛋白家族中最重要的一种, 它在细胞内的大量表达可以明显改善细胞的生存能力, 提高对环境胁迫的耐受性。为探讨热胁迫对二化螟Chilo suppressalis幼虫热休克蛋白70表达的影响, 采用RT-PCR及RACE技术从二化螟血淋巴细胞中克隆了热休克蛋白70基因全长cDNA序列。该基因全长2 102 bp, 开放阅读框 (open reading frame, ORF)为1 959 bp, 编码652个氨基酸; 5′非编码区（untranslated region, UTR)为81 bp, 3′UTR为62 bp。从该基因推导的氨基酸序列与其他昆虫的同源序列比较有很高的相似性（73%~97%)。实时定量PCR显示二化螟HSP70基因能被热胁迫诱导表达, 幼虫血淋巴细胞的HSP70基因在36℃时表达量最高。流式细胞术研究发现HSP70在蛋白质水平上的表达变化与在mRNA水平上高度一致, 说明二化螟HSP70基因在转录及翻译水平上受到热应激的调节。     

热激蛋白90与中红侧沟茧蜂的发育关系

根据热激蛋白90(the heat shock proteins90,Hsp90)基因序列的保守性,设计引物,采用PCR结合RACE扩增的方法克隆得到中红侧沟茧蜂Microplitis mediatorHaliday Hsp90cDNA全序列,并用半定量RT-PCR的方法研究Hsp90与中红侧沟茧蜂发育及滞育间的关系。结果表明,滞育条件和非滞育条件下,热激和冷激都能诱导Hsp90的表达;不论在滞育还是非滞育条件下,Hsp90表达量均在2龄初期最高,随发育时间的增加逐渐降低,成虫期表达量又显著升高;滞育褐茧在4℃保存初期,Hsp90呈现高表达且随保存时间的增加而降低。     

Molecular cloning and expression analysis of heat-shock protein 70 in orange-spotted grouper Epinephelus coioides following heat shock and Vibrio alginolyticus challenge

In this study, the complementary (c)DNA encoding heat-shock protein 70 (Hsp70) of orange-spotted grouper Epinephelus coioides (OsgHsp70) was cloned. OsgHsp70 was 2206 bp and encoded 652 amino acids with predicted molecular mass of 70·89 kDa and theoretical isoelectric point of 5·48. Three Hsp70 family signatures, bipartite nuclear localization signal sequence (NLS) and cytoplasmic characteristic motif (EEVD) were observed in the OsgHsp70, which shared high similarity in amino-acid sequences with the Hsp70 gene of other vertebrates. The results indicated that the OsgHsp70 is a member of the heat-shock protein 70 family. The Hsp70 messenger (m)RNA expressions were quantified by real-time PCR following heat shock, bacterial infection and immunization with formalin-killed Vibrio alginolyticus, a kind of bacterial pathogen that causes septicaemia. Hsp70 mRNA expression in gill, kidney, spleen, thymus gland, muscle and total-blood samples increased at first and then decreased gradually following heat shock. A similar time-dependent pattern was observed following V. alginolyticus pathogen challenge, in which Hsp70 mRNA expression peaked at 24 h after live bacterial infection and 3 days after dead bacterial vaccination. The results indicated that the Hsp70 gene was inducible and involved in the fish immune response.     

Purification and characterization of porcine testis 90-kDa heat shock protein (HSP90) as a substrate for various protein kinases

We purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90. Compared with the commercial anti-HSP90 antibody, the polyclonal antibody raised in this study could specifically detect the testis HSP90 and immunoprecipitate HSP90 from tissue homogenates or cell extracts. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [-³²P]ATP/Mg²⁺ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [-³²P]ATP·Mg²⁺ alone but could be phosphorylated by various protein kinases, including PKA, CKII, kinase FA/GSK-3 , and AK. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK >> kinase FA/GSK-3 .     

