HSP90, HSPA8, HIF-1 alpha and HSP70-2 polymorphisms in breast cancer: a case–control study

This case control study aims to investigate the role of HSP90 Gln488His (C?>?G), HSP70-2 P1/P2, HIF-1 alpha C1772T and HSPA8 intronic 1541–1542delGT polymorphisms as potential risk factors and/or prognostic markers for breast cancer. 113 consecutive incident cases of histologically confirmed ductal breast cancer and 124 healthy cases were recruited. The above mentioned polymorphisms were genotyped; multivariate logistic regression was performed. HSP90 GG (His/His) genotype was associated with elevated breast cancer risk. Similarly, the allele dose–response model pointed to increase in breast cancer risk per G allele. HSP70-2 P1/P2, HSPA8 intronic 1541–1542delGT and HIF-1 alpha polymorphisms were not associated with breast cancer risk, as evidenced by the dose–response allele models. The positive association between HSP90 G allele and breast cancer risk seemed to pertain to both premenopausal and postmenopausal women. With respect to survival analysis, none of the aforementioned polymorphisms was associated with either disease-free survival or overall survival. HSP90α Gln488His polymorphism seems to be a risk factor for breast cancer. On the other hand, our study did not point to excess risk conferred by HSPA8 1541–1542delGT, Hsp70-2 P1/P2 and HIF-1α C1772T.     

Knockdown of Heat Shock Proteins HSPA6 (Hsp70B’) and HSPA1A (Hsp70-1) Sensitizes Differentiated Human Neuronal Cells to Cellular Stress

Heat shock proteins are involved in cellular repair and protective mechanisms that counter characteristic features of neurodegenerative diseases such as protein misfolding and aggregation. The HSPA (Hsp70) multigene family includes the widely studied HSPA1A (Hsp70-1) and the little studied HSPA6 (Hsp70B’) which is present in the human genome and not in mouse and rat. The effect of knockdown of HSPA6 and HSPA1A expression was examined in relation to the ability of differentiated human SH-SY5Y neuronal cells to tolerate thermal stress. Low dose co-application of celastrol and arimoclomol, which induces Hsps, enhanced the ability of differentiated neurons to survive heat shock. Small interfering RNA (siRNA) knockdown of HSPA6 and HSPA1A resulted in loss of the protective effect of co-application of celastrol/arimoclomol. More pronounced effects on neuronal viability were apparent at 44 °C heat shock compared to 43 °C. siRNA knockdown suggests that HSPA6 and HSPA1A contribute to protection of differentiated human neuronal cells from cellular stress.     

HSP70单克隆抗体的制备

利用DEAE 离子交换和ATP 亲和层析法从热休克的大鼠肝脏中分离纯化了热休克蛋白 70 (HSP70 )。用它做抗原免疫小鼠 ,通过分离脾淋巴细胞和细胞融合技术 ,得到了杂交瘤细胞 ;用酶联免疫吸附测定法筛选出了阳性克隆 ;经过多次亚克隆、抗体的大量制备及分离纯化 ,得到了抗HSP70的单克隆抗体。用此一抗和Western免疫印迹技术分析了C6 大鼠神经胶质瘤细胞中HSP70的表达。     

分子伴侣HSP70研究进展

分子伴侣ＨＳＰ７０在原核、真核细胞均已发现,它们具有共同的生化特征,在细胞内各自分布在特定的区域。其主要生物学功能是促进新生多肽链的正确折叠,对于分子重排、解离聚集蛋白和新生多肽的膜运输也具有重要的辅助作用。     

HSP70分子伴侣系统研究进展

综述了HSP70分子伴侣系统的晶体结构、功能及作用机理方面的研究进展.HSP70分子伴侣能够帮助细胞内新生蛋白的折叠和跨膜运输、蛋白质多聚体结构的装配和解装配,并能在胁迫下维持蛋白质的特殊构象,防止未折叠的蛋白质变性和使聚集的蛋白质溶解复性.所有这些活性均依赖于ATP调节的HSP70与底物蛋白中的疏水片段的相互作用.     

