Preventing apoptotic cell death by a novel small heat shock protein

NCBI database analysis indicated that the human C1orf41 protein (small heat shock-like protein-Hsp16.2) has sequence similarity with small heat shock proteins (sHsps). Since sHsps have chaperone function, and so prevent aggregation of denatured proteins, we determined whether Hsp16.2 could prevent the heat-induced aggregation of denatured proteins. Under our experimental conditions, recombinant Hsp16.2 prevented aggregation of aldolase and glyceraldehyde-3-phosphate dehydrogenase, and protected Escherichia coli cells from heat stress indicating its chaperone function. Hsp16.2 also formed oligomeric complexes in aqueous solution. Hsp16.2 was found to be expressed at different levels in cell lines and tissues, and was mainly localized to the nucleus and the cytosol, but to a smaller extent, it could be also found in mitochondria. Hsp16.2 could be modified covalently by poly(ADP ribosylation) and acetylation. Hsp16.2 over-expression prevented etoposide-induced cell death as well as the release of mitochondrial cytochrome c and caspase activation. These data suggest that Hsp16.2 can prevent the destabilization of mitochondrial membrane systems and could represent a suitable target for modulating cell death pathways.     

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.     

高温对水稻叶片蛋白质表达的影响

为明确高温对耐热性不同水稻品种叶片蛋白质表达的影响,以耐热性不同的2个籼稻品种双桂1号(不耐热)和黄华占(耐热)为材料,分别于苗期、减数分裂期及抽穗(始穗后0—10d)和灌浆早期(始穗后11—20d)进行高温处理,之后取材并采用双向凝胶电泳技术研究高温对不同水稻品种叶片蛋白质表达的影响。结果表明,高温胁迫导致叶片中蛋白质的变化呈4种状况:新蛋白质的产生,一些蛋白质表达量上调,一些蛋白质的表达被抑制,一些蛋白质表达量下调。蛋白质表达变化在两品种以及4个处理时期的表现不同,总体表现为在热敏感品种中表达谱发生变化的蛋白质总数高于耐热品种。质谱分析表明,差异蛋白质主要涉及光合作用和信号转导,该类蛋白质在热敏感品种中表现为不表达或表达量下降,而在耐热品种则表现为有新诱导的蛋白质的产生或表达量上调,表明参与光合作用和信号转导的蛋白质在水稻耐热机制中发挥了重要作用。     

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.     

Interaction of a small heat shock protein with light-harvesting cyanobacterial phycocyanins under stress conditions

Phycobiliproteins such as phycocyanins are the most abundant proteins found in cyanobacteria which are assembled to form the phycobilisome. Here, we showed that a small heat shock protein, HspA, interacts directly with phycocyanins from the cyanobacterium Synechococcus sp. strain PCC 7942 in vitro and suppresses inactivation of their light-harvesting functions due to heat denaturation in the presence of hydrogen peroxide. Under the denaturing conditions, phycobilisomes were de-assembled to lighter complexes and then aggregated. HspA associated with phycocyanins in the dissociated complexes, and suppressed the aggregation. The specific interaction between a small heat shock protein and phycocyanins was further supported by the fact that HspA and alpha-crystallin protected isolated phycocyanins from denaturation, while HtpG and lysozyme did not. The maximum protection was observed at a molar ratio of four HspA monomer per one phycocyanin (alpha beta) monomer.     

Altered gene expression in plants with constitutive expression of a mitochondrial small heat shock protein suggests the involvement of retrograde regulation in the heat stress response

Heat stress can negatively affect crop productivity. One way in which plants attempt to alleviate the effects of heat stress is to induce the expression of genes encoding heat shock proteins (HSPs), including small HSPs (sHSPs). We produced transgenic lines of Arabidopsis thaliana expressing a transgene encoding a maize mitochondrial sHSP, ZmHSP22. The transgene, under the control of the cauliflower mosaic virus 35S promoter, is constitutively highly expressed in these lines. As demonstrated by confocal immunofluorescence microscopy and analyses of isolated mitochondria, ZmHSP22 is directed to the mitochondria of Arabidopsis and is processed into the mature form. These transgenic lines demonstrated altered expression of nuclear genes encoding the endogenous mitochondrial sHSP, AtHSP23.6, chloroplast localized AtHSP25.3, class I cytosolic AtHSP17.4, cytosolic AtHSP70-1 and chloroplast localized AtHSP70-6, but not cytosolic AtHSP70-15, following exposure to heat stress. This suggests that the expression of HSPs can be affected by heat-induced mitochondrial retrograde regulation. Three-week-old plants from the transgenic Arabidopsis lines expressing ZmHSP22 have increased thermotolerance, as measured by the maintenance of higher leaf mass following successive days with short periods of heat stress.     

