A structural model of the Sgt2 protein and its interactions with chaperones and the Get4/Get5 complex

The insertion of tail-anchored transmembrane (TA) proteins into the appropriate membrane is a post-translational event that requires stabilization of the transmembrane domain and targeting to the proper destination. Sgt2 is a heat-shock protein cognate (HSC) co-chaperone that preferentially binds endoplasmic reticulum-destined TA proteins and directs them to the GET pathway via Get4 and Get5. Here, we present the crystal structure from a fungal Sgt2 homolog of the tetratrico-repeat (TPR) domain and part of the linker that connects to the C-terminal domain. The linker extends into the two-carboxylate clamp of the TPR domain from a symmetry-related molecule mimicking the binding to HSCs. Based on this structure, we provide biochemical evidence that the Sgt2 TPR domain has the ability to directly bind multiple HSC family members. The structure allows us to propose features involved in this lower specificity relative to other TPR containing co-chaperones. We further show that a dimer of Sgt2 binds a single Get5 and use small angle x-ray scattering to characterize the domain arrangement of Sgt2 in solution. These results allow us to present a structural model of the Sgt2-Get4/Get5-HSC complex.     

The Effect of Temperature Stress on Development and Heat-shock Protein Expression in Larval Green Sturgeon (Acipenser mirostris)

Water temperature is an important environmental variable influencing the distribution and health of coldwater fishes such as the green sturgeon, Acipenser medirostris. In this study, we investigated if larval sturgeon were able to tolerate or recover from acute, non-lethal temperature stress that commonly causes deformed notochords, and sought to identify the role of heat-shock proteins (hsp) in stress tolerance. The hsp response is one of the most important cellular mechanisms to prevent the damaging effects of thermal cellular stress, and differences in the ability to over-express hsps during stressful conditions may be associated with an organism’s vulnerability and the extent of thermal injury. In this study, newly hatched larvae were maintained at 17°C (control), or exposed to (a) 26°C for 3 d then maintained at 17°C until yolk-sac absorption or (b) 26°C until yolk-sac absorption. Individuals with deformed notochords were counted, and hsp60, 72, 78 and 89 were analyzed in both normal and deformed larvae by western blotting. Approximately 33% of fish developed curved notochords within the first 3 d of exposure to 26°C. After transfer to cool water 16.5% showed deformities at stage 45, suggesting a significant number of larvae had recovered. Hsp levels remained elevated for at least 9 days after termination of heat-exposure. Overall, percentage of deformed larvae, and hsp72/hsp78 levels were highest in fish continuously exposed to 26°C until yolk-sac absorption. Deformed individuals had significantly higher expression levels of hsp72 and hsp78, and lower hsp60 levels than normal larvae. We conclude that expression of hsp72 and hsp78 and potentially hsp60 are linked to phenotypic variation in the response and vulnerability of green sturgeon larvae to thermal stress.     

The 60- and 70-kDa heat-shock proteins and their correlation with cardiovascular risk factors in postmenopausal women with metabolic syndrome

We investigated the association between circulating levels of 60 and 70 kDa heat-shock proteins (HSP60 and 70) and cardiovascular risk factors in postmenopausal women with or without metabolic syndrome (MetS). This cross-sectional study included 311 Brazilian women (age ≥45 years with amenorrhea ≥12 months). Women showing three or more of the following diagnostic criteria were diagnosed with MetS: waist circumference (WC) ≥88 cm, blood pressure ≥130/85 mmHg, triglycerides ≥150 mg/dl, high-density lipoprotein (HDL) <50 mg/dl, and glucose ≥100 mg/dl. Clinical, anthropometric, and biochemical parameters were collected. HSP60, HSP70, antibodies to HSP60 and HSP70, and C-reactive protein (CRP) levels were measured in serum. Student''s t test, Kruskal–Wallis test, chi-square test, and Pearson correlation were used for statistical analysis. Of the 311 women, 30.9 % (96/311) were diagnosed with MetS. These women were, on average, obese with abdominal fat deposition and had lower HDL values as well as higher triglycerides and glucose levels. Homeostasis model assessment-insulin resistant (HOMA-IR) test values in these women were compatible with insulin resistance (P < 0.05). CRP and HSP60 concentrations were higher in women with MetS than in women without MetS (P < 0.05). HSP60, anti-HSP70, and CRP concentrations increased with the number of features indicative of MetS (P < 0.05). There was a significant positive correlation between anti-HSP70 and WC, blood pressure and HOMA-IR, and between CRP and WC, blood pressure, glucose, HOMA-IR, and triglycerides (P < 0.05). In postmenopausal women, serum HSP60 and anti-HSP70 concentrations increased with accumulating features of the metabolic syndrome. These results suggest a greater immune activation that is associated with cardiovascular risk in postmenopausal women with metabolic syndrome.     