HSP70抗原呈递分子的研究进展

热休克蛋白７０（ＨＳＰ７０）是广泛存在于原核和真核细胞中的一组高度保守的蛋白质分子家族,其主要功能是促进与维持新生多肽的正确折叠,并且调节细胞的生长、发育、分化、死亡。ＨＳＰ７０还是免疫系统识别的主要抗原,在抗原的加工、处理和呈递以及效应Ｔ细胞的激活过程中扮演着重要角色。来自同源肿瘤成份的ＨＳＰ治疗小鼠肿瘤具有明显的效果,是肿瘤肽的高效传递系统,是ＣＤ＾＋８Ｔ细胞激活的佐剂。了解其功能对某些疾病发     

脑缺血后HSP70表达的研究进展

Ritosso ( 1 962 )首先发现环境温度高于正常生理温度 4℃～ 6℃时 ,染色体上会出现特殊的膨突( Puffs) ,称其为热休克现象。后来 Tissieres等发现染色体的这一膨突与细胞中的一类特殊蛋白质的合成有关 ,并将这一类特殊蛋白质称为热休克蛋白 ( Heatshock protein,HSP)或热应激蛋白。近年来研究发现缺血性细胞损伤可以出现 HSP的表达 [1] ,还发现 HSP对受损细胞有保护作用。1　 HSP的分类　　 70 k Da家族是 HSP中最保守和最主要的一类 ,包括 68、70、72及 78k Da的 HSP。其中 HSP70是在缺血性脑损伤中研究最广泛的一种。氨基酸顺…     

Death by chaperone: HSP90, HSP70 or both?

HSP70 family members are highly conserved proteins that function as molecular chaperones. Their principle role is to aid protein folding and promote the correct cellular localisations of their respective substrates. The function of HSP70 isoforms can be exhibited independently or with the HSP90 chaperone system in which HSP70 is important for substrate recruitment. In addition to their chaperone role, HSP70 isoforms promote cell survival by inhibiting apoptosis at multiple points within both the intrinsic and extrinsic cell death pathways. Consistent with this cytoprotective function, increased expression of HSP70 isoforms is commonly associated with the malignant phenotype. We recently reported that dual silencing of the major constitutive (HSC70) and inducible (HSP72) isoforms of HSP70 in cancer cells could phenocopy the effects of a pharmacologic HSP90 inhibitor to induce proteasome-dependent degradation of HSP90 client proteins CRAF, CDK4 and ERBB2. This was accompanied by a G1 cell cycle arrest and extensive apoptosis which was not seen in non-tumorigenic human cell lines. Here we discuss the possible implications of our research for the development of HSP70 family modulators which offer not only the possibility of inhibiting HSP70 activity but also the simultaneous inhibition of HSP90, resulting in extensive tumour-specific apoptosis.     

重金属对HeLa细胞中热激蛋白70(HSP70)表达的影响

热激蛋白(HSPs)是受热等因素刺激后而诱导产生的蛋白质,是一类可以调节应激反应并且保护机体防止细胞损伤的蛋白质,在机体的应激反应中具有重要作用。它们作为一般标志物被广泛应用于环境监测中。CdCl2,Cu2+,Zn2+这三种重金属是普遍存在的环境污染物,对人体和动物的一些主要器官造成损伤。以HeLa细胞(子宫肿瘤细胞)为材料,采用不同浓度的CdCl2,Cu2+,Zn2+三种重金属物质诱导细胞,并利用免疫荧光染色(IFS),SDS-PAGE,Western blotting和RT-PCR四种手段分别从基因和蛋白质的水平来研究重金属对HSP70表达的影响。结果表明,三种金属对HSP70表达的影响程度为CdCl2>Zn2+>Cu2+,且HSP70的产生量与重金属的浓度呈正相关。通过研究,以建立一种对HSPs的表达更有效的检测手段用于以后的研究。     

HSP70系统分析与ER的起源

HSP 70是迄今研究过的进化上最保守的蛋白质之一,是分子伴侣的主要成分,对蛋白的跨膜转运及特定构象的维持等起着重要作用。对其序列进行系统分析表明,古细菌,真细菌和真核细胞内的HSP 70虽具有很强的相似性,但各具特点。真核细胞各区室(细胞器)的HSP 70顺序也既有共性,又有特性。根据ER HSP 70的特点,可推测出ER起源于真核细胞诞生的早期阶段,并由此引起细胞内的区室化,这是真核细胞区别于原核细胞的本质特征之一。     