Synthesis and biological evaluation of novobiocin analogues as potential heat shock protein 90 inhibitors

Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors that target this site and to define structure–activity relationships (SARs) for this class of compounds, we have synthesized twenty seven 3-amido-7-noviosylcoumarin analogues starting from NB and CA. These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibition of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of luciferase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR study revealed that a substantial increase in biological activity can be achieved by introduction of an indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and 13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90-binding agents, but with greater potency than NB.     

Synthesis and biological evaluation of C-ring truncated deguelin derivatives as heat shock protein 90 (HSP90) inhibitors

Based on the lead compound L-80 (compound 2), a potent heat shock protein 90 (HSP90) inhibitor, a series of C-ring truncated deguelin analogs were designed, synthesized and evaluated for Hypoxia Inducible Factor-1α (HIF-1α) inhibition as a primary screening method. Their structure–activity relationship was investigated in a systematic manner by varying the A/B ring, linker and D/E ring, respectively. Among the synthesized inhibitors, compound 5 exhibited potent HIF-1α inhibition in a dose-dependent manner and significant antitumor activity in human non-small cell lung carcinoma (H1299), with better activities than L-80. It also inhibited in vitro hypoxia-mediated angiogenic processes in human retinal microvascular endothelial cells (HRMEC). The docking study of 5 showed a similar binding mode as L-80: it occupied the C-terminal ATP-binding pocket of HSP90, indicating that the anticancer and antiangiogenic activities of 5 were derived from HIF-1α destabilization by inhibiting the C-terminal ATP-binding site of hHSP90.     

Protection of red snapper (Lutjanus sanguineus) against Vibrio alginolyticus with a DNA vaccine containing flagellin flaA gene

Aims: The main aims of this study were to construct a DNA vaccine containing flagellin flaA gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of pcDNA‐flaA as a DNA vaccine candidate for red snapper (Lutjanus sanguineus). Methods and Results: Plasmid DNA encoding flagellin flaA gene (designated as pcDNA‐flaA) was used as a DNA vaccine to immunize red snapper. The distribution, expression and immunoprotection of the DNA vaccine were analysed in tissues of the red snapper by PCR, RT‐PCR and challenge test. PCR results indicated that pcDNA‐flaA distributed in liver, spleen, kidney, gill and injection site muscle at 7–28 days after vaccination. RT‐PCR results indicated that the flaA gene was expressed in all above tissues of vaccinated fish at 7–28 days after vaccination. In addition, fish receiving the DNA vaccine developed a protective response to live V. alginolyticus challenge 28 days post inoculation, the relative per cent survival (RPS) was 88%. Conclusions: This study showed that injection of pcDNA‐flaA induced an efficient, systemic and antigen‐specific immune response in red snapper, which makes it an effective vaccine candidate against V. alginolyticus infection. Significance and Impact of the Study: The finding that red snapper does adequately respond to pcDNA‐flaA intramuscular injection makes pcDNA‐flaA a promising candidate for DNA vaccine treatment. Furthermore, the availability of red snapper for foreign gene expression represents a useful model to develop effective prophylactic strategies and opens new perspectives for the treatment of bacterial pathogens of marine cultured fish.     

热激蛋白90在植物发育和疾病抗性中的作用

相对分子质量90000的热激蛋白（heatshock protein,HSP90）是真核细胞必需的分子伴侣。拟南芥中HSP90有7个成员,其中AtHSP90-1、AtHSP90-2、AtHSP90-3和AtHSP90-4组成细胞质亚族;AtHSP90-5、AtHSP90-6、AtHSP90-7分别位于叶绿体、线粒体和内质网。HSP90分子伴侣复合物在植物发育和对外部刺激应答中非常重要,尤其是在抗性（resistance R）蛋白介导的抵抗病毒侵入的过程中起重要作用。     

Expression of heat shock protein 90 at the cell surface in human neuroblastoma cells

