Bollinger, K. | bioRxiv Science

bioRxiv2022

Defining the Ligand-dependent Interactome of the Sigma 1 Receptor

Sigma 1 Receptor (S1R) is a therapeutic target for a wide spectrum of pathological conditions ranging from neurodegenerative diseases to cancer and COVID-19. S1R is ubiquitously expressed throughout the visceral organs, nervous, immune and cardiovascular systems. It is proposed to function as a ligand-dependent molecular chaperone that modulates multiple intracellular signaling pathways. The purpose of this study was to define the S1R interactome under native conditions and upon binding to well-characterized ligands. This was accomplished by fusing the biotin ligase, Apex2, to the C terminus of S1R. Cells stably expressing S1R-Apex or a GFP-Apex control were used to map specific protein interactions. Biotinylated proteins were labeled under native conditions and in a ligand dependent manner, then purified and identified using quantitative mass spectrometry. Under native conditions, S1R biotinylates over 200 novel proteins, many of which localize within the endomembrane system (ER, Golgi, secretory vesicles) and function within the secretory pathway. Under conditions of cellular exposure to either S1R agonist or antagonist, results show enrichment of proteins integral to secretion, extracellular matrix formation, and cholesterol biosynthesis. Notably, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) displays increased binding to S1R under conditions of treatment with Haloperidol, a well-known S1R antagonist; whereas Low density lipoprotein receptor (LDLR) binds more efficiently to S1R upon treatment with (+)-Pentazocine ((+)-PTZ), a classical S1R agonist. Our results are consistent with the postulated role of S1R as an intracellular chaperone and further suggest important and novel functionalities related to cholesterol metabolism and biosynthesis.

cell biology↗

bioRxiv2022

The outer surface protease, SepM, is required for blp locus activation in three of the four most common pherotypes of Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) is an important human pathogen that primarily resides in the nasopharynx. To persist in this polymicrobial environment, pneumococcus must compete with other members of the bacterial community. Competition is mediated in part by the action of the blp locus which encodes a variable array of bacteriocins and their associated immunity proteins. The locus is controlled by a two-component regulatory system that senses the extracellular concentration of the peptide pheromone, BlpC. There are four major pherotypes of BlpC that can be found in most pneumococcal genomes. Here, we show that the protease, SepM, is required for activation of three of the four major pherotypes. The only SepM independent BlpC type is 9AA shorter than the SepM-dependent peptides, consistent with a cleavage event at the C-terminal end. The processing event occurs following secretion and removal of the C terminal region is required for binding to the histidine kinase receptor. Synthetic truncated peptides or full-length peptides pre-incubated with SepM-expressing bacteria can upregulate the blp locus independent of SepM. We show that SepM-independent peptides accumulate in the supernatant of secreting cells at low levels suggesting a role for the tail in peptide secretion, stability or solubility and demonstrating a significant tradeoff for SepM-independence. ImportanceStreptococcus pneumoniae is an important cause of disease in humans that occurs when the bacteria in the nasopharynx bypasses host defenses to invade deeper tissues. Colonization fitness thus represents an important initial step in pathogenesis. S. pneumoniae produces antimicrobial peptides called bacteriocins which provide a competitive advantage over neighboring bacteria in the nasopharynx. The blp locus encodes a variable array of bacteriocins that participate in competition. Here, we demonstrate that activation of the blp locus requires a surface protease that activates the blp signal peptide. There are naturally occurring signal peptides that do not require cleavage, but these are characterized by poor secretion. We describe an additional, previously unappreciated activation step in the control of bacteriocin production in S. pneumoniae.

microbiology↗