褐飞虱HSP70与HSP90基因特性及温度诱导表达

[目的]研究不同温度条件下,褐飞虱HSP70和HSP70基因的表达变化,探索HSP蛋白在褐飞虱对温度胁迫适应中的作用。[方法]利用同源克隆方法获得HSP70A和HSP70基因序列,实时荧光定量PCR(qRT-PCR)检测HSPs基因在不同温度诱导下的表达量。[结果]褐飞虱HSP70A基因包含1 896 bp的ORF,编码631个氨基酸; HSP70基因包含2 193 bp的ORF,编码730个氨基酸。HSP70A在38℃高温下,表达量出现不同程度下降; HSP70B和HSP70在32℃及38℃高温下能够被诱导高表达。在低温诱导下,HSP70A及HSP70B的表达出现不同程度下降(HSP70A在15℃处理6 h及10℃处理2 h例外),HSP70表达量在低温下为显著或者极显著上升。[结论] HSP70B和HSP70在褐飞虱的高温胁迫下起重要的作用;在低温下则主要通过提高HSP70的表达来保护机体内细胞的正常生理代谢。     

荷斯坦牛HSP70-1基因遗传多态性与乳腺炎抗性关系

以253头中国荷斯坦奶牛为研究对象, 检测HSP70-1基因的多态性, 并分析其多态性与中国荷斯坦牛体细胞评分(Somatic cell score, SCS)的相关性。首先以PCR-SSCP法寻找HSP70-1基因编码区的突变, 并通过测序确定突变的类型, 根据突变类型寻找合适的内切酶, 最终采用PCR-RFLP方法鉴定实验牛基因型; 然后分析基因多态性与中国荷斯坦牛SCS的相关性。结果表明HSP70-1基因的1 623 bp处产生G→A→C突变, 2 409 bp处产生G→A突变, 两位点都是沉默突变, 未引起氨基酸序列的改变; 经χ2 适合性检验, 中国荷斯坦牛在两个位点均未达到Hardy-Weinberg平衡状态; 同时, 群体基因座不同基因型与SCS相关分析的结果表明, 2409位点基因型与SCS相关性不显著(P>0.05), 1623位点基因型与SCS相关性显著(P<0.05), CC型SCS显著低于AG、GG型(P<0.05), CC基因型为乳腺炎抗性基因型。在中国荷斯坦奶牛群体中, HSP70-1基因CC基因型可作为改良奶牛乳腺炎抗性性状的分子遗传标记。     

中国辣椒热激蛋白HSP70基因家族分析

以中国辣椒(Capsicum chinense Jacq.)基因组数据为基础,采用生物信息学方法对中国辣椒HSP70基因家族进行全基因组鉴定分析。结果显示,中国辣椒全基因共鉴定得到20个HSP70基因,编码蛋白序列长度为516~854 aa,分子量大小为56.21~94.26 kD。系统进化分析结果表明,中国辣椒HSP70基因可分为A、B、C 3个亚家族。比较转录组分析结果显示,有16个HSP70基因对热胁迫有不同程度的响应。     

棉花HSP70基因的克隆与原核表达

以抗旱性较强的棉花品种‘KK1543’为材料,采用RT-PCR技术克隆了1个棉花HSP70基因,命名为GhHSP70。研究结果表明:GhHSP70基因开放阅读框长1 997bp,编码648个氨基酸,GhHSP70蛋白相对分子量为70.94kD,等电点为4.83。氨基酸序列比对和系统进化树分析发现,GhHSP70与欧洲大叶杨HSP70的亲缘关系最近,氨基酸序列一致性达到96.4%。为进一步分析基因的功能,构建原核表达载体pGEX-4T-1-GhHSP70,并在大肠杆菌中异源表达。SDS-PAGE分析表明所表达蛋白与预期蛋白大小一致,重组蛋白在37℃,0.2mmol/L IPTG诱导2h时表达量最大。研究为进一步研究棉花HSP70基因功能奠定基础。     