In addition to the activity of heat shock protein 90 (Hsp90/HSPC) as a chaperone, some recent studies have reported expression of Hsp90 at the cell surface in certain types of cancer and nervous system cells. We study the expression of Hsp90 at the cell surface in human neuroblastoma (NB69) cells. Immunofluorescence experiments labeling with anti-Hsp90 antibodies on both nonpermeabilized cells and live cells detected Hsp90 at the cell surface. Hsp90 was also identified in a membrane fraction from subcellular fractionation. Cell-surface Hsp90 was significantly more expressed in undifferentiated proliferative spherical neuroblastoma cells than in differentiated flattened cells. In addition, spherical cells were significantly more sensitive to Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin compared to flattened cells. This paper describes the first evidence of cell-surface Hsp90 expression in a cancer cell line from nervous tissue and may indicate a novel target for anti-tumoral agents.     

Tissue-specific expression of heat shock proteins of the mouse in the absence of stress

The steady-state levels of four members of the heat shock proteins families (HSP84, HSC73, HSP71, and HSP25) were examined by immunoblot analysis of several different tissues of young and adult mice in the absence of stress. These hsps were detected in all tissues but their level was variable. The levels of HSC73 and HSP84 varied only slightly between different tissues in either young or adult mice, with the exception of skin where these hsps were found in reduced amounts. In contrast, the stress-inducible member of the HSP70 family, HSP71, was found to be expressed in all tissues but in amounts which differed by as much as two orders of magnitude between tissues. In general, the levels of both HSP71 and HSP25 were found to be tissue dependent, with higher levels found in tissues such as stomach, intestine, colon and bladder, tissues which are exposed to toxic environmental or metabolic products, and which may concentrate these substances by water resorption and/or be exposed to them for longer periods. The levels of HSP71 and HSP25 were generally positively correlated both in young and adult mice although this correlation was not found in certain tissues such as kidney, testes, and bone. Tissues of young mice contained lower amounts of HSP25 and HSP71 than were found in the same tissues from adults. We conclude that hsps are expressed in all tissues of the mouse in the absence of stress and that some organs, particularly those exposed to potentially toxic metabolites, show a higher level of expression of HSP71 and HSP25. © 1993Wiley-Liss, Inc.     

Cloning and differential expression of three heat shock protein genes associated with thermal stress from the wolf spider Pardosa pseudoannulata (Araneae: Lycosidae)

Pardosa pseudoannulata is the main predatory natural enemy of crop pests in a paddy ecosystem. When P. pseudoannulata is exposed to unfavorable temperature conditions, the response of heat shock proteins could resist the damage, and is therefore, conducive to the organism’s rapid adaptation to the surrounding stress environment. In this study, we explored the roles of hsp70 and hsp90 genes in response to heat stress, using the rapid amplification of cDNA ends technique and cloned full-length cDNAs of Pphsp70, Pphsp83, and Pphsp90. The mRNA expression levels of the three genes under different temperature stresses (25, 28, 31, 34, 37, 40, and 43 °C) and with different duration stresses (4, 8, 12, 16, and 20 h) were analyzed by quantitative real-time polymerase chain reaction. The full-length cDNA of Pphsp70, Pphsp83, and Pphsp90 was 2331 base pair (bp), 2466 bp, and 2663 bp, respectively. Phylogenetic analysis of amino acid sequences of Pphsp70, Pphsp83, and Pphsp90 showed that the sequences had high homology with that of other spiders. The mRNA expression of all three genes was extremely significantly up-regulated at 43 °C. Moreover at 43 °C, the expression of all three genes in both female and male spiders at the duration of 4 h was the highest compared to that of other stress duration groups. Therefore, it can be inferred that the three genes of P. pseudoannulata play a crucial protective role in resistance in a high-temperature environment.     

