革兰氏阴性菌分泌蛋白质的途径

革兰氏阴性菌分泌蛋白质的途径方深高（复旦大学生化系,上海２００４３３）关键词革兰氏阴性菌,蛋白质分泌途径革兰氏阴性菌能分泌胞外蛋白,例如Ｅ·ｃｏｌｉ分泌溶血素,Ｅｒ·ｃｈｒｙｓａｎｔｈｅｍｉ分泌果胶酶、纤维素酶、多聚半乳糖醛酸酶、溶血素、白细胞毒等胞...     

以SecA为靶点的新型抗绿脓杆菌药物细胞水平筛选模型的建立和应用

Sec途径(即分泌途径secretion pathway)是蛋白质转运的主要途径.其中,最为关键的组分之一是SecAATP酶,是蛋白质转运途径中的"动力泵",通过ATP的水解循环驱使蛋白质前体穿过细菌内膜,在细菌中是不可缺少的.我们推测抑制SecAATP酶活性的化合物.必然会在一定程度上抑制蛋白质的转运和分泌.通过绿脓杆菌与大肠杆菌SecA蛋白的互补作用,利用本实验室构建的高效表达SecA蛋白的基因工程菌,建立了SecA蛋白ATP酶活性抑制剂的细胞水平筛选模型.利用所纯化的绿脓杆菌SecA蛋白的ATP酶活测定体系,验证了所建立的细胞水平筛选模型具有一定的特异性.研究结果表明其中两个酯相组分在细胞水平和蛋白水平均具有活性,值得进行深入的研究.     

大肠杆菌Sec依赖性蛋白质转运途径

与真核细胞蛋白质外运方式不同,大肠杆菌分泌蛋白的合成和跨内膜转运是不偶联的,对于一些小分子蛋白质（例如噬菌体Ｍ１３外壳蛋白）不需要其它分子协助能自发保持松散构象从核糖体定位到质膜,而对于大的蛋白质分子,尤其含有疏水结构域或疏水信号肽,需要其它分子伴侣的协助以保持转运感受状态才能被有效转运。除了这种依赖Ｓｃｃ（ｓｅｃｒｅｔｏｒｙ）蛋白的一般分泌途径以外,还存在其它Ｓｅｃ非依赖性的Ｔａｔ途径等。本文对大肠杆菌Ｓｅｃ依赖性蛋白质转运途径进行综述。     

细菌蛋白分泌途径的研究进展

蛋白质分泌对于细菌的生长和繁殖具有至关重要的作用.在革兰氏阴性菌中,蛋白质分泌包括两步和一步分泌途径,主要涉及Sec、SRP和Tat途径.近年来发现I-Ⅳ型途径也参与胞内蛋白质转运.主要介绍革兰氏阴性茵蛋白分泌的机制及生理意义.对于细菌蛋白分泌机制的深入研究将为细茵蛋白质分泌工程和病原微生物的防治带来有益启示.     

以SecA蛋白为“动力泵”的细菌Sec蛋白转运途径

细菌细胞中,三分之一的蛋白质是在合成后被转运到细胞质外才发挥功能的.其中大多数蛋白是通过Sec途径(即分泌途径secretion pathway)进行跨膜运动的.Sec转运酶是一个多组分的蛋白质复合体,膜蛋白三聚体SecYEG及水解ATP的动力蛋白SecA构成了Sec转运酶的核心.整合膜蛋白SecD,SecF和vajC形成了一个复合体亚单位,可与SecYEG相连并稳定SecA蛋白的膜结合形式.SecB是蛋白质转运中的伴侣分子,可以和很多蛋白质前体结合.SecM是由位于secA基因上游的secM基因编码的,可调节SecA蛋白的合成量,维持细胞在不同环境条件下的正常生长.新生肽链的信号肽被高度保守的SRP特异性识别.伴侣分子SecB通过与细胞膜上的SecA二聚体特异性结合将蛋白质前体引导至Sec转运途径,起始转运过程.结合蛋白质前体的SecA与组成转运通道的SecYEG复合体具有较高的亲和性.SecA经历插入和脱离细胞内膜SecYEG通道的循环,为转运提供所需的能量,每一次循环可推动20多个氨基酸的连续跨膜运动.     

