小热休克蛋白的结构和功能

小热休克蛋白（small heat shock protein,sHSP）几乎存在于所有生物体中,其主要结构是一个保守的α晶体蛋白(α-crystallin)结构域,由约90个氨基酸残基组成,与其相邻的是可变的N端域. N端域能够调节低聚体形成、亚单位动力学及其与底物的结合.sHSP能够与细胞内各组分（蛋白质、细胞核、细胞骨架元件、膜）进行相互作用,以维持细胞的稳定.小热休克蛋白家族成员的共同特点是特殊丝氨酸残基上的磷酸化,磷酸化作用对于受到胁迫时的细胞非常重要.由MAPKAP激酶 2/3和p38参与的级联反应能够诱导sHSP发生磷酸化,从而调节sHSP的低聚体状态,而低聚体状态和sHSP的生物学功能密切相关.本文介绍sHSP的结构特征和细胞内的作用底物,并讨论sHSP被不同的蛋白激酶磷酸化及磷酸化作用对低聚体状态和伴侣活性的影响.     

热激蛋白ClpB的结构和功能

ClpB是HSP100／Clp蛋白家族的一员,具有分子伴侣功能,通过溶解热胁迫下的蛋白聚集体,减少热激对细胞的伤害,ClpB与细胞的耐热性紧密相关。近年来,ClpB在植物中的功能成为研究的热点,但大多局限在植物胞质ClpB的研究,而对细胞器ClpB的研究较少。文章介绍了热激蛋白ClpB的结构、功能和植物热激蛋白ClpB的研究现状。     

植物热激蛋白90的结构和功能

文章从结构和功能两个方面介绍植物热激蛋白90的研究进展。     

Effect of trifluoperazine on in vitro ATP synthesis by Mycobacterium leprae

The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae . M. leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M. leprae . This incubation was carried out in the absence and presence of different concentrations of trifluoperazine. Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation. TFP inhibited ATP levels in M. leprae and this inhibitory effect was marginal at 2.5 μg ml⁻¹ (35% inhibition), highly significant at 5 μg ml⁻¹ (87% inhibition) and almost total at 10 μg ml⁻¹ (98.5% inhibition). This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier.     

结核杆菌小分子热休克蛋白Hsp16.3的高效自发再折叠和再组装

来自结核杆菌的小分子热休克蛋白Hsp16.3以九聚体的形式存在.用三种不同的强变性条件(100℃加热15 min,12 mol/L脲或8 mol/L盐酸胍处理4 h)将Hsp16.3变性, 然后通过冷却或透析使之复性,并利用孔径梯度聚丙烯酰胺凝胶电泳和圆二色性光谱比较了变性-复性前后Hsp16.3的各个层次高级结构.结果显示,变性的Hsp16.3几乎可以完全恢复至天然构象,这表明小分子热休克蛋白Hsp16.3具有很强的自发折叠和组装能力.     

Detection and Architecture of Small Heat Shock Protein Monomers

Background

Small Heat Shock Proteins (sHSPs) are chaperone-like proteins involved in the prevention of the irreversible aggregation of misfolded proteins. Although many studies have already been conducted on sHSPs, the molecular mechanisms and structural properties of these proteins remain unclear. Here, we propose a better understanding of the architecture, organization and properties of the sHSP family through structural and functional annotations. We focused on the Alpha Crystallin Domain (ACD), a sandwich fold that is the hallmark of the sHSP family.

Methodology/Principal Findings

We developed a new approach for detecting sHSPs and delineating ACDs based on an iterative Hidden Markov Model algorithm using a multiple alignment profile generated from structural data on ACD. Using this procedure on the UniProt databank, we found 4478 sequences identified as sHSPs, showing a very good coverage with the corresponding PROSITE and Pfam profiles. ACD was then delimited and structurally annotated. We showed that taxonomic-based groups of sHSPs (animals, plants, bacteria) have unique features regarding the length of their ACD and, more specifically, the length of a large loop within ACD. We detailed highly conserved residues and patterns specific to the whole family or to some groups of sHSPs. For 96% of studied sHSPs, we identified in the C-terminal region a conserved I/V/L-X-I/V/L motif that acts as an anchor in the oligomerization process. The fragment defined from the end of ACD to the end of this motif has a mean length of 14 residues and was named the C-terminal Anchoring Module (CAM).

Conclusions/Significance

This work annotates structural components of ACD and quantifies properties of several thousand sHSPs. It gives a more accurate overview of the architecture of sHSP monomers.     

热休克蛋白70的主要功能和应用前景

热休克蛋白70(HSP70)是生物体在各种应激条件下产生的蛋白之一,具有维持细胞自身稳定等多种生物学功能.随着研究的深入,在生物学的功能不断被发现的同时,HSP70的应用前景也变得越来越广泛.     

