Heat shock protein DnaK — Substrate of actin-specific bacterial protease ECP32

It has been found that actin-specific bacterial protease ECP32 cleaves prokaryotic heat shock protein DnaK, which belongs to the family of heat shock proteins with molecular weight 70 kDa. We propose a new one-step method for DnaK purification using heat treatment. The technique yields ∼1 mg of partially purified DnaK from 25 g of wet bacterial biomass. Polyclonal antibodies against DnaK were obtained. The degree of ECP32 catalyzed proteolysis of partially purified DnaK and that of DnaK in initial cell extracts was compared.     

鸭疫里默氏菌DnaK、OmpA和OmpA-DnaK蛋白的免疫原性

【背景】鸭疫里默氏菌(Riemerella anatipestifer,RA)可引起鸭等多种禽类败血症和浆膜炎,给禽养殖业造成严重经济损失。蛋白疫苗是预防RA感染的重要策略之一。目前,有关RA重组蛋白免疫原性报道较少,且其应用也受到单一蛋白抗原诱导的特异性免疫反应不足的限制。【目的】探究分子伴侣DnaK、外膜蛋白A (outer membrane protein A,OmpA)和OmpA-DnaK蛋白疫苗在鸭体内诱导的免疫应答,评估其免疫原性,为RA疫苗抗原研发提供依据。【方法】克隆DnaK和OmpA基因并分别与pET-32a(+)载体相连,利用限制性酶切位点Nco I和Bam H I将OmpA连接至DnaK基因上游,经原核表达和纯化制得重组蛋白DnaK、OmpA和OmpA-DnaK。3种重组蛋白分别皮下免疫雏鸭2次,检测其血清抗体滴度、淋巴细胞增殖和细胞因子(IL-2和IL-4)水平;以RA-GH5肌肉注射攻毒,检查其组织病理学变化及免疫保护率。【结果】成功表达了DnaK、OmpA和OmpA-DnaK重组蛋白,分子量分别约为90、60和130 kDa。3种蛋白疫苗均能诱导宿主产生体液...     

DnaK蛋白扭链残基突变体影响其ATPase活性的分子动力学模拟研究

大肠杆菌分子伴侣蛋白Dna K氮端核苷酸结合域(NBD,nucleotide-binding domain)的II-A和II-B子域之间的一些高度保守的扭链残基突变后(I202A,S203A,G223A,L227A,G228A),其ATPase活性也发生变化原因不清楚。我们通过同源建模的方法构建NBD与小分子ATP相互作用的各蛋白模型,使用分子动力学模拟方法研各模型的结构变化并尝试找出其与ATPase活性变化的关系。结果表明,除L227A外,所有突变模型T11烃基与ATP-γ磷酸基团间的距离与活性变化间具有明显规律;但是所有模型中,能影响与Dna J结合,从而影响ATPase活性的β220(214-221)部分的紧致性变化符合规律,进一步的蛋白对接实验证实了这一点,所以这些扭链残基突变体可能主要是通过这两个部分的变化,引起ATPase活性的改变。     

绵羊肺炎支原体DnaK B细胞抗原表位预测及其蛋白结构分析

以绵羊肺炎支原体(Mo)热休克蛋白Hsp70(DnaK蛋白)氨基酸序列为基础,运用DNAStar软件和在线服务器等生物信息学分析工具,在同源性分析的基础上,通过Jameson-Wolf法、Kyte-Doolittle法、Emini法、Karplus-Schulz法及Welling法分别预测其抗原指数、亲水性、柔韧性、表面可极性等参数,综合分析、预测了该蛋白的B细胞抗原表位,并进行了蛋白的二级结构预测和3D结构模拟。结果表明,Mo贵州分离株的Hsp70蛋白与其它可致羊发病支原体的Hsp70蛋白同源性较低,说明该蛋白具有良好的特异性;Mo Hsp70蛋白整体抗原性较好,同时呈现较规则的空间结构,其中以C末端较稳定,区域也最大(第394-598区段),为优势B细胞抗原表位区域。     

赤红球菌SD3全基因组测序及其热休克蛋白DnaK的表达分析

红球菌属微生物因其自身较强的有机物耐受性和较宽的降解谱,能够适应多种生境而被广泛应用于生物脱硫、石油污染修复、有毒有机化合物降解、污水处理等领域。本研究利用单分子PacBio测序技术,对一株耐有机溶剂的赤红球菌SD3 (Rhodococcus ruber SD3)全基因组进行测序并进行生物信息学分析。该菌株的全基因组长度大约为5.37 Mb,GC含量为70.63%,GenBank序列登录号为CP029146。使用Barrnap0.4.2和tRNAscan-SEv1.3.1软件对基因组中包含的rRNA基因和tRNA基因进行预测,发现有12个rRNA基因和53个tRNA基因。利用Glimmer3.02软件对该基因组进行基因预测,共得到5 120个编码蛋白的基因。将预测的蛋白序列同时与KEGG、STRING和GO三类数据库进行Blastp比对,共计2 836个蛋白基因获得COG功能注释,并且注释得到3 130条GO功能条目和2 190条KEGG通路条目。此外,基于荧光定量PCR的分析表明在甲苯和苯酚胁迫下,赤红球菌SD3中热休克蛋白DnaK的表达分别上调了29.87倍和3.93倍。这些研究结果为赤红球菌的遗传改造和揭示赤红球菌的有机溶剂耐受性机制提供了理论依据。     

A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor ��32 of Escherichia coli

The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70''s ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ³², which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ³² facilitates RB43 bacteriophage proliferation is discussed.     

Complementation studies of the DnaK–DnaJ–GrpE chaperone machineries from <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>, both in vivo and in vitro

The marine bacterium Vibrio harveyi is a potential indicator organism for evaluating marine environmental pollution. The DnaK–DnaJ–GrpE chaperone machinery of V. harveyi has been studied as a model of response to stress conditions and compared to the Escherichia coli DnaK system. The genes encoding DnaK, DnaJ and GrpE of V. harveyi were cloned into expression vectors and grpE was sequenced. It was found that V. harveyi possesses a unique organization of the hsp gene cluster (grpE–gltP–dnaK–dnaJ), which is present exclusively in marine Vibrio species. In vivo experiments showed that suppression of the E. coli dnaK mutation by V. harveyi DnaK protein was weak or absent, while suppression of the dnaJ and grpE mutations by V. harveyi DnaJ and GrpE proteins was efficient. These results suggest higher species-specificity of the DnaK chaperone than the GrpE and DnaJ cochaperones. Proteins of the DnaK chaperone machinery of V. harveyi were purified to homogeneity and their efficient cooperation with the E. coli chaperones in the luciferase refolding reaction and in stimulation of DnaK ATPase activity was demonstrated. Compared to the E. coli system, the purified DnaK–DnaJ–GrpE system of V. harveyi exhibited about 20% lower chaperoning activity in the luciferase reactivation assay. ATPase activity of V. harveyi DnaK protein was at least twofold higher than that of the E. coli model DnaK but its stimulation by the cochaperones DnaJ and GrpE was significantly (10 times) weaker. These results indicate that, despite their high structural identity (approximately 80%) and similar mechanisms of action, the DnaK chaperones of closely related V. harveyi and E.coli bacteria differ functionally.