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首页> 外文学位 >Studies on Escherichia coli membrane protein biogenesis: Mechanism of signal peptide peptidase A and the influence of YidC depletion on cellular processes.
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Studies on Escherichia coli membrane protein biogenesis: Mechanism of signal peptide peptidase A and the influence of YidC depletion on cellular processes.

机译：大肠杆菌膜蛋白生物发生的研究：信号肽肽酶A的机制以及YidC耗竭对细胞过程的影响。

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摘要

Escherichia coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein but does not label the S409A mutant with an alanine substituted for the essential serine. These results are consistent with Ser 409 being directly involved in the proteolytic mechanism. Remarkably, additional site-directed mutagenesis studies showed that none of the lysines or histidine residues in the carboxyl-terminal protease domain (residues 326-549) is critical for activity, suggesting this domain lacks the general base residue required for proteolysis. In contrast, we found that E. coli SppA has a conserved lysine (K209) in the N-terminal domain (residues 56-316) that is essential for activity and important for activation of S409 for reactivity toward the FP-biotin inhibitor and is conserved in those other bacterial SppA proteins that have an N-terminal domain. We also performed alkaline phosphatase fusion experiments that establish that SppA has only one transmembrane segment (residues 29-45) with the C-terminal domain (residues 46-618) protruding into the periplasmic space. These results support the idea that E. coli SppA is a Ser-Lys dyad protease, with the Lys recruited to the amino-terminal domain that is itself not present in most known SppA sequences.;YidC depletion affects membrane protein insertion and leads to a defect in the growth of the E. coli cell. We analyzed global changes in gene expression upon YidC depletion to determine the importance of YidC for cellular functions. We used a gene-chip method to compare the transcriptome of JS71 (control) and JS7131 (yidC depletion strain). Of the more than 4300 identified genes, 163 were up-regulated and 99 genes down-regulated upon YidC depletion, including genes which are responsible for DNA/RNA repair, energy metabolism, various transporters, proteases and chaperones, stress response and translation and transcription functions. Real-time PCR was performed on selected genes to confirm the results. Specifically, we found up-regulation of the genes encoding the energy transduction proteins F1Fo ATP synthase and cytochrome bo3 oxidase due to perturbation in the assembly when YidC was depleted. We also determined that the high level induction of the PspA stress protein under YidC depletion conditions is roughly 10-fold higher than the activation due to the addition of protonophore CCCP which dissipates the proton motive force. In addition, the gene chip data reveals the Cpx stress pathway is activated upon YidC depletion. The data provides better understanding toward YidC's function for a number of celluar processes.
机译：大肠杆菌信号肽肽酶A（SppA）是一种丝氨酸蛋白酶，通过信号肽酶处理从输出蛋白中进行蛋白水解去除后，会裂解信号肽。我们在这里介绍了羧基末端域中所有SppA保守丝氨酸的定点诱变研究结果，显示只有Ser 409对酶活性至关重要。另外，我们显示丝氨酸水解酶抑制剂FP-生物素抑制SppA并修饰蛋白质，但未用取代必需丝氨酸的丙氨酸标记S409A突变体。这些结果与Ser 409直接参与蛋白水解机制一致。值得注意的是，另外的定点诱变研究表明，羧基末端蛋白酶结构域（残基326-549）中的赖氨酸或组氨酸残基都不对活性至关重要，这表明该结构域缺乏蛋白水解所需的一般碱基残基。相反，我们发现大肠杆菌SppA在N端结构域（残基56-316）具有保守的赖氨酸（K209），这对于活性至关重要，对于激活S409对FP-生物素抑制剂的反应性也很重要，并且在具有N末端结构域的其他细菌SppA蛋白中保守。我们还进行了碱性磷酸酶融合实验，该实验确定SppA仅具有一个跨膜片段（29-45位残基），其C端结构域（46-618位残基）伸入周质空间。这些结果支持了大肠杆菌SppA是一种Ser-Lys dyad蛋白酶，而Lys被募集到其自身在大多数已知SppA序列中不存在的氨基末端结构域的想法.YidC耗竭影响膜蛋白的插入并导致大肠杆菌细胞生长缺陷。我们分析了YidC耗尽后基因表达的全球变化，以确定YidC对细胞功能的重要性。我们使用基因芯片方法比较了JS71（对照）和JS7131（yidC耗竭菌株）的转录组。在超过4300个已鉴定的基因中，YidC耗尽后上调了163个基因，下调了99个基因，包括负责DNA / RNA修复，能量代谢，各种转运蛋白，蛋白酶和分子伴侣，应激反应以及翻译和转录的基因功能。对选定的基因进行了实时PCR，以确认结果。具体来说，我们发现由于YidC耗尽时装配中的扰动，编码能量转导蛋白F1Fo ATP合酶和细胞色素bo3氧化酶的基因上调。我们还确定，在YidC耗尽条件下，PspA应激蛋白的高水平诱导约比激活高10倍，这是由于添加了质子载体CCCP（其消散了质子原动力）引起的。此外，基因芯片数据显示，Cip应激途径在YidC耗尽时被激活。数据可以更好地理解许多蜂窝工艺的YidC功能。

著录项

  • 作者

    Wang, Peng.;

  • 作者单位

    The Ohio State University.;

  • 授予单位 The Ohio State University.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 140 p.
  • 总页数 140
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:20

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