Dynamics and Composition of Small Heat Shock Protein Condensates and Aggregates

Small heat shock proteins (sHSPs) are essential ATP-independent chaperones that protect the cellular proteome. These proteins assemble into polydisperse oligomeric structures, the composition of which dramatically affects their chaperone activity. The biomolecular consequences of variations in sHSP ratios, especially inside living cells, remain elusive. Here, we study the consequences of altering the relative expression levels of HspB2 and HspB3 in HEK293T cells. These chaperones are partners in a hetero-oligomeric complex, and genetic mutations that abolish their mutual interaction are associated with myopathic disorders.HspB2 displays three distinct phenotypes when co-expressed with HspB3 at varying ratios. Expression of HspB2 alone leads to formation of liquid nuclear condensates, while shifting the stoichiometry towards HspB3 resulted in the formation of large solid-like aggregates. Only cells co-expressing HspB2 with a limited amount of HspB3 formed fully soluble complexes that were distributed homogeneously throughout the nucleus. Strikingly, both condensates and aggregates were reversible, as shifting the HspB2:HspB3 balance in situ resulted in dissolution of these structures.To uncover the molecular composition of HspB2 condensates and aggregates, we used APEX-mediated proximity labelling. Most proteins interact transiently with the condensates and were neither enriched nor depleted in these cells. In contrast, we found that HspB2:HspB3 aggregates sequestered several disordered proteins and autophagy factors, suggesting that the cell is actively attempting to clear these aggregates. This study presents a striking example of how changes in the relative expression levels of interacting proteins affects their phase behavior. Our approach could be applied to study the role of protein stoichiometry and the influence of client binding on phase behavior in other biomolecular condensates and aggregates.     

刺山柑70 kD热休克蛋白基因克隆及原核表达

以干旱区特有的抗逆性植物刺山柑为材料,利用RT-PCR结合cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)方法,克隆了一个HSP70基因,将该基因进行原核表达,通过对大肠杆菌进行温度胁迫实验,并计算存活率来验证该基因对细胞的保护作用.结果表明:该基因全长2 202 bp,开放阅读框共1 950 bp,编码649个氨基酸,表达产物分子量为71.044 6 kD;序列分析表明,该基因属于HSP70基因家族,将该基因命名为CsHSP70,并提交到GenBank,登录号为EU574936.构建了CsHSP70基因的原核表达载体,并使重组质粒pGEX-4T-2-CsHSP70在大肠杆菌中异源表达,对大肠杆菌进行温度胁迫实验结果显示,在高温(50℃)和低温(4℃)条件下,转重组质粒的大肠杆菌在两种胁迫下存活率均高于对照,说明在温度胁迫下CsHSP70对大肠杆菌细胞具有保护作用.     

嗜热古菌S.solfataricus小热激蛋白基因的克隆及表达

目的:从嗜热古菌Sulfolobus solfataricus中克隆一种新的小热激蛋白SsHsp14.1的基因,并研究其表达和生物活性。方法:用PCR技术以S.solfataricus基因组为模板扩增得到目的基因序列片段,并将其克隆到pET-28a(+)中,转化到E coli BL21(DE3)中经IPTG诱导表达,纯化后对产物进行生物活性测定。结果:从菌株S.solfataricus中克隆出目的基因,该基因的编码框由375个碱基组成,编码的蛋白质由124个氨基酸组成。含该质粒的大肠杆菌经诱导表达了一个与预期理论值相符的约14kDa的蛋白,利用亲和层析和凝胶柱分离纯化了重组蛋白。试验证明纯化后的重组SsHsp14.1具有分子伴侣活性,重组蛋白在体内表达时能提高E.coli细胞的耐热性。结论:成功克隆SsHsp14.1基因并表达出蛋白,并明确了其分子伴侣活性,为该热激蛋白的研究和应用奠定基础。     

Late Onset of the Serological Response against the 18 kDa Small Heat Shock Protein of Mycobacterium ulcerans in Children

A previous survey for clinical cases of Buruli ulcer (BU) in the Mapé Basin of Cameroon suggested that, compared to older age groups, very young children may be less exposed to Mycobacterium ulcerans. Here we determined serum IgG titres against the 18 kDa small heat shock protein (shsp) of M. ulcerans in 875 individuals living in the BU endemic river basins of the Mapé in Cameroon and the Densu in Ghana. While none of the sera collected from children below the age of four contained significant amounts of 18 kDa shsp specific antibodies, the majority of sera had high IgG titres against the Plasmodium falciparum merozoite surface protein 1 (MSP-1). These data suggest that exposure to M. ulcerans increases at an age which coincides with the children moving further away from their homes and having more intense environmental contact, including exposure to water bodies at the periphery of their villages.     

