Identification and characterization of a single-stranded DNA-binding protein from the archaeon Methanococcus jannaschii

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria and eukarya. We report here the identification and characterization of the SSB of an archaeon, Methanococcus jannaschii. The M. jannaschii SSB (mjaSSB) has significant amino acid sequence similarity to the eukaryotic SSB, replication protein A (RPA), and contains four tandem repeats of the core single-stranded DNA (ssDNA) binding domain originally defined by structural studies of RPA. Homologous SSBs are encoded by the genomes of other archaeal species, including Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus. The purified mjaSSB binds to ssDNA with high affinity and selectivity. The apparent association constant for binding to ssDNA is similar to that of RPA under comparable experimental conditions, and the affinity for ssDNA exceeds that for double-stranded DNA by at least two orders of magnitude. The binding site size for mjaSSB is ≈20 nucleotides. Given that RPA is related to mjaSSB at the sequence level and to Escherichia coli SSB at the structural level, we conclude that the SSBs of archaea, eukarya, and bacteria share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.     

Circulating heat shock protein and heat shock protein antibody levels in established hypertension

OBJECTIVE: Serum Hsp60 and anti-Hsp65 antibody levels are raised in subjects with borderline hypertension, and there is an association between circulating Hsp60 levels and early atherosclerosis. Given the recognized relationship between hypertension and atherosclerosis, this study determined heat shock protein and heat shock protein antibody levels in subjects with established hypertension. METHODS: Samples from 111 men with hypertension were obtained from the European Lacidipine study on Atherosclerosis and samples from 75 normotensive controls were taken from a population-screening programme (diastolic pressure, 95 and 80 mmHg, respectively). Hsp60, Hsp70 and anti-human Hsp60, anti-human Hsp70 and anti-mycobacterial Hsp65 antibody levels were measured by enzyme immunoassay. Intima-media thickness (I-M) and the presence of carotid atherosclerosis were determined by ultrasonography. RESULTS: Hsp60, Hsp70 and anti-Hsp60 antibody levels in hypertension were similar to those in normotensive controls, whereas anti-Hsp70 and anti-Hsp65 antibody levels were elevated ( 0.001). Hsp60 levels and atherosclerosis were not associated. Anti-Hsp70 and anti-Hsp65 antibody levels were both associated with hypertension, independently of age, smoking habits and blood lipids. CONCLUSIONS: This study demonstrates elevated levels of selected heat shock protein antibodies in subjects with hypertension. Although the association between heat shock protein antibody levels and human cardiovascular stress/disease appears to be robust, the relationship of the latter with heat shock protein levels is more complex. Further studies are required before the factors inducing, and the clinical significance of, circulating heat shock proteins can be evaluated.     

Cytosolic heat shock protein 60, apoptosis,and myocardial injury

BACKGROUND: Heat shock proteins (HSPs) are well known for their ability to "protect" the structure and function of native macromolecules, particularly as they traffic across membranes. Considering the role of key mitochondrial proteins in apoptosis and the known antiapoptotic effects of HSP27 and HSP72, we postulated that HSP60, primarily a mitochondrial protein, also exerts an antiapoptotic effect. Methods and Results- To test this hypothesis, we used an antisense phosphorothioate oligonucleotide to effect a 50% reduction in the levels of HSP60 in cardiac myocytes, a cell type that has abundant mitochondria. The induced decrease in HSP60 precipitated apoptosis, as manifested by the release of cytochrome c, activation of caspase 3, and induction of DNA fragmentation. Antisense treatment was associated with an increase in bax and a decrease in bcl-2 secondary to increased synthesis of bax and degradation of bcl-2. A control oligonucleotide had no effect on these measurements. We further demonstrated that cytosolic HSP60 forms a macromolecular complex with bax and bak in vitro suggesting that complex formation with HSP60 may block the ability of bax and bak to effect apoptosis in vivo. Lastly, we show that as cytosolic (nonmitochondrial) HSP60 decreases, a small unbound fraction of bax appears and that the amount of bax associated with the mitochondria and cell membranes increases. CONCLUSIONS: These results support a key antiapoptotic role for cytosolic HSP60. To our knowledge, this is the first report suggesting that interactions of HSP60 with bax and/or bak regulate apoptosis.     

旋毛虫热激蛋白的研究进展

旋毛虫热激蛋白(heat shock protein,HSP)是一个高度保守的蛋白质家族,具有特殊的生物学作用,开展HSP的研究对旋毛虫病的免疫诊断和免疫预防具有重要意义.该文对旋毛虫与宿主HSP水平的变化、旋毛虫HSP的分离纯化、克隆表达及其检测方法等方面的研究进展进行了综述.     

Identification of heat shock protein 27 as a radioresistance-related protein in nasopharyngeal carcinoma cells

Purpose

To identify the proteins involved in radioresistance in nasopharyngeal cancer (NPC) cells.