马铃薯WRKY2基因的克隆和非生物逆境下的表达模式

马铃薯WRKY2基因的功能尚未见有报道,WRKY蛋白是近年来发现的一类重要转录因子家族,它们在植物应对生物胁迫、非生物胁迫和生长发育过程中起到关键的调控作用。该研究采用电子克隆法获得马铃薯WRKY2基因,该基因的编码区长度1 065 bp,编码355氨基酸,序列分析表明,该蛋白属于WRKY家族第二组成员,锌指结构为C-X5-C-X23H-X-H。构建系统发育树结果表明它与番茄WRKY7亲缘关系较近,氨基酸序列相似性达96%,与烟草中WRKY蛋白的相似性为86%,利用原核表达法在大肠杆菌中获得该蛋白。通过凝胶阻滞实验证明,该蛋白在体外能结合W-box元件,而且这种结合能被冷探针所竞争,同时也表明St WRKY2不能结合含有突变W-box DNA片段,证明St WRKY2与W-box结合具有特异性。通过实时荧光定量PCR技术分析该基因在根、茎和叶中的表达量,结果表明该基因主要在根中表达,其次是叶和茎。为进一步研究该基因可能参与的生理功能,对马铃薯组培苗进行10μmol/L低磷、10μmol/L低钾、200 mmol/L Na Cl、400 mmol/L PEG溶液和4℃低温处理,处理时间6 h,实时荧光定量PCR的结果表明该基因在低磷处理后表达量明显下降,在200 mmol/L Na Cl和400 mmol/L PEG处理6 h后表达量明显升高,但在10μmol/L低钾和4℃低温处理后表达量与对照相比无明显变化。说明St WRKY2能响应低磷、Na Cl和PEG这三种非生物逆境胁迫。研究结果可为进一步深入研究马铃薯WRKY2基因的功能奠定基础。     

热胁迫对双孢蘑菇抗氧化酶及热激蛋白基因的差异表达的影响

抗氧化酶和热激蛋白是双孢蘑菇Agaricus bisporus抵御逆境胁迫的重要蛋白,高温胁迫下菌丝会通过二者基因的差异表达来减少对自身的损伤.通过对双孢蘑菇菌丝进行40℃热胁迫处理0-120min后发现,随着热胁迫时间延长,菌丝生长速度降低、气生菌丝增多和菌丝分叉明显.转录组分析抗氧化酶和热激蛋白基因差异表达发现,在...     

The chaperonin-related protein Tcm62p ensures mitochondrial gene expression under heat stress

Tcm62p, distantly related to chaperonins, is required for the assembly of succinate dehydrogenase in mitochondria of Saccharomyces cerevisiae and was proposed to exert chaperone activity. We demonstrate here crucial functions of Tcm62p under heat stress. It ensures mitochondrial gene expression at elevated temperatures and prevents heat-aggregation of the ribosomal subunit Var1p. Similar to chaperonins, Tcm62p forms a high molecular mass protein complex of approximately 850 kDa in the mitochondrial matrix space. These results suggest a more general chaperone function of Tcm62p in mitochondria.     

褐飞虱热胁迫下内参基因的筛选及热激蛋白基因表达分析（英文）

【目的】褐飞虱Nilaparvata lugens(Stl)是为害水稻的重要害虫之一,温度是影响其暴发、迁飞的主要环境因子之一。本研究旨在探讨研究褐飞虱对高温胁迫适应性的热激蛋白基因表达调控模式。【方法】分别以不同的高温(30℃~40℃)处理褐飞虱雌、雄虫1 h和2 h,利用荧光定量PCR技术检测其体内的β-actin 1,β-actin2,β-actin3,28S rRNA,18S rRNA和α-2-tubulin 6个内参基因的表达量,用geN orm和BestK eeper软件分析确定最稳定表达的内参基因,并检测热胁迫后hsp70和hsp90基因在处理褐飞虱成虫体内的表达模式。【结果】geN orm软件分析结果表明,热胁迫后褐飞虱内参基因稳定性在雌虫体内为:β-actin1=β-actin328S rRNAα-2-tubulin18S rRNAβ-actin2;在雄虫体内为:β-actin1=β-actin3α-2-tubulin28S rRNA18S rRNAβ-actin2。BestK eeper软件分析结果显示,在热胁迫的雌、雄虫体内β-actin1均最稳定,18S rRNA次之,β-actin2最不稳定。两种软件分析结果基本一致。以β-actin1为校正内参基因,荧光定量PCR分析hsp70和hsp90在不同热胁迫条件下的表达模式,结果表明,各高温处理下hsp70表达量与对照26℃下的表达量没有显著性差异;而hsp90基因表达模式表现为被高温诱导上调表达,在雌、雄虫体内表达量达到最高的处理条件分别为40℃和38℃处理2 h。【结论】β-actin1基因可以作为热胁迫下褐飞虱雌雄虫体内基因表达模式分析的校正内参基因使用。褐飞虱hsp90基因能被高温诱导表达,该基因可能在褐飞虱适应热胁迫过程中起着重要的作用。     