细菌Ⅲ型分泌系统

1 .细菌分泌机制概述致病细菌和宿主细胞相互作用的特点是将致病因子定位到细菌表面或分泌到胞外。虽然其分泌的细菌蛋白质数量和种类都很多且具有范围很广的作用 (包括蛋白质水解、溶血、细胞毒性、蛋白质的磷酸化和去磷酸化 ) ,但是只存在几种途径把这些蛋白质从细菌的细胞质运输到胞外。在革兰氏阴性细菌中已发现四种蛋白质分泌途径 ,称为Ｉ、ＩＩ、ＩＩＩ、ＩＶ型分泌机制。另外 ,第五种大分子分泌途径和质粒的接合转移有关。根癌农杆菌Ｔ ＤＮＡ的转移 ,百日咳杆菌毒素的分泌 ,称之为Ｖ型分泌 ,这方面的研究很少[1] 。ＩＩ型和ＩＶ型是…     

Caspase-1介导的蛋白质分泌机制新进展

此前多数观点认为内质网一高尔基体运输是经典的分泌型蛋白质分泌出胞的途径。它们要先翻译出一段信号肽,这段信号肽将mRNA连同核糖体转移到内质网上并使合成的蛋白质保存于内质网内,而后通过高尔基体分泌出胞。     

泛肽系统的组成和功能(一) 一 系统组成 底物识别与蛋白质泛肽化

泛肽(Ubiquitin 简称Ub)是一个由76个氨基酸残基组成的非常保守的小蛋白质。泛肽依赖性的蛋白质降解途径(Ubiquitin-dependebt proteiytic pathway)是目前已知的最重要的、有高度选择性的蛋白质降解途径。泛肽系统由Ub、Ub活比酶、Ub结合酶、Ub-蛋白质连接酶、Ub-C末端水解酶和26S蛋白酶体组成。本文详细地介绍了泛肽系统各个组成部分的种类、结构与功能,蛋白质泛肽化及其降解机制和底物识别模式。     

氯霉素和四环素阻碍细菌蛋白分泌研究进展

氯霉素和四环素发挥活性的一个途径就是阻碍细菌蛋白质的分泌,其分泌功能是由其氨基端的信号序列决定的,该序列能将蛋白质引导到由SecY,E,G和A组成的转运蛋白复合体上。蛋白的转运还取决于融合蛋白的折叠特点,蛋白质转运到周质后的错误折叠可导致毒素聚集体形成,快速折叠还会使转运复合体发生拥堵,使所有的蛋白质分泌都受到抑制,导致细胞死亡。抗生素氯霉素和四环素处理细菌后会导致转运复合体中SecY的降解,造成致命的蛋白拥堵。现就抗生素氯霉素和四环素的干扰细菌蛋白质合成的作用机制以及导致SecY的降解来发挥阻碍细菌蛋白质分泌活性的一个新模式进行概述,以期为探讨新的靶向细菌的治疗方法提供科学依据。     

蛋白质的翻译后加工3.蛋白质定位在细胞内的信号

蛋白质的翻译后加工３．蛋白质定位在细胞内的信号王克夷（中国科学院上海生物化学研究所,上海２０００３１）关键词细胞内定位,信号肽细胞内有大量蛋白质。那么哪些蛋白质被保留在细胞内,而不被分泌呢？研究表明,蛋白质分泌到细胞外一定要通过内质网（ＥＲ）。一些分...     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Secretion of TEM beta-lactamase with signal sequences isolated from the chromosome of Lactococcus lactis subsp. lactis

With TEM beta-lactamase as a reporter gene, a set of expression-secretion-promoting fragments were isolated from the chromosome of Lactococcus lactis subsp. lactis. The fact that only translocated beta-lactamase renders cells resistant to ampicillin allowed direct ampicillin selection with an Escherichia coli vector (pKTH33). The clones showing the greatest ampicillin resistance were subcloned onto a replicon capable of replication in lactic acid bacteria (pVS2), and the nucleotide sequences of the relevant fragments were determined. The structure of the secretion-promoting fragments in general resembled that of gram-positive true signal sequences, with a strongly positively charged N terminus, a long hydrophobic core, and a putative signal peptidase recognition site. The promoterlike sequences preceding the signal sequences matched well with those of previously published lactococcal promoters. In addition to E. coli, the functioning of these expression-secretion cassettes was studied in three gram-positive hosts: Bacillus subtilis, L. lactis, and Lactobacillus plantarum. Efficient expression and secretion of TEM beta-lactamase into the culture medium of each gram-positive host was obtained. Furthermore, when a strain of L. lactis subsp. lactis showing increased sensitivity to lysozyme was compared with a standard laboratory strain, threefold-higher secreted enzyme activities were detected.     