Methods

Sublethal ionizing radiation was applied to establish a radioresistant NPC cell line from its parental NPC cell line CNE1. Clonogenic survival assay, cell growth assay and flow cytometry analysis were used to examine the difference of radiosensitivity in the radioresistant CNE1 cells (CNE1-IR) and control CNE1 cells. Comparative proteomics was performed to identify the differential proteins in the two cell lines. Association of HSP27, one of upregulated proteins in CNE1-IR cells, with NPC cell radioresistance was selected for further investigation using antisense oligonucleotides (ASOs), clonogenic survival assay, Hoechst 33258 staining of apoptotic cells and MTT assay of cell viability.

Results

Radioresistant NPC cell line CNE1-IR derived from its parental cell line CNE1 was established. Thirteen differential proteins in the CNE1-IR and CNE1 cells were identified by proteomics, and differential expression of HSP27, one of identified proteins, was selectively confirmed by western blot. Inhibition of HSP27 expression by HSP27 ASOs decreased clonogenic survival and cell viability and increased cell apoptosis of CNE1-IR cells after irradiation, that is, enhanced radiosensitivity of CNE1-IR cells.

Conclusion

The data suggest that HSP27 is a radioresistant protein in NPC cells, and its upregulation may be involved in the NPC radioresistance.     

Metabolite regulation of heat shock protein levels.

When murine L929 cells are briefly exposed to elevated temperature (43 degrees C) they preferentially synthesize heat shock proteins (HSPs) of Mrs 85,000 and 69,000. By the criteria of two-dimensional polyacrylamide gel electrophoresis and partial proteolytic peptide mapping, the Mr 85,000 HSP is indistinguishable from the major cytoplasmic protein whose synthesis and intracellular level were shown previously to be suppressed by glucose deprivation. The mechanism regulating the Mr 85,000 HSP levels is quite sensitive, operating in the range of 0-50 microM glucose. In glucose-free medium, synthesis is at first suppressed but returns to a high level after 3 days as levels of the protein decrease. Synthesis and level of the Mr 69,000 HSP also were affected by glucose deprivation, but this protein appeared to be a complex of several isoelectric and Mr species, so the effects were not as dramatic. Chase experiments show that the half-lives of both the Mr 85,000 and Mr 69,000 HSPs are reduced by a factor of 2.0-2.5 after 5 days of glucose deprivation. The half-life of the Mr 69,000 HSP also was reduced by glutamine deprivation, whereas that of the Mr 85,000 HSP was essentially unaffected. This increase in turnover appears to be sufficient to account for the reduced intracellular level, thus suggesting that glucose sustains high HSP levels mainly by decreasing degradation of the proteins. Although the function of the HSPs is not known, these data support the concept that they have important roles in the general cellular economy and do not function merely as "stress" proteins.     

热休克蛋白与肿瘤的研究进展

热休克蛋白（heat shock proteins,HSP）是细胞暴露在致死性环境刺激下产生的一种功能高度保守的蛋白质,是一种应激蛋白,广泛存在于原核生物及真核生物中。其作为分子伴侣通过协助蛋白质的适当折叠,保护细胞免受外界环境的损害,并参与细胞的生长分化、新陈代谢及信号转导;此外,     

Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease

Huntington's disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAG/polyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2/HSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43-74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2/HSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2/HSDJ in protein folding.     

Alternative bacterial two-component small heat shock protein systems

Small heat shock proteins (sHsps) are molecular chaperones that prevent the aggregation of nonnative proteins. The sHsps investigated to date mostly form large, oligomeric complexes. The typical bacterial scenario seemed to be a two-component sHsps system of two homologous sHsps, such as the Escherichia coli sHsps IbpA and IbpB. With a view to expand our knowledge on bacterial sHsps, we analyzed the sHsp system of the bacterium Deinococcus radiodurans, which is resistant against various stress conditions. D. radiodurans encodes two sHsps, termed Hsp17.7 and Hsp20.2. Surprisingly, Hsp17.7 forms only chaperone active dimers, although its crystal structure reveals the typical α-crystallin fold. In contrast, Hsp20.2 is predominantly a 36mer that dissociates into smaller oligomeric assemblies that bind substrate proteins stably. Whereas Hsp20.2 cooperates with the ATP-dependent bacterial chaperones in their refolding, Hsp17.7 keeps substrates in a refolding-competent state by transient interactions. In summary, we show that these two sHsps are strikingly different in their quaternary structures and chaperone properties, defining a second type of bacterial two-component sHsp system.     

Small heat shock proteins of Drosophila associate with the cytoskeleton.