菜粉蝶热激蛋白70(HSP70)基因的分子特性及其对杀虫剂胁迫的响应

【目的】明确菜粉蝶Pieris rapae热激蛋白70(HSP70)基因的分子特性及其对杀虫剂胁迫的响应,为探索菜粉蝶HSP70基因在抵御杀虫剂胁迫中的功能提供前期基础。【方法】利用同源检索方法,从菜粉蝶转录组数据中鉴定HSP70基因;使用生物信息学方法分析HSP70基因的分子特性;采用实时荧光定量PCR技术分析HSP70基因在菜粉蝶不同发育阶段(2-5龄幼虫、蛹和雌雄成虫)、4龄幼虫不同组织(中肠、马氏管、脂肪体和体壁)以及LD₂₀剂量高效氯氟氰菊酯(0.12 ng/μL)和氯虫苯甲酰胺(1.04 ng/μL)胁迫下4龄幼虫体内的表达谱。【结果】从菜粉蝶转录组中鉴定了3个HSP70基因(PrHsp70-1, PrHsp70-2和PrHsp70-3) (GenBank登录号分别为MW691114, MW691115和MW691116),它们分别编码628, 630和653个氨基酸,分子量分别为68.7, 69.2和71.7 kD。生物信息学分析结果表明,3个PrHSP70蛋白均为胞质蛋白,且均含有HSP70家族的特征序列。PrHsp70-1和PrHsp70-2无内含子,而PrHsp70-3含有1个内含子。随着幼虫龄期的增加,PrHsp70-1和PrHsp70-2的表达量也不断上调,但在蛹期和成虫期下调;PrHsp70-3在供试的不同发育阶段样本中的表达量无显著差异。PrHsp70-1高量表达于幼虫脂肪体,PrHsp70-2高量表达于幼虫中肠,PrHsp70-3在幼虫体壁和脂肪体中表达量均较高。LD₂₀剂量高效氯氟氰菊酯和氯虫苯甲酰胺均能诱导3个PrHsp70基因显著上调表达,但不同基因的响应速度有差异;此外,LD₂₀剂量高效氯氟氰菊酯处理24 h后,PrHsp70-3显著下调表达。【结论】PrHsp70基因可能在菜粉蝶生长发育以及抵御杀虫剂胁迫中均有重要作用。     

COMMD1 Promotes pVHL and O2-Independent Proteolysis of HIF-1α via HSP90/70

Background

The Copper Metabolism MURR1 Domain containing 1 protein COMMD1 has been associated with copper homeostasis, NF-κB signaling, and sodium transport. Recently, we identified COMMD1 as a novel protein in HIF-1 signaling. Mouse embryos deficient for Commd1 have increased expression of hypoxia/HIF-regulated genes i.e. VEGF, PGK and Bnip3. Hypoxia-inducible factors (HIFs) are master regulators of oxygen homeostasis, which control angiogenesis, erythropoiesis, glycolysis and cell survival/proliferation under normal and pathologic conditions. Although HIF activity is mainly controlled by ubiquitination and protein degradation by the von Hippel Lindau (pVHL) tumor suppressor gene other mechanisms have recently been identified that regulate HIF signaling independently of pVHL.

Principal Findings

Here we characterized the mechanism by which COMMD1 regulates HIF-1α protein degradation. We show that COMMD1 competes with the chaperone heat shock protein HSP90β for binding to the NH₂-terminal DNA-binding and heterodimerization domain of HIF-1α to regulate HIF-1α stability together with HSP70. Inhibition of HSP90 activity with 17-Allylamino-17-demethoxygeldanamycin (17-AAG) increased COMMD1-mediated HIF-1α degradation independent of ubiquitin and pVHL.

Conclusion/Significance

These data reveal a novel role for COMMD1 in conjunction with HSP90β/HSP70 in the ubiquitin and O₂-independent regulation of HIF-1α.     

Heteromeric complexes of heat shock protein 70 (HSP70) family members,including Hsp70B′, in differentiated human neuronal cells

Human neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have been termed “protein misfolding disorders.” Upregulation of heat shock proteins that target misfolded aggregation-prone proteins has been proposed as a potential therapeutic strategy to counter neurodegenerative disorders. The heat shock protein 70 (HSP70) family is well characterized for its cytoprotective effects against cell death and has been implicated in neuroprotection by overexpression studies. HSP70 family members exhibit sequence and structural conservation. The significance of the multiplicity of HSP70 proteins is unknown. In this study, coimmunoprecipitation was employed to determine if association of HSP70 family members occurs, including Hsp70B′ which is present in the human genome but not in mouse and rat. Heteromeric complexes of Hsp70B′, Hsp70, and Hsc70 were detected in differentiated human SH-SY5Y neuronal cells. Hsp70B′ also formed complexes with Hsp40 suggesting a common co-chaperone for HSP70 family members.