Post-translocational protein folding in Gram-positive bacteria

项目摘要 从真核生物到原核生物，正确的蛋白质折叠对细胞功能至关重要。二硫键形成 有助于整个蛋白质折叠过程，稳定结构并防止降解。 促进蛋白质正确折叠的二硫键形成机器在真核生物中得到了很好的认识， 革兰氏阴性菌。相比之下，一个主要的二硫键形成途径最近才被确定， 革兰氏阳性放线菌口腔放线菌、白喉棒状杆菌和棒状杆菌 matruchotii。在这些生物体中，一种名为MdbA的膜结合巯基-二硫键氧化还原酶催化 输出蛋白质的易位折叠。重要的是，mdbA的遗传破坏消除了 粘附性皮利和生物膜形成，改变细胞形态，并减弱细菌毒力。尽管如此， 放线菌细胞科普压力和蛋白质错误折叠的能力还不清楚。为了解决这一基本问题， 为了解决这个问题，我们开始分析放线菌的蛋白质组，发现大多数PBP都含有2个或更多个 有趣的是，C.白喉导致细胞形态缺陷， 与mdbA突变相似。通过遗传学方法，我们筛选了可行的抑制突变体， C.白喉mdbA突变体细胞在非允许温度下生长。我们偶然发现 另一种硫醇-二硫键氧化还原酶，我们将其命名为TsdA（tsd，用于温度敏感的dsb-形成）。 初步研究表明，TsdA含有在MdbA中发现的硫氧还蛋白样折叠，这表明TsdA可能 作为一个专门的二硫键形成机器，以应对细胞压力。最后，我们发现了一个潜在的 蛋白质二硫键异构酶，可以作为一个保护系统，以拯救错误折叠的蛋白质。正如我们 继续使用A。oris和C.白喉作为实验模型，在这个更新的应用，通过使用 结合遗传学、生物化学和生物膜测定以及晶体学的多学科方法，我们的目标是 为了检测放线菌中氧化蛋白折叠和细胞壁生物合成之间的分子偶联， 阐明由补偿性巯基氧化还原酶介导的氧化蛋白质折叠的机制 机器响应压力，并阐明蛋白质二硫键异构化的途径， 放线菌。 .

PROJECT SUMMARY From eukaryotes to prokaryotes, proper protein folding is essential to cellular function. Disulfide bond formation contributes to the overall protein folding process, stabilizing structures and protecting against degradation. Disulfide bond-forming machines that facilitate proper protein folding are well recognized in eukaryotes and Gram-negative bacteria. In contrast, a major disulfide bond-forming pathway has only recently been identified in the Gram-positive Actinobacteria Actinomyces oris, Corynebacterium diphtheriae, and Corynebacterium matruchotii. In these organisms, a membrane-bound thiol-disulfide oxidoreductase named MdbA catalyzes post- translocational folding of exported proteins. Importantly, genetic disruption of mdbA abrogates assembly of adhesive pili and biofilm formation, alters cell morphology, and attenuates bacterial virulence. Nonetheless, how actinobacterial cells cope with stress and protein misfolding is not well understood. To address this fundamental question, we began to analyze the proteomes of Actinobacteria and found that most PBPs harbor 2 or more cysteines; intriguingly, deletion of pbp1A or pbp1B in C. diphtheriae resulted in a cell morphology defect that mirrors that of mdbA mutations. With a genetic approach, we then screened for viable suppressor mutants when C. diphtheriae mdbA mutant cells grown at non-permissive temperatures. Serendipitously, we discovered another thiol-disulfide oxidoreductase, which we named TsdA (tsd for temperature-sensitive dsb-forming). Preliminary studies reveal that TsdA contains a thioredoxin-like fold found in MdbA, suggesting that TsdA may serve as a specialized disulfide bond-forming machine to encounter cell stress. Finally, we identified a potential protein disulfide bond isomerase that may serve as a safeguarding system to rescue misfolded proteins. As we continue employing A. oris and C. diphtheriae as experimental models in this renewal application, by using a multidisciplinary approach that combines genetics, biochemical and biofilm assays, and crystallography, we aim to examine the molecular coupling between oxidative protein folding and cell wall biosynthesis in Actinobacteria, to elucidate the mechanism of oxidative protein folding mediated by a compensatory thiol-oxidoreductase machine in response to stress, and to elucidate a pathway for protein disulfide bond isomerization in Actinobacteria. .