A Model for Small Heat Shock Protein Inhibition of Polyglutamine Aggregation

Polyglutamine (polyQ) repeat expansions that lead to the formation of amyloid aggregates are linked to several devastating neurodegenerative disorders. While molecular chaperones, including the small heat shock proteins (sHsp), play an important role in protection against protein misfolding, the aberrant protein folding that accompanies these polyQ diseases overwhelms the chaperone network. By generating a model structure to explain the observed suppression of spinocerebellar ataxia 3 (SCA3) by the sHsp αB-crystallin, we have identified key vulnerabilities that provide a possible mechanism to explain this heat shock response. A docking study involving a small bioactive peptide should also aid in the development of new drug targets for the prevention of polyQ-based aggregation.     

被子植物小热激蛋白家族的比较基因组学分析

小热激蛋白(sHSP)是一类重要的响应外界环境变化以及调控植物生长发育的蛋白家族。基于在睡莲(Nymphaea colorata)、水稻(Oryza sativa)、拟南芥(Arabidopsis thaliana)和葡萄(Vitis vinifera)中分别鉴定到的33个NcsHSPs、24个OssHSPs、17个A...     

Measurement of ATP generation and decay in Mycobacterium leprae in vitro

The intracellular ATP content of Mycobacterium leprae isolated from armadillo tissue was approximately 1.5 X 10(-16) g per bacillus. During in vitro incubation of bacilli at 4 degrees C, 33 degrees C or 37 degrees C there was an exponential decrease in ATP content, the rate depending on the medium and the temperature. M. leprae incorporated phosphate into ATP and into other nucleotide materials during in vitro incubation.     

人热休克蛋白70和热休克固有蛋白70的基因克隆和表达

目的:克隆人热休克蛋白70(HSP70)和热休克固有蛋白70(HSC70)基因,并在大肠杆茵中表达,获得重组蛋白.方法:用RT-PCR法从HepG2细胞中扩增HSP70及HSC70cDNA序列.测序后,将相应的cDNA插入pRSET-A表达载体,在大肠杆菌中表达,重组蛋白纯化后用SDS-PAGE及Western Blotting分析.结果:DNA序列结果显示.本研究所获得的HSP70及HSC70 cDNA序列与参考序列一致.将全长cDNA分别插入表达质粒后,转化BL21(DE3)细菌,在IPTG的诱导下,表达产物SDS-PAGE显示相应的分子量(70kDa)位置有明显的蛋白条带.Western Blotting结果证实了其为目的蛋白,经镍树脂柱纯化,获得了相应的重组多肽.结论:成功构建了原核表达重组质粒HSP70-pRSET-A和HSC70-pRSET-A,并获得了纯化的重组人HSP70和HSC70蛋白,为进一步研究这两种蛋白的结构、功能及临床应用奠定了基础.     

甜椒细胞质小分子量热激蛋白基因(CaHSP18)的cDNA克隆与表达

用RT-PCR和RACE-PCR技术,从热激处理的甜椒叶片总RNA中扩增出了细胞质小分子量热激蛋白(sHSP)全长779 bp的cDNA基因序列,包含一个480 bp开放阅读框,编码159个氨基酸.Southern杂交结果表明在甜椒基因组中有该基因的小的多基因家族.Northern结果显示该基因在甜椒根、茎、叶中的表达受热激和低温的诱导.原核表达分析表明该基因在高温以及低温条件下可以提高大肠杆菌的生存能力.     

Heat Shock Protein 70 Family: Multiple Sequence Comparisons, Function, and Evolution

The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport. They are abundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts). A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications. Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences. These two groups also miss a signature segment [about 25 amino acids (aa) long] present in all other HSP70 species (Gupta and Golding 1993). We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences. Consensus sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms]. All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons. The global individual consensus ``matches' 87% with the consensus of consensuses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters. These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function. We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed. The E. histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences. This appears to be at variance with the hypothesis that E. histolytica rather recently lost its mitochondrial organelle. T. vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins. Received: 29 January 1998 / Accepted: 14 May 1998     

Effect of Inhibitors on Phenoloxidase of Mycobacterium leprae

Previous results had shown that the human leprosy bacilli possess a phenoloxidase, which, when compared with the enzyme from mammalian and plant sources, seemed unique in the range of substrates utilized and in the nature of the products formed. The effect of several inhibitors on the enzyme in Mycobacterium leprae was tested. Compounds which bind copper were found to be more effective than substrate analogues. Diethyldithiocarbamate penetrated the bacillus and completely suppressed its phenolase activity. Diasone (a derivative of diaminodiphenylsulfone used in the treatment of leprosy) proved to be a potent inhibitor of phenoloxidase of mammalian and plant origin. However, it was less efficient in the case of M. leprae. A biochemical peculiarity of M. leprae was observed in its ability to metabolize mimosine and penicillamine. These compounds produced total inhibition of tyrosinase in melanoma extract and of mushroom tyrosinase. Nontoxic inhibitors of phenoloxidase in the leprosy bacilli may be of value in developing a rational approach to chemotherapy of the disease.