Identification of a novel cognate cytosolic Hsp70 gene (MnHsc70-2) from oriental river prawn Macrobrachium nipponense and comparison of its expressions with the first cognate Hsc70 (MnHsc70-1) under different stresses

The 70-kDa family of heat-shock proteins (Hsp70) plays an important role in the host immunity, which is widely expressed in eukaryotic cells as a major chaperone protein. In the present study, the full-length complementary DNA (cDNA) of a second cognate cytosolic Hsp70 family member (MnHsc70-2) was cloned and characterized from Macrobrachium nipponense, which is an economically and nutritionally important crustacean. The cDNA was 2,717 bp, containing an open reading frame (ORF) of 1,950 bp, which encodes a protein of 649 amino acids with a theoretical molecular weight of 71.1 kDa and an isoelectric point of 5.27. Sequence alignment showed that the MnHsc70-2 shared 75–97 % identity with other heat-shock proteins. Compared to the previously identified cognate Hsp70 (MnHsc70-1) in M. nipponense, MnHsc70-2 showed quite different expression profiles under unstressed conditions in all tested tissues, including the hemocytes, heart, hepatopancreas, gill, intestine, nerve, and muscle. The phylogenetic analysis demonstrated that MnHsc70-2 showed the closest relationship with MnHsc70-1. Heat-inducibility assays showed that two isolated messenger RNAs (mRNAs) displayed different expression profiles in both the hepatopancreas and gill tissues. MnHsc70-1 mRNA expression level decreased at first and then increased to the normal level, whereas MnHsc70-2 mRNA level increased at first and then decreased. The expressions of two MnHsc70s showed substantial obvious heat-inducible regulation in both the hepatopancreas and gill. Under bacterial challenge by Aeromonas hydrophila, both MnHsc70-1 and MnHsc70-2 mRNA level was up-regulated moderately. The results suggested that two cognate Hsc70s may play essential functions in mediating responses to heat-shock and bacterial challenge.     

热休克蛋白调控机制

热休克蛋白是生物体体应对温度、pH、渗透压等不利环境刺激时合成的一种保护蛋白。在环境应激时,调控因子可以在转录水平上调控热休克基因的表达,恢复或加速清除细胞内已经变性的蛋白质,使细胞处于稳态并产生耐受性。大量研究发现,热休克调控因子对微生物应激耐受性发挥重要作用,具有广阔的应用前景。综述了6类热休克调控因子的调控机制以及相互作用,对调控因子HrcA、σ^B和CtsR进行了重点阐述,旨在为进一步构建热休克调控网络提供有价值的参考。     

汉滩病毒核蛋白与热休克蛋白GRP94、HSP27的相互作用

为研究汉滩病毒(Hantaan virus, HTNV)感染诱导乳鼠脑组织热休克蛋白GRP94、HSP27与病毒蛋白的相互关系,选出生2～3d的昆明乳鼠实验性感染汉滩病毒,取8d后发病乳鼠脑组织部分制石蜡切片,用免疫组化结合共聚焦显微镜检测组织中病毒抗原及GRP94、HSP27的表达,部分制匀浆液,用ELISA、免疫共沉淀方法分析病毒抗原和GRP94、HSP27的关系.结果示汉滩病毒感染诱导乳鼠脑组织神经细胞高表达GRP94且与细胞内病毒抗原有共定位关系,但未见HSP27诱导高表达;免疫共沉淀显示汉滩病毒核心抗原(HINV-NP)与GRP94、HSP27呈非共价复合物形式存在.该结果为进一步探讨HSPs在病毒感染复制中的作用以及抗病毒感染方面提供了有意义的实验资料.     