韭菜迟眼蕈蚊热休克蛋白BoHsp70的克隆及热胁迫下的表达分析

[目的]本研究旨在克隆韭菜眼蕈蚊Bradysia odoriphaga热休克蛋白Hsp70基因,并对其进行序列和表达模式分析,以及探讨该基因在韭菜迟眼蕈蚊生长发育及响应温度胁迫方面的作用.[方法]选择韭菜迟眼蕈蚊温度转录组中高温下表达上调的Hsp70序列,设计其基因引物扩增序列,构建qRT-PCR检测体系分析该虫在短时高温热激(30、32、34和36℃;1、2、4、6、8、10和12 h)和高温热激后不同恢复时间(25℃;1 h、2 h)下的Hsp70表达谱.[结果]获得韭菜迟眼蕈蚊Hsp70基因cDNA全长序列并命名为BoHp70(GeneBank登录号:MW250640),包含1 971 bp的开放阅读框,编码656个氨基酸,具有真核生物Hsp70基因家族的3个保守序列,同时在C-末端具有KDEL序列,推测其属于内质网型热休克蛋白.BLAST分析和氨基酸序列系统发育分析结果显示,韭菜迟眼蕈蚊与双翅目蝇类昆虫Hsp70聚类为一个分支.BoHsp70在韭菜迟眼蕈蚊体内不同发育阶段中都有表达,雄成虫体内的表达量高于雌成虫,且在雌雄成虫头部表达量的差异显著.高温胁迫可诱导BoHsp70表达,并在诱导1-2 h内达到最高水平.在30、32和34℃热激条件下随热激时间的增加,BoHsp70表达量呈下降趋势,而在36℃热激下, BoHsp70表达水平不变.韭菜迟眼蕈蚊在解除高温热激后,BoHsp70表达水平随着恢复时间的增长而下降.[结论]韭菜迟眼蕈蚊可以通过调节体内Hsp70的表达来应对不良的环境温度.     

Identification of a Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress

Elucidating the mechanisms underlying the response and resistance to high-temperature stress in the Lepidoptera is essential for understanding the effect of high-temperature on the regulation of gene expression. A tag (CATGAACGTGAAGAGATTCAG) matching the predicted gene BGIBMGA005823-TA in SilkDB identified the most significant response to high-temperature stress in a screen of the heat-treated digital gene expression library of Bombyx mori (B. mori) (Unpublished data). BLAST and RACE showed that the gene is located on chromosome 5 and has an open reading frame (ORF) of 741 bp. Phylogenetic analysis found that B. mori small heat shock protein 27.4 (BmHSP27.4) is in an evolutionary branch separate from other small heat shock proteins. Expression analysis showed that BmHsp27.4 is highly expressed in brain, eyes and fat bodies in B. mori. Its mRNA level was elevated at high-temperature and this increase was greater in females. The ORF without the signal peptide sequence was cloned into vector pET-28a(+), transformed and over-expressed in Escherichia coli Rosetta (DE3). Western blotting and immunofluorescence analysis with a polyclonal antibody, confirmed that the level of protein BmHSP27.4 increased at a high-temperature, in accordance with its increased mRNA level. In this study, BmHsp27.4 was identified as a novel B. mori gene with an important role in response to high-temperature stress.     

Oxidative stress, heat shock and drought differentially affect expression of a tobacco protein phosphatase 2C

A protein phosphatase 2C (PP2C)-homologous cDNA was isolated from Nicotiana tabacum (NtPP2C1). The deduced protein sequence of 416 amino acids showed the highest degree of similarity to the PP2C of Arabidopsis thaliana (AtPP2CA) implicated in abscisic acid signalling. The expression of NtPP2C1 was strongly induced by drought, but repressed by oxidative stress and heat shock. It is suggested that NtPP2C1 operates at the junction of drought, heat shock and oxidative stress.     

线粒体内膜解偶联蛋白UCP1在大肠杆菌中的活性表达

线粒体的呼吸耗氧偶联着ATP的合成,而位于线粒体内膜上的跨膜蛋白解偶联蛋白(uncoupling protein,UCP)能够破坏这种偶联关系.在大肠杆菌中表达了有生物活性的鼠源解偶联蛋白1(rUCP1).重组rUCP1的表达导致大肠杆菌宿主细胞生长变慢;在电子显微镜下观察免疫标记的结果显示,重组rUCP1主要表达在细菌膜上;同时将rUCP1重构到脂质体中也能够测到质子转运活性.这些结果说明,真核生物UCP1能够在原核生物中表达出有生物活性的形式,且能纯化得到足量的rUCP1蛋白用于进一步的结构生物学研究.