A Module for Analyzing Interactomes via APEX-MS Integrated into PatternLab for Proteomics.

Proximity labeling techniques, such as APEX-MS, provide valuable insights into proximal interactome mapping; however, the verification of biotinylated peptides is not straightforward. With this as motivation, we present a new module integrated into PatternLab for proteomics to enable APEX-MS data interpretation by targeting diagnostic fragment ions associated with APEX modifications. We reanalyzed a previously published APEX-MS data set and report a significant number of biotinylated peptides and, consequently, a confident set of proximal proteins. As the module is part of the widely adopted PatternLab for proteomics software suite, it offers users a comprehensive, easy, and integrated solution for data analysis. Given the broad utility of the APEX-MS technique in various biological contexts, we anticipate that our module will be a valuable asset to researchers, facilitating and enhancing interactome studies. PatternLab's APEX, including a usage protocol, is available at http://patternlabforproteomics.org/apex.

Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic Actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. These bacteria have multi-layered cell walls composed of the mycolyl-arabinogalactan-peptidoglycan complex and a polar growth mode, thus requiring tight coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the coiled-coil protein Wag31. Here, using C. glutamicum, we report the discovery of two divisome members: a gephyrin-like repurposed molybdotransferase (Glp) and its membrane receptor (GlpR). Our results show how cell cycle progression requires interplay between Glp/GlpR, FtsZ and Wag31, showcasing a crucial crosstalk between the divisome and elongasome machineries that might be targeted for anti-mycobacterial drug discovery. Further, our work reveals that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis, similar to the gephyrin/GlyR system that mediates synaptic signalling in higher eukaryotes through network organization of membrane receptors and the microtubule cytoskeleton.

Cys mutants as tools to study the oligomerization of the pore-forming toxin sticholysin I.

Sticholysin I (StI) is a water-soluble protein with the ability to bind membranes where it oligomerizes and forms pores leading to cell death. Understanding the assembly property of this protein may be valuable for designing potential biotechnological tools, such as stable or structurally defined nanopores. In order to get insights into the stabilization of StI oligomers by disulfide bonds, we designed and characterized single and double cysteine mutants at the oligomerization interface. The oligomer formation was induced in the presence of lipid membranes and visualized by SDS-PAGE. The contribution of the oligomeric structures to the membrane binding and pore-forming capacities of StI was assessed. Single and double cysteine introduction at the protein-protein oligomerization interface does not considerably affect the conformation and function of the monomeric protein. In the presence of membranes, a cysteine double mutation at positions 15 and 59 favored formation of different size oligomers stabilized by disulfide bonds. The results of this work highlight the relevance of these positions (15 and 59) to be considered for developing biosensors based on nanopores from StI.