分子伴侣的研究进展

文章综述了分子伴侣特别是HSP70分子伴侣系统的结构、功能、作用机理及应用方面的研究进展。分子伴侣能结合和稳定另一种蛋白质的不稳定构象，促进新生多肽链的正确折叠，因而在辅助蛋白质复性以及免疫保护等方面有很重要的作用。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。     

植物组蛋白分子伴侣研究进展

染色质作为真核生物遗传信息的载体,其基本结构与功能单位是核小体．细胞核内与DNA相关的生理反应如DNA复制,需要核小体的去组装以利于各类细胞因子与DNA结合,再进行核小体的重新组装以重建有功能的染色质结构,这些过程需要组蛋白分子伴侣的介导．目前的研究结果显示,组蛋白分子伴侣对于染色质结构稳定和基因表达调控非常重要．文中对植物组蛋白分子伴侣的研究进展,及其在植物生长发育过程中所发挥的作用进行综述．     

热休克蛋白的研究进展

在不利的环境中,各种有机体都有其共同对应的分子反应,即正常基因的表达抑制和一组特殊基因——热休克基因的激活和表达,导致热休克蛋白的大量产生,热休克蛋白主要作为分子伴侣而参与蛋白质的折叠、转运及组装等过程,能恢复或加速清除细胞内已变性的蛋白质而稳定细胞结构,细胞产生热耐受。随着对热休克蛋白研究的不断深入,在生物工程和医学等方面的应用前景十分广阔。     

分子伴侣GroEL对青霉素G酰化酶亚基折叠的影响

采用Tac启动子控制表达质粒，在不同的宿主细胞中表达了青霉素G酰化酶（PAC），检测这些菌株所表达的PAC活性，分析细胞内分子伴侣GroEL含量，PAC翻译后加工为α，β亚基的状况，以及它们之间的关系，结果表明：质粒pKK-SP在不同宿主中表达时，翻译后加工状况有明显差异，单位质量细胞所表达的PAC活性与翻译后加工效率相关，且与细胞内分子伴侣GroEL在菌体总蛋白中含量正相关，同时也阐明了亚基的折叠成为翻译后加工过程的限制步骤，细胞内分子伴侣GroEL有助于PAC亚基的折叠和稳定。     

肌小节组装研究进展

肌小节是横纹肌的基本功能单位,是由肌动蛋白、肌球蛋白和各种相关蛋白质组装而成的高度有序的结构。在肌小节组装过程中,Z带、M带以及一系列相关蛋白的正确组装是维持肌肉运动的关键,研究肌小节组成蛋白的折叠和组装机制对于了解肌肉疾病的病因和进行有针对性地治疗非常重要。本文综述了肌小节中的主要组分以及组装过程的研究进展,认为目前仍对肌小节骨架的装配、收缩复合体的功能及肌小节组装相关分子伴侣与疾病的关系等的相关研究不够深入。因此,未来还需从肌球蛋白结合蛋白、肌联蛋白、分子伴侣等与疾病的关系方面开展进一步的研究,为肌肉相关疾病的治疗寻找新的思路和解决办法。     

葡萄糖调节蛋白GRP78研究进展

葡萄糖调节蛋白78(glucose regulated p rotein78 kD,GRP78)又称免疫球蛋白重链结合蛋白(immunoglobulin heavy chain binding protein,Bip),是位于内质网上重要的分子伴侣,属热休克蛋白70家族的一员.GRP78分子及其DNA分子序列结构在许多生物物种中高度保守.GRP78在内质网中参与阻止内质网新生肽聚集、调节内质网钙稳态、抗内质网相关性细胞凋亡,以及启动未折叠蛋白反应等细胞生命过程.GRP78生物学功能的研究已经引起生物学家的广泛重视.     

拟南芥AtJ2基因植物超量表达载体的构建

在胁迫条件下,分子伴侣可以稳定蛋白质结构,防止蛋白质凝聚变性,并修复受伤害蛋白.J蛋白是一类重要的分子伴侣.AtJ2是拟南芥(Arabidopsis thaliana)J蛋白中的一种.为研究该基因的功能,提取了拟南芥幼苗的总RNA,经过RT-PCR获得了AtJ2的全长.并将其构建入表达载体pMD中,得到重组质粒pMD/AtJ2.通过农杆菌(Agrobacterium tumefaciens)将此重组质粒转化入拟南芥,得到了转基因植株,为后续该基因的功能研究奠定基础.     

植物寒冻抗性的分子机理研究进展

概述了植物冷激蛋白的生理作用及其基因表达的调控，阐释了植物寒冻损伤和寒冻抗性的分子机理．植物冷激蛋白的生理作用有：1．具有生物膜系统的保护作用；2．有蛋白质、酶和RNA分子伴侣的作用；3．具有抗冻和抗脱水活性．冷激蛋白基因是一类调节基因，可感受低温、脱水、ABA等多种不同的信号刺激．冷激蛋白基因的表达存在着信号转导、转录和转录后等多个不同层次的调控．     

热休克蛋白70的生物学功能研究进展

热休克蛋白是熟休克反应中转录合成的一组特殊糖蛋白.其中HSP70是最保守和最重要的一种蛋白质,HSP70具有多种生物学功能,包括分子伴侣功能,参与肿瘤免疫,抗细胞凋亡功能,提高细胞的应激耐受性,促进细胞增殖等等.本文着重对HSP70生物学功能研究的进展作一综述.     

