Post-translocational protein folding in Gram-positive bacteria

项目概要 从真核生物到原核生物，正确的蛋白质折叠对于细胞功能至关重要。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。 促进正确蛋白质折叠的二硫键形成机器在真核生物中得到了广泛认可 革兰氏阴性细菌。相比之下，主要的二硫键形成途径最近才被发现 革兰氏阳性放线菌放线菌、白喉棒状杆菌和棒状杆菌 马特鲁霍蒂。在这些生物体中，一种名为 MdbA 的膜结合硫醇二硫化物氧化还原酶催化后 输出蛋白质的易位折叠。重要的是，mdbA 的遗传破坏会消除 粘附菌毛和生物膜的形成，改变细胞形态，并减弱细菌毒力。尽管如此，如何 放线菌细胞应对压力和蛋白质错误折叠的机制尚不清楚。为了解决这个根本问题 问题之后，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 半胱氨酸；有趣的是，白喉杆菌中 pbp1A 或 pbp1B 的缺失导致了细胞形态缺陷， 反映了 mdbA 突变。通过遗传方法，我们随后筛选了可行的抑制突变体 白喉棒状杆菌 mdbA 突变细胞在不允许的温度下生长。无意中我们发现 另一种硫醇二硫化物氧化还原酶，我们将其命名为 TsdA（tsd 表示温度敏感的 DSB 形成）。 初步研究表明，TsdA 含有 MdbA 中发现的硫氧还蛋白样折叠，这表明 TsdA 可能 作为特殊的二硫键形成机器来应对细胞压力。最后，我们确定了一个潜在的 蛋白质二硫键异构酶可作为拯救错误折叠蛋白质的保护系统。正如我们 在此更新申请中，通过使用 结合遗传学、生化和生物膜测定以及晶体学的多学科方法，我们的目标 检查放线菌中氧化蛋白折叠和细胞壁生物合成之间的分子耦合， 阐明补偿性硫醇氧化还原酶介导的氧化蛋白质折叠机制 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. .