菱形蛋白酶是普遍保守的并且促进细胞膜内或邻近细胞膜的肽键的蛋白水解。虽然真核菱形蛋白酶已被证明具有独特的细胞作用,原核成员的特征要少得多。第一次,我们证明,霍乱弧菌表达两种活性菱形蛋白酶,在不同的位点切割共享的底物,导致加工蛋白质的差异定位。先前发现菱形蛋白酶菱形分选酶(RssP)可加工称为GlyGly-CTERM的新型C末端结构域,如通过其通过霍乱弧菌细胞包膜转运过程中对细胞外丝氨酸蛋白酶VesB的作用所证明的。这里,我们表征了RssP和GlpG的底物特异性,普遍保守的细菌菱形蛋白酶。我们表明RssP与GlpG具有不同的切割特异性,VesB的GlyGly-CTERM内的特定残基将其靶向GlpG上的RssP,允许有效的蛋白水解。RssP在其跨膜结构域内切割VesB,而GlpG在GlyGly-CTERM之前的无序环中切割膜外部。RssP对VesB的切割最初将VesB靶向细菌细胞表面,随后,外膜囊泡,而GlpG裂解导致分泌,完全溶解的VesB。总的来说,这项工作建立在对菱形蛋白水解的分子理解的基础上,并为额外的菱形底物识别提供了基础,同时也证明了RssP在含有GlyGly-CTERM的蛋白质成熟中的独特作用。
目的:尽管对真核细胞同源物的深入了解,细菌菱形蛋白酶的研究相对较少。我们的研究旨在了解两种菱形蛋白酶在霍乱弧菌中的功能。这项工作具有重要意义,因为它将帮助我们更好地了解菱形蛋白酶的整体催化机理,并为一个独特的亚家族赋予特定的作用,该亚家族的功能是处理霍乱弧菌和其他病原菌分泌的效应分子的子集。
Rhomboid proteases are universally conserved and facilitate the proteolysis of peptide bonds within or adjacent to cell membranes. While eukaryotic rhomboid proteases have been demonstrated to harbor unique cellular roles, prokaryotic members have been far less characterized. For the first time, we demonstrate that Vibrio cholerae expresses two active rhomboid proteases that cleave a shared substrate at distinct sites, resulting in differential localization of the processed protein. The rhomboid protease rhombosortase (RssP) was previously found to process a novel C-terminal domain called GlyGly-CTERM, as demonstrated by its effect on the extracellular serine protease VesB during its transport through the V. cholerae cell envelope. Here, we characterize the substrate specificity of RssP and GlpG, the universally conserved bacterial rhomboid proteases. We show that RssP has distinct cleavage specificity from GlpG, and specific residues within the GlyGly-CTERM of VesB target it to RssP over GlpG, allowing for efficient proteolysis. RssP cleaves VesB within its transmembrane domain, whereas GlpG cleaves outside the membrane in a disordered loop that precedes the GlyGly-CTERM. Cleavage of VesB by RssP initially targets VesB to the bacterial cell surface and, subsequently, to outer membrane vesicles, while GlpG cleavage results in secreted, fully soluble VesB. Collectively, this work builds on the molecular understanding of rhomboid proteolysis and provides the basis for additional rhomboid substrate recognition while also demonstrating a unique role of RssP in the maturation of proteins containing a GlyGly-CTERM.
OBJECTIVE: Despite a great deal of insight into the eukaryotic homologs, bacterial rhomboid proteases have been relatively understudied. Our research aims to understand the function of two rhomboid proteases in Vibrio cholerae. This work is significant because it will help us better understand the catalytic mechanism of rhomboid proteases as a whole and assign a specific role to a unique subfamily whose function is to process a subset of effector molecules secreted by V. cholerae and other pathogenic bacteria.