Fractionation of heat-shocked Drosophila melanogaster Kc cells reveals that both the small heat shock proteins (hsp28, -26, -23, and -22) and vimentin-like intermediate filament proteins (IFPs) are abundantly represented in the nuclear fraction. Cofractionation of the IFPs with nuclei is due to the collapse of the IFP network against the nucleus upon heat shock, raising the possibility that cofractionation of the small hsps is by a similar mechanism. Indirect immunofluorescence supports this possibility. In salivary glands, both the hsps and the IFPs are cytoplasmic after mild-to-moderate heat shocks and only enter the nucleus upon severe--indeed, lethal--shocks. Double-label experiments with Schneider line 2 cells show that the IFPs and small hsps colocalize to the same perinuclear aggregates in 70% of the cells examined. Thus, the small hsps are associated with the cytoskeleton rather than with nuclear structures.     

A novel pH2 control on the expression of flagella in the hyperthermophilic strictly hydrogenotrophic methanarchaeaon Methanococcus jannaschii

The methanarchaeon, Methanococcus jannaschii, a hyperthermophilic, autotrophic, and strictly hydrogenotrophic inhabitant of submarine hydrothermal vents, was cultivated in a reactor at two hydrogen partial pressure (p(H(2))) values, 178 kPa (high) and 650 Pa (ultralow), and the cells were subjected to a comparative proteome analysis. From these studies, it was discovered that, when p(H(2)) was high and the cell density was low (a combination representing a hydrogen-excess condition), the cells possessed very low or undetectable levels of four flagella-related polypeptides (FlaB2, FlaB3, FlaD, and FlaE); electron microscopic examination showed that most of these cells were devoid of flagella. Flagella synthesis occurred when hydrogen became limiting either at high cell density under high p(H(2)) or at low cell density under low p(H(2)). The results from a p(H(2))-shift experiment corroborated the above observations. The p(H(2))-dependent changes in the levels of two methanogenic enzymes (MTD and HMDX) were as expected, and thus they served as internal controls. To our knowledge, this is the first example for the regulation of expression of flagella by hydrogen in any domain of life and for a control of any kind on flagella synthesis in the archaea. Our work also provides the only known example for each of the following: (i) the pure culture cultivation of a methanogen at an ultralow, near ecologically relevant p(H(2)); (ii) experimental functional genomics for M. jannaschii; and (iii) the use of proteomics with M. jannaschii.     

Estrogenic regulation of uterine 90-kilodalton heat shock protein

Recently two lines of evidence have implicated that cellular heat shock proteins (hsp) may play a role in steroid hormonal regulation of target tissues. One is the demonstration that cellular 90K hsp (hsp-90) can complex with steroid receptors in vitro and inhibit their ability to interact with DNA, and second, the demonstration that in avian oviduct sex steroids can regulate the synthesis of hsp-108. As yet, there is no report that sex steroids can regulate hsp-90 synthesis, especially in mammalian tissues. In these studies we have examined the estrogenic regulation of murine uterine hsp-90. We report that ovariectomy reduces the uterine concentration of hsp-90, and estradiol causes a time-dependent increase in uterine hsp-90 as early as 4 h after steroid administration, reaching a maximum increase of 4-fold between 18-24 h. The effect is specific to estrogens and not elicited by other steroid hormones. It is also target tissue specific, such that it is seen with uterus and vagina and does not occur in nontarget tissue for estradiol, such as spleen. The possible physiological significance of estrogenic stimulation of uterine hsp-90 has been discussed.     

热休克蛋白60在扩张型心肌病中的意义

目的探讨心肌组织中热休克蛋白60(HSP60)在扩张型心肌病(DCM)发生、发展中的作用。方法应用免疫组化方法检测15例DCM患者及10例对照者心肌组织中HSP60的表达和分布。结果DCM患者心肌组织中HSP60的表达较对照组明显增加(P<0.05),而且发现HSP60阳性细胞主要局限于心肌纤维的连接区域。结论DCM患者心肌组织中HSP60的过高表达,可能与DCM的发生发展有关。     

热休克蛋白及其在血吸虫研究中的进展

热休克蛋白(HSP)是生物界普遍存在的一类高度保守蛋白质,在生物体正常状态下有一定浓度的表达,当受到不同理化及病理因素刺激时,生物体合成这类蛋白质的能力将大大增加。HSP具有重要的生理功能,如参与相关蛋白质的折叠、亚基的装配、蛋白质的降解修复和细胞内物质运输等,以保护生物体体细胞免受内外不良因素的损害。该蛋白的生物学功能十分广泛,具有分子伴侣作用并参与抗原提呈过程。自发现以来,人们对果蝇、细菌、寄生虫、哺乳动物和人等各种生物的HSP进行了广泛的研究,已成为抗寄生虫感染中研究的热点之一。本文对其特点、功能和在血吸虫研究中的进展做一综述。