Browsing by Subject "C9orf72 Protein"
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Item A Chemically Induced Colitis Screen Reveals the Necessity for Membrane Traffic in Intestinal Homeostasis(2019-03-21) McAlpine, William Elliott; Winter, Sebastian E.; Beutler, Bruce; Burstein, Ezra; Schmid, Sandra; Scherer, PhilippInflammatory bowel disease is most commonly a complex disorder caused by the interaction of environmental and genetic aberrations. Under normal conditions, a genetic program actively prevents inflammatory bowel disease, preventing invasion of microbes without permitting severe inflammation of the gut. To identify genes that maintain this balance, we performed a sensitized screen of 49,420 third generation (G3) germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires, bearing 104,658 coding/splicing mutations. We induced mild mucosal damage in these mice by orally administering dextran sodium sulfate (DSS) and found mutations that led to diarrhea and weight loss under these conditions. Causative mutations were mapped concurrently with screening using an automated mapping procedure. Among 114 DSS phenotypes identified and mapped, 36 have been validated by CRISPR/Cas9 targeting. Three vesicle trafficking genes, Myo1d, Smcr8, and Tvp23b, were selected for mechanistic evaluation. MYO1D is a class I myosin that binds both actin and lipid. MYO1D localizes to the basolateral membrane of enterocytes and functions in the intestinal epithelium to protect against colitis. SMCR8, along with C9ORF72 and WDR41, is a member of a tripartite complex that functions as a guanine exchange factor. SMCR8 localizes to the lysosome, and its absence results in perturbations to endocytic and phagocytic pathways. Hyperactivation of endosomal Toll-like receptors in Smcr8-/- mice causes spontaneous inflammation, and hyperactivation of multiple pathways contributes to DSS susceptibility. TVP23B is a trans-Golgi protein that binds YIPF6. Both TVP23B and YIPF6 are necessary for the formation of secretory granules in goblet and Paneth cells of the intestinal epithelium. These studies reveal non-redundant molecules required for the return of normal physiologic balance within the intestine after DSS insult.Item Toxic PRn Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Block Nuclear Import and Export(2017-03-02) Shi, Kevin; Nijhawan, Deepak; McKnight, Steven L.; Rizo-Rey, José; Rosen, Michael K.Expansion of the (GGGGCC)n hexanucleotide repeat within the first intron of the C9orf72 gene is the mutation that leads to the most prevalent heritable form of amyotrophic lateral sclerosis (ALS). The expanded repeat is aberrantly transcribed from both sense and anti-sense strands relative to the C9orf72 gene, and both transcripts are translated in an ATG-independent manner to yield five distinct poly-dipeptides. Expression of either the glycine:arginine (GRn) or proline:arginine (PRn) poly-dipeptide in Drosophila leads to neurodegneration of the eye, and when introduced into culturing medium, synthetic forms of both peptides lead to the death of human cells. We recently identified PRn poly-dipeptide interacting partners in vivo using an unbiased proteomics approach, demonstrating that the nuclear pore complex (NPC) as a major binding target. The biological significance of the PRn peptide-NPC interaction became apparent when we observed major defects in both mature mRNA export to the cytoplasm and protein import into the nucleus after cells were treated with PRn. The functional nucleocytoplasmic transport defect caused by PRn peptide was due to binding of the peptide to the central channel of the NPC as visualized by super-resolution microscopy. The NPC central channel is comprised of phenylalanine-glycine (FG) domain nucleoporins, and these proteins are essential for the NPC's active transport and passive size-exclusion permeability barrier functions. Purified FG domains of Nup54 and Nup98, both identified in our proteomics study as PRn targets, polymerized into structurally labile, cross-β sheet fibers under physiological conditions. Several lines of evidence suggested that the polymerized state of FG domains is relevant to intact NPC. First, PRn peptides only bound to polymerized Nup54 and Nup98 and not to the soluble versions of either protein, potentially representing how PRn binds to the FG-rich central channel. Second, the aliphatic alcohol 1,6-hexanediol (HD) selectively disrupted the permeability barrier of the NPC, while 2,5-hexanediol had no effect. Similarly, 1,6-HD can effectively solubilize Nup FG polymers in vitro, while 2,5-HD had no effect on polymer stability. Finally, PRn binding to the NPC abrogated the disruption of the permeability barrier by 1,6-HD. Similarly, PRn also protected the Nup FG polymers from solubilization by 1,6-HD. Our study has elucidated a major mechanism by which the C9orf72 expansion associated PRn poly-dipeptide inhibits the transport of macromolecules in and out of the nucleus, leading to a major disruption of cellular physiology. Our results support a model in which the FG domains of the NPC exist in equilibrium between the polymerized and unpolymerized states. By binding to polymerized FG domains and stabilizing them, the PRn peptide is understood to shift the equilibrium toward the polymerized state, with consequent blockage of nuclear transport. The aliphatic alcohol 1,6-hexanediol has the opposite effect, shifting the equilibrium toward the unstructured state of FG domains and disrupting the permeability barrier. The effects of the PRn peptide and aliphatic alcohols on nuclear pore function represent extreme conditions. More subtle changes in the equilibrium between structured and unstructured FG domains may regulate nuclear pore function in living cells.