Post-translocational protein folding in Gram-positive bacteria

项目总结 从真核生物到原核生物，适当的蛋白质折叠对细胞功能至关重要。二硫键的形成 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制在真核生物和 革兰氏阴性细菌。相反，一种主要的二硫键形成途径直到最近才在 革兰氏阳性放线菌、白喉棒状杆菌和棒状杆菌 Matruchotii。在这些生物中，一种名为MDBA的膜结合的硫醇-二硫代氧化还原酶催化后- 出口蛋白质的易位折叠。重要的是，MDBA的基因破坏废除了 粘附性菌毛和生物膜的形成，改变细胞形态，并减弱细菌的毒力。尽管如此，如何 放线细菌细胞应对压力，蛋白质错误折叠还不是很清楚。要解决这一根本问题 问题，我们开始分析放线杆菌的蛋白质组，发现大多数多酚类化合物含有2个或更多 有趣的是，白喉隐翅虫中pbp1A或pbp1B的缺失导致了一种细胞形态缺陷， 这反映了MDBA突变的情况。利用遗传方法，我们随后在以下情况下筛选出可行的抑制突变体 在不允许的温度下生长的白喉杆菌MDBA突变细胞。偶然的是，我们发现 另一种是硫醇二硫化物氧化还原酶，我们将其命名为TsdA(TSD，温度敏感型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 作为一种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，可以作为保护系统来拯救错误折叠的蛋白质。因为我们 在这一更新应用中，继续使用奥里斯和白喉轮虫作为实验模型，通过使用 结合遗传学、生物化学和生物膜分析以及结晶学的多学科方法，我们的目标是 为了研究放线杆菌氧化蛋白质折叠和细胞壁生物合成之间的分子偶联， 一种代偿性硫醇氧化还原酶介导蛋白质氧化折叠的机制 机器应激，并阐明蛋白质二硫键异构化的途径。 放线杆菌。 。

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. .