Investigation of B,C-ring truncated deguelin derivatives as heat shock protein 90 (HSP90) inhibitors for use as anti-breast cancer agents

On the basis of deguelin, a series of the B,C-ring truncated surrogates with N-substituted amide linkers were investigated as HSP90 inhibitors. The structure activity relationship of the template was studied by incorporating various substitutions on the nitrogen of the amide linker and examining their HIF-1α inhibition. Among them, compound 57 showed potent HIF-1α inhibition and cytotoxicity in triple-negative breast cancer lines in a dose-dependent manner. Compound 57 downregulated expression and phosphorylation of major client proteins of HSP90 including AKT, ERK and STAT3, indicating that its antitumor activity was derived from the inhibition of HSP90 function. The molecular modeling of 57 demonstrated that 57 bound well to the C-terminal ATP-binding pocket in the open conformation of the hHSP90 homodimer with hydrogen bonding and pi-cation interactions. Overall, compound 57 is a potential antitumor agent for triple-negative breast cancer as a HSP90 C-terminal inhibitor.     

环链棒束孢hsp90基因的克隆及冷热胁迫下的表达特征分析

通过本地Blast筛选转录组数据库方法,首次克隆了环链棒束孢热休克蛋白90基因全长cDNA序列,命名为Ichsp90（GenBank登录号KT944289）。克隆结果表明,该序列含有2 284个碱基,包括一个含2 097个碱基的开放阅读框,编码699个氨基酸,推测蛋白的分子量为79.23kDa,等电点（pI）为4.86,且含有5个Hsp90家族特征基序和胞质特征序列MEEVD,推导的氨基酸序列与其他丝状真菌相似性在92%-96%之间。用qRT-PCR方法分析了冷热胁迫下,该基因在环链棒束孢中的相对表达情况,结果表明：在4℃冷胁迫下15min检测到Ichsp90表达量下降到最低点,为对照的-1.8倍;随后表达量开始上升,至120min表达量是对照的1.07倍。在39℃高温胁迫下,60min Ichsp90表达量达到最高峰,为对照样品的5.02倍;随后表达量开始下降,至110min为对照样品的2.46倍。因此推测,Ichsp90基因在环链棒束孢抵抗外界温度胁迫中发挥重要的作用。     

Differential expression pattern of heat shock protein 70 gene in tissues and heat stress phenotypes in goats during peak heat stress period

It has been established that the synthesis of heat shock protein 70 (Hsp70) is temperature-dependent. The Hsp70 response is considered as a cellular thermometer in response to heat stress and other stimuli. The variation in Hsp70 gene expression has been positively correlated with thermotolerance in Drosophila melanogaster, Caenorhabditis elegans, rodents and human. Goats have a wide range of ecological adaptability due to their anatomical and physiological characteristics; however, the productivity of the individual declines during thermal stress. The present study was carried out to analyze the expression of heat shock proteins in different tissues and to contrast heat stress phenotypes in response to chronic heat stress. The investigation has been carried out in Jamunapari, Barbari, Jakhrana and Sirohi goats. These breeds differ in size, coat colour and production performance. The heat stress assessment in goats was carried out at a temperature humidity index (THI) ranging from 85.36–89.80 over the period. Phenotyping for heat stress susceptibility was carried out by combining respiration rate (RR) and heart rate (HR). Based on the distribution of RR and HR over the breeds in the population, individual animals were recognized as heat stress-susceptible (HSS) and heat stress-tolerant (HST). Based on their physiological responses, the selected animals were slaughtered for tissue collection during peak heat stress periods. The tissue samples from different organs such as liver, spleen, heart, testis, brain and lungs were collected and stored at ?70 °C for future use. Hsp70 concentrations were analyzed from tissue extract with ELISA. mRNA expression levels were evaluated using the SYBR green method. Kidney, liver and heart had 1.5–2.0-fold higher Hsp70 concentrations as compared to other organs in the tissue extracts. Similarly, the gene expression pattern of Hsp70 in different organs indicated that the liver, spleen, brain and kidney exhibited 5.94, 4.96, 5.29 and 2.63-fold higher expression than control. Liver and brain tissues showed the highest gene expression at mRNA levels as compared to kidney, spleen and heart. HST individuals had higher levels of mRNA level expression than HSS individuals in all breeds. The Sirohi breed showed the highest (6.3-fold) mRNA expression levels as compared to the other three breeds, indicating the better heat stress regulation activity in the breed.