麻疯树小热激蛋白JcHSP-17.5的纯化与活性鉴定

将实验室已有的pET32a-JcHSP17.5重组载体,经测序验证后,转入大肠杆菌BL21(DE3)plysS中,不同温度条件下,用不同浓度异丙基硫代-D-半乳糖苷(IPTG)诱导重组蛋白表达,优化表达条件;选择镍离子亲和层析柱(Ni-NTA)纯化JcHSP-17.5蛋白,SDS-PAGE电泳检测蛋白纯度;通过热激聚合反应来鉴定蛋白的分子伴侣活性.结果表明,17℃,200r/min,0.5mM IPTG诱导13h为JcHSP-17.5蛋白的最佳诱导表达条件,使用200mmol/L咪唑缓冲液纯化蛋白后,每500mL菌液可得到1.345mg的电泳纯蛋白,该蛋白在高温(45℃)条件下能够阻止苹果酸脱氢酶(MDH)发生聚合反应,表现出较强的分子伴侣活性.     

Heat-shock protein 90 inCandida albicans

Researches on Candidal heat-shock protein 90 (HSP90) in recent years are summarized.Candida albicans is a commensal pathogen in human and animals. In immunocompromised individuals it behaves as an opportunist pathogen, giving rise to superficial or systemic infections. Systemic candidosis is a common cause of death among immunocompromised and debilitated patients, in which the mortality is as high as 70%. HSP90 is now recognized as an immunodominant antigen inC. albicans and plays a key role in systemic candidosis as a molecular chaperone. The 47-ku peptide is the breakdown product of HSP90. Patients who has recovered from systemic candidosis produce high titre of antibodies to 47-ku antigen, whereas the fatal cases have little antibody or falling titres. The three commonest epitopes of candidal HSP90 have been mapped, epitopes C, B and H. Epitopes C and H are immunogenic. The antibody probes of both epitopes may be developed into a new serological test agents for systemic candidosis due to rather high specificity and sensitivity. The recent results establish HSP90 as an ATP-dependent chaperone that is involved in the folding of cell regulatory proteins and in the refolding of stress-denatured polypeptides. Some researches on fungal HSP90 and the treatment of patients with candidosis are reviewed as well.     

Heat-shock protein 90 in Candida albicans

Researches on Candidal heat-shock protein 90 (HSP90) in recent years are summarized.Candida albicans is a commensal pathogen in human and animals.In immunocompromised individuals it behaves as an opportunist pathogen,giving rise to superficial or systemic infections.Systemic candidosis is a common cause of death among immunocompromised and debilitated patients,in which the mortality is as high as 70%.HSP90 is now recognized as an immunodominant antigen in C.albicans and plays a key role in systemic candidosis as a molecular chaperone.The 47-ku peptide is the breakdown product of HSP90.Patients who has recovered from systemic candidosis produce high titre of antibodies to 47-ku antigen,whereas the fatal cases have little antibody or falling titres.The three commonest epitopes of candidal HSP90 have been mapped,epitopes C,B and H.Epitopes C and H are immunogenic.The antibody probes of both epitopes may be developed into a new serological test agents for systemic candidosis due to rather high specificity and sensitivity.The recent results establish HSP90 as an ATP-dependent chaperone that is involved in the folding of cell regulatory proteins and in the refolding of stress-denatured polypeptides.Some researches on fungal HSP90 and the treatment of patients with candidosis are reviewed as well.     

NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration

Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.     

A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1

There is evidence to suggest that components of archaebacteria are evolutionarily related to cognates in the eukaryotic cytosol. We postulated that the major heat-shock protein of the thermophilic archaebacterium, Sulfolobus shibatae, is a molecular chaperone and that it is related to an as-yet unidentified chaperone component in the eukaryotic cytosol. Acquired thermotolerance in S. shibatae correlates with the predominant synthesis of this already abundant protein, referred to as thermophilic factor 55 (TF55). TF55 is a homo-oligomeric complex of two stacked 9-membered rings, closely resembling the 7-membered-ring complexes of the chaperonins, groEL, hsp60 and Rubisco-binding protein. The TF55 complex binds unfolded polypeptides in vitro and has ATPase activity-features consistent with its being a molecular chaperone. The primary structure of TF55, however, is not significantly related to the chaperonins. On the other hand, it is highly homologous (36-40% identity) to a ubiquitous eukaryotic protein, t-complex polypeptide-1 (TCP1). In Saccharomyces cerevisiae, TCP1 is an essential protein that may play a part in mitotic spindle formation. We suggest that TF55 in archaebacteria and TCP1 in the eukaryotic cytosol are members of a new class of molecular chaperones.     

新型核苷碱基卤化酶AcmX的原核表达和结晶实验

在链霉菌中发现了一类能对核苷碱基进行特异位点卤化修饰的新型卤化酶,可用于核苷类药物的卤代优化。对新型核苷碱基卤化酶晶体结构的解析,是阐明其生物催化的机理,建立生物催化卤化制备核苷类药物的最直接手段。为了获得一个卤化酶AcmX的晶体结构,首先通过大肠杆菌原核表达制备AcmX蛋白样品,但只能得到没有活性的AcmX的蛋白包涵体。通过构建AcmX、分子伴侣蛋白共表达系统,注意控制合适的分子伴侣蛋白表达水平,并通过高分辨率的分子筛凝胶层析步骤,去除混入AcmX样品的分子伴侣蛋白GroEL,摸索出表达和纯化高质量AcmX蛋白样品的方法,并将蛋白样品成功用于结晶实验。这不仅为解析卤化酶AcmX的晶体结构打下坚实的基础,还可将本研究的方法用于其他表达困难的蛋白的制备。     

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.     

Crystal structure of chaperone protein PapD reveals an immunoglobulin fold

The chaperone protein PapD mediates assembly of pili in Escherichia coli. Its polypeptide chain folds into two immunoglobulin-type domains that are homologous in sequence to the human lymphocyte differentiation antigen Leu-1/CD5.