Secretion of TEM beta-lactamase with signal sequences isolated from the chromosome of Lactococcus lactis subsp. lactis.

With TEM beta-lactamase as a reporter gene, a set of expression-secretion-promoting fragments were isolated from the chromosome of Lactococcus lactis subsp. lactis. The fact that only translocated beta-lactamase renders cells resistant to ampicillin allowed direct ampicillin selection with an Escherichia coli vector (pKTH33). The clones showing the greatest ampicillin resistance were subcloned onto a replicon capable of replication in lactic acid bacteria (pVS2), and the nucleotide sequences of the relevant fragments were determined. The structure of the secretion-promoting fragments in general resembled that of gram-positive true signal sequences, with a strongly positively charged N terminus, a long hydrophobic core, and a putative signal peptidase recognition site. The promoterlike sequences preceding the signal sequences matched well with those of previously published lactococcal promoters. In addition to E. coli, the functioning of these expression-secretion cassettes was studied in three gram-positive hosts: Bacillus subtilis, L. lactis, and Lactobacillus plantarum. Efficient expression and secretion of TEM beta-lactamase into the culture medium of each gram-positive host was obtained. Furthermore, when a strain of L. lactis subsp. lactis showing increased sensitivity to lysozyme was compared with a standard laboratory strain, threefold-higher secreted enzyme activities were detected.     

欧芹苯丙氨酸脱氨酶cDNA在乳酸乳球菌中的表达研究

将欧芹（Ｐｅｔｒｏｓｅｌｉｎｕｍｃｒｉｓｐｕｍ）苯丙氨酸脱氨酶（ＰＡＬ）ｃＤＮＡ亚克隆到组成型表达载体ｐＭＧ３６ｅ启动子Ｐ３２下游,电穿孔法转化乳酸乳球菌,获得有ＰＡＬ表达活性的乳酸乳球菌工程菌（ｐＭＧ３６ｅＰＡＬ／Ｌ．ｌａｃｔｉｓＭＧ１３６３）。通过递归ＰＣＲ合成了一段１２０ｂｐ的调控片段,用以将ｐＭＧ３６ｅ改造为分泌型表达载体ｐＸＨＳ,以翻译偶联的方式表达ＰＡＬ,可使ＰＡＬ的Ｎ末端带上ｕｓｐ４５信号肽,结果亦检测到ＰＡＬ酶活性。自行分离克隆了乳酸乳球菌热休克蛋白基因ｄｎａＪ的启动子区域,构建了热诱导表达载体ｐＸＨＪ,获得ＰＡＬ热诱导表达工程菌（ｐＸＨＪＰＡＬ／Ｌ．ｌａｃｔｉｓＩＬ１４０３）,经３０℃至３７℃热诱导,可使ＰＡＬ表达活性提高至２倍。本文还就乳酸乳球菌ＰＡＬ工程菌在经典型苯丙酮尿症防治中的应用进行了分析和讨论     

Gene cloning, functional expression and secretion of the S-layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a in Lactococcus lactis

The ~93-kDa surface layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a forms a regular crystalline array providing a nanopatterned matrix for the future display of biologically relevant molecules. Lactococcus lactis NZ9000 was established as a safe expression host for the controlled targeted production of SgsE based on the broad host-range plasmid pNZ124Sph, into which the nisA promoter was introduced. SgsE devoid of its signal peptide-encoding sequence was cloned into the new vector and purified from the cytoplasm at a yield of 220 mg l- of expression culture. Secretion constructs were based on the signal peptide of the Lactobacillus brevis SlpA protein or the L. lactis Usp45 protein, allowing isolation of 95 mg of secreted rSgsE l-1. N-terminal sequencing confirmed correct processing of SgsE in L. lactis NZ9000. The ability of rSgsE to self-assemble in suspension and to recrystallize on solid supports was demonstrated by electron and atomic force microscopy.