Brogan Anna P, Habib Cameron, Hobbs Samuel J, Kranzusch Philip J, Rudner David Z
Department of Microbiology, Harvard Medical School, Boston, MA 02115.
Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02115.
bioRxiv. 2023 Jun 27:2023.06.27.546760. doi: 10.1101/2023.06.27.546760.
Gram-positive bacteria use SigI/RsgI-family sigma factor/anti-sigma factor pairs to sense and respond to cell wall defects and plant polysaccharides. In this signal transduction pathway involves regulated intramembrane proteolysis (RIP) of the membrane-anchored anti-sigma factor RsgI. However, unlike most RIP signaling pathways, site-1 cleavage of RsgI on the extracytoplasmic side of the membrane is constitutive and the cleavage products remain stably associated, preventing intramembrane proteolysis. The regulated step in this pathway is their dissociation, which is hypothesized to involve mechanical force. Release of the ectodomain enables intramembrane cleavage by the RasP site-2 protease and activation of SigI. The constitutive site-1 protease has not been identified for any RsgI homolog. Here, we report that RsgI's extracytoplasmic domain has structural and functional similarities to eukaryotic SEA domains that undergo autoproteolysis and have been implicated in mechanotransduction. We show that site-1 proteolysis in and Clostridial RsgI family members is mediated by enzyme-independent autoproteolysis of these SEA-like (SEAL) domains. Importantly, the site of proteolysis enables retention of the ectodomain through an undisrupted ß-sheet that spans the two cleavage products. Autoproteolysis can be abrogated by relief of conformational strain in the scissile loop, in a mechanism analogous to eukaryotic SEA domains. Collectively, our data support the model that RsgI-SigI signaling is mediated by mechanotransduction in a manner that has striking parallels with eukaryotic mechanotransducive signaling pathways.
SEA domains are broadly conserved among eukaryotes but absent in bacteria. They are present on diverse membrane-anchored proteins some of which have been implicated in mechanotransducive signaling pathways. Many of these domains have been found to undergo autoproteolysis and remain noncovalently associated following cleavage. Their dissociation requires mechanical force. Here, we identify a family of bacterial SEA-like (SEAL) domains that arose independently from their eukaryotic counterparts but have structural and functional similarities. We show these SEAL domains autocleave and the cleavage products remain stably associated. Importantly, these domains are present on membrane-anchored anti-sigma factors that have been implicated in mechanotransduction pathways analogous to those in eukaryotes. Our findings suggest that bacterial and eukaryotic signaling systems have evolved a similar mechanism to transduce mechanical stimuli across the lipid bilayer.
革兰氏阳性菌利用SigI/RsgI家族的σ因子/抗σ因子对来感知细胞壁缺陷和植物多糖并做出反应。在这个信号转导途径中，涉及膜锚定抗σ因子RsgI的调节性膜内蛋白水解（RIP）。然而，与大多数RIP信号通路不同，RsgI在细胞膜外质侧的1型切割是组成性的，并且切割产物保持稳定结合，从而阻止了膜内蛋白水解。该途径中的调节步骤是它们的解离，据推测这涉及机械力。胞外结构域的释放使得能够被RasP 2型蛋白酶进行膜内切割并激活SigI。尚未鉴定出任何RsgI同源物的组成性1型蛋白酶。在这里，我们报告RsgI的胞外结构域与经历自蛋白水解并参与机械转导的真核生物SEA结构域具有结构和功能上的相似性。我们表明，枯草芽孢杆菌和梭菌RsgI家族成员中的1型蛋白水解是由这些类SEA（SEAL）结构域的不依赖酶的自蛋白水解介导的。重要的是，蛋白水解位点通过跨越两个切割产物的完整β折叠使胞外结构域得以保留。自蛋白水解可以通过消除可裂环中的构象应变来消除，其机制类似于真核生物SEA结构域。总体而言，我们的数据支持这样的模型，即RsgI-SigI信号传导是通过机械转导介导的，其方式与真核生物机械转导信号通路有显著的相似之处。
SEA结构域在真核生物中广泛保守，但在细菌中不存在。它们存在于多种膜锚定蛋白上，其中一些与机械转导信号通路有关。已发现许多这些结构域会进行自蛋白水解，并且在切割后保持非共价结合。它们的解离需要机械力。在这里，我们鉴定出一个细菌类SEA（SEAL）结构域家族，它们独立于其真核生物对应物产生，但具有结构和功能上的相似性。我们表明这些SEAL结构域会自切割，并且切割产物保持稳定结合。重要的是，这些结构域存在于膜锚定抗σ因子上，这些抗σ因子与类似于真核生物中的机械转导途径有关。我们的发现表明，细菌和真核生物信号系统已经进化出一种类似的机制来跨脂质双层转导机械刺激。
