Investigation of Mechanically Labile Type III Secretion Protein Effectors
Investigation of Mechanically Labile Type III Secretion Protein Effectors
复制标题
机械不稳定的 III 型分泌蛋白效应器的研究
DOI：
10.1016/j.bpj.2019.11.2802
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发表时间：
2020
影响因子：
3.4
通讯作者：
Sousa, Marcelo C.
中科院分区：
文献类型：
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作者：
DaPron, Katherine E.;Fink, Morgan;LeBlanc, Marc-Andre;Edwards, Devin T.;Perkins, Thomas T.;Sousa, Marcelo C.
革兰氏阴性致病菌已经进化出复杂的机制来分泌蛋白质效应物，这有助于建立复制生态位和感染。 III 型分泌系统 (T3SS) 就是这样的复合体之一，它跨越细菌膜并与宿主质膜结合。像注射器一样，T3SS 可以通过其针头将这些蛋白质效应物直接注射到宿主细胞中。由于针很窄，蛋白质效应子必须在分泌前展开，然后在宿主细胞中重新折叠。这种限制对效应器提出了独特的结构挑战，其中它们需要机械不稳定才能有效展开，但也需要热力学稳定，以便它们在宿主细胞内恢复到折叠状态。 Sousa 实验室之前使用单分子力谱 (SMFS) 进行的研究发现，尽管有有序的折叠，但其中一些效应器在很小的力下就能机械地展开。然而，仍然需要对其非分泌同源物进行进一步的表征和比较。该项目旨在彻底表征蛋白质效应器与其非分泌同源物相比的机械不稳定性，工程师称具有相当机械不稳定性的同源物，并通过活细胞测定可视化其分泌动态。
Gram-negative pathogenic bacteria have evolved complex machinery to secrete protein effectors, which facilitate establishing a replicative niche and infection. One such complex, the Type III Secretion System (T3SS), spans both bacterial membranes and engages host plasma membrane. Like a syringe, the T3SS can thus inject these protein effectors through its needle directly into the host cell. Due to the narrowness of the needle, protein effectors must be unfolded prior to secretion and then refold once in the host cell. This constraint poses a unique structural challenge for effectors, in which they need to be mechanically unstable to unfold efficiently but also thermodynamically stable, so that they return to their folded state inside the host cell. Previous work in the Sousa lab using single molecule force spectroscopy (SMFS) discovered some of these effectors mechanically unfold under very little force despite ordered folds. However, further characterization and comparisons to their non-secreted homologs is still required. This project aims to thoroughly characterize the mechanical instability of protein effectors compared to their non-secreted homologues, engineer said homologs with comparable mechanical lability, and visualize their secretion dynamics with a live cell assay.