Hsh载体对外源基因在E. coli中高效表达的机制探讨

pHsh是根据大肠杆菌的热休克反应构建而成的新型表达载体, 受σ32调控。正常E. coli细胞的整个热休克反应持续时间约12 min, 而在携带有外源基因的高拷贝pHsh的E. coli细胞中, 外源基因却能持续高效表达4?10 h。为探求外源基因高效表达的机制, 以一个编码木聚糖酶的外源基因为代表, 首先研究了质粒拷贝数对木聚糖酶表达的影响, 接着通过Western-blot检测了携带质粒pHsh-xynIII和对照组携带pLac-xynIII的E. coli细胞在非诱导条件下(30 °C)和诱导条件下(30 °C→42 °C)胞内σ32的差异, 最后测定了不同温度下(30 °C、37 °C、42 °C、30 °C→42 °C)携带质粒(pHsh-xynIII)的E. coli细胞内稳定状态下热休克的水平(以木聚糖酶活性表征)。研究结果表明外源基因在pHsh中的高效表达是与3个方面密切相关的: pHsh质粒的高拷贝数增加了外源基因的剂量; pHsh的存在使E. coli细胞内σ32的水平较正常E. coli细胞显著增加了, 并最终增强了E. coli的热休克反应; 诱导状态下带有pHsh重组质粒的E. coli细胞内稳定状态下的热休克水平明显高于其它温度的水平。     

不同纬度螅状独缩虫耐热能力及Hsp70 mRNA表达水平的比较

本文以游泳体的形成作为虫体对热激响应的指征,比较了不同纬度 (西安:东经10 8°98′,北纬34°25′;武汉:东经114°34′,北纬30°57′) 两地螅状独缩虫 (Carc hesium polypinum) 的耐热能力,发现武汉地区螅状独缩虫 (WH株) 形成游泳体的热激温度 ( 36℃) 高于西安地区 (XA株) 的33℃,说明前者的耐热能力较强;进一步利用实时PCR技术分析不同热激温度下两地螅状独缩虫热休克蛋白Hsp70 mRNA的表达变化发现,XA株Hs p70 mRNA热激后高表达水平的阈值温度为28℃,而WH株的为33℃,表明在螅状独缩虫中, Hsp70 mRNA表达的阈值温度与它们的耐热能力之间存在正相关.从而在分子水平阐明了螅状独缩虫WH株较XA株具有更高耐热能力的原因,这为研究纤毛虫野生种群热休克蛋白Hsp7 0 mRNA表达与栖息环境温度间的关系,进而了解原生动物温度适应的分子机理提供了线索[动物学报 54(3):525-530,2008].     

Overexpression of 27-kDa heat-shock protein in MCF-7 breast cancer cells: effects on lymphocyte-mediated killing by natural killer and γδ T cells

Overexpression of the heat-shock protein hsp27 protein in primary breast cancers has been associated with early relapse in women with breast cancer. This study was designed to determine the role of the hsp27 protein in lymphocyte recognition of estrogen-receptor(ER)-positive breast cancer cells and to assess the effect of hsp27 expression on lymphocyte-mediated lysis. The hsp27 cDNA was inserted into the pHbAPr-1-neo plasmid expression vector and driven by the constitutive actin promoter. The ER-positive MCF-7 human breast cancer cell line was then transfected with this vector and the resulting clonal cell lines were confirmed to overexpress hsp27. hsp27-transfected clonal cell lines stimulated the proliferation of fresh peripheral blood lymphocytes (PBL) significantly better than control cells transfected with the expression vector alone. When clonal T cell lines were utilized as effectors, hsp27-transfected cell lines were significantly better targets for lysis than a control-transfected MCF-7 cell line. In contrast, hsp27-transfected cell lines had no increase in susceptibility to lymphokine-activated-killer- or natural-killer-mediated lysis. These results suggest that overexpression of the hsp27 protein in ER-positive MCF-7 cells stimulated the proliferation of fresh PBL and the lysis of MCF-7 cells by T cell clones.