ABSTRACT: The soil bacterium Flavobacterium johnsoniae was grown on agar plates with or without pectin,bacteria were harvested, lysed, and subjected to LC-MS/MS analysis
Project description:Adult beta cells in the pancreas are the sole source of insulin in our body. Beta cell loss or increased demand for insulin, impose metabolic challenges because adult beta cells are generally quiescent and infrequently re-enter the cell division cycle. miR-17-92/106b is a family of proto-oncogene microRNAs, that regulate proliferation in normal tissues and in cancer. Here, we employ mouse genetics to demonstrate a critical role for miR-17-92/106b in glucose homeostasis and in controlling insulin secretion. Mass spectrometry analysis was performed on miR-17-92LoxP/LoxP;106-25-/- MEF lysate, without or with CRE-Adenovirus. miR-17-92LoxP/LoxP;106-25+/+ MEFs with GFP-Adenovirus served as controls. We demonstrate that miR-17-92/106b regulate the adult beta cell mitotic checkpoint and that miR-17-92/106b deficiency results in reduction in beta cell mass in-vivo. Furthermore, protein kinase A (PKA) is a new relevant molecular pathway downstream of miR-17-92/106b in control of adult beta cell division and glucose homeostasis. Therefore, contributes to the understanding of proto-oncogene miRNAs in the normal, untransformed endocrine pancreas, and illustrates new genetic means for regulation of beta cell mitosis and function by non-coding RNAs.
Project description:Flavobacterium johnsoniae is a free-living member of the Bacteroidota phylum found in soil and water. It is frequently used as a model species for studying a type of gliding motility dependent on the type IX secretion system (T9SS). O-glycosylation has been reported in several Bacteroidota species and the O-glycosylation of S-layer proteins in Tannerella forsythia was shown to be important for certain virulence features. In this study we characterised the O-glycoproteome of F. johnsoniae and identified 325 O-glycosylation sites within 226 glycoproteins. The structure of the major glycan was found to be a hexasaccharide with the sequence Hex–(Me-dHex)–Me-HexA–Pent–HexA–Me-HexNAcA. Bioinformatic localisation of the glycoproteins determined 68 inner membrane proteins, 60 periplasmic proteins, 26 outer membrane proteins, 57 lipoproteins and 9 proteins secreted by the T9SS. The glycosylated sites were predominantly located in the periplasm where they are postulated to be beneficial for protein folding/stability. Six proteins associated with gliding motility or the T9SS were demonstrated to be O-glycosylated.
Project description:Secretion Systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we use deletion of the T9SS motor protein to trap the T9SS Flavobacterium johnsoniae in the process of substrate transport. CryoEM analysis of purified substrate-bound T9SS translocons revealed an extended translocon compared to previous analyses. The translocon core is augmented by a five-subunit periplasmic structure incorporating the proteins SprE, PorD, and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate-carrier protein complex from the translocon is the energy-requiring step in T9SS transport.