Scramblases for protein glycosylation

用于蛋白质糖基化的 Scramblases

• 批准号: 10420706
• 负责人: ANANT K MENON
• 金额: $ 56.34万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10420706
• 关键词: 2019-nCoV; Alkynes; Antibodies; Azides; Benzophenones; Biochemical; Bioinformatics; Biological; Biological Assay; Carrier Proteins; Cell Adhesion Molecules; Cells; Cellular biology; Characteristics; Complement; Complex; Congenital disorders of glycosylation; Consequentialism; Cytoskeleton; Data; Defect; Dengue; Disease; Distant; Dolichol; Dolichol Monophosphate Mannose; Dystroglycan; Embryo; Endoplasmic Reticulum; Eukaryota; Extracellular Matrix; Face; Future; G-Protein-Coupled Receptors; GPI Membrane Anchors; GTP-Binding Protein alpha Subunits, Gs; Genome; Glucose; Glycolipids; Glycoproteins; Glycosylphosphatidylinositols; Goals; Hematopoietic; Hereditary Disease; Ion Channel; Isomerism; Learning; Link; Lipids; Malaria; Mammals; Mannose; Mediating; Membrane; Membrane Proteins; Methods; Microsomes; Molecular; Mus; Muscular Dystrophies; Neurologic Symptoms; Organism; Phenotype; Phylogenetic Analysis; Physiological; Plant Resins; Polysaccharides; Positioning Attribute; Post-Translational Protein Processing; Procedures; Process; Protein Glycosylation; Proteins; Proteomics; Reaction; Reporter; Side; Source; Specificity; Structure; Testing; Trypanosoma; Universities; Vesicle; Virus; Virus Diseases; Work; Yeasts; analog; base; biophysical techniques; crosslink; experience; experimental study; genomic locus; glycosylation; human disease; human stem cells; in silico; in vivo; innovation; interest; mutant; novel; paroxysmal nocturnal hemoglobinuria; phospholipid scramblase; protein function; reconstitution; secretory protein; sugar

Protein glycosylation is essential in all eukaryotes, from disease-causing protists such as malaria, to yeast and mammals. Secretory proteins are N-glycosylated, O- and C-mannosylated, and/or glycosylphosphatidylinositol (GPI)-anchored as they enter the lumen of the endoplasmic reticulum (ER). Yeast that cannot synthesize N- glycoproteins or GPI-proteins are inviable, and mice with the same defects die as embryos. Glycosylation is important in dengue and SARS-CoV-2 viral infections, and defects in glycosylation cause human disease. Thus, deficient O-mannosylation of dystroglycan is a cause of muscular dystrophy and GPI deficiency in hematopoietic human stem cells underlies the hemolytic disease paroxysmal nocturnal hemoglobinuria. Congenital Disorders of Glycosylation (CDGs) are severe inherited diseases with neurological symptoms. Protein glycosylation reactions require the glycolipids mannosyl- and glucosyl-phosphoryl dolichol (MPD, GPD) to act as sugar donors in the lumen of the ER. As these lipids are synthesized on the cytoplasmic side, they must be flipped across the ER membrane to function in the lumen, a process requiring specific transporters, termed scramblases, that have yet to be identified. Assays of the two scramblases in microsomes and reconstituted vesicles, using natural lipids and short-chain analogs as reporters, reveal that transport is bidirectional, ATP-independent, and highly structure specific, discriminating between structural isomers. We will identify the MPD and GPD scramblases using chemo-proteomic and bioinformatic approaches. Deploying novel photo-clickable probes synthesized by the Häner group (University of Bern) we will determine the MPD and GPD interactomes, that we hypothesize will include the scramblases. Our preliminary results validate this approach: the MPD probe functions in ER mannosylation and photo-identifies specific yeast microsomal proteins. Photo-adducted proteins will be identified by quantitative proteomics and tested for scramblase activity in our reconstitution-based assays. Promising candidates will be validated in vivo by evaluating phenotypes of yeast mutants. For GPD scramblase we will also identify candidates via phylogenetic profiling, a bioinformatics method for assignment of protein function. This approach complements the photo- identification strategy and has already yielded a list of GPD scramblase candidates for testing. This is a consequential proposal to discover critical players in ER protein glycosylation. Our extensive experience in studying scramblases puts us in a strong position to tackle this objective. We discovered the scramblase activity of Class A GPCRs and were the first to show lipid scrambling by a TMEM16 ion channel. We now deploy in silico, biochemical and biophysical methods to elucidate their mechanism. We will use this expertise in future work to reveal the molecular mechanism of structure-specific lipid scrambling mediated by the MPD and GPD scramblases that we predict to be distinct from that of the currently known phospholipid scramblases. At a biological level, our discoveries will reveal new genetic loci associated with CDGs.

蛋白质糖基化在所有真核生物中都是必不可少的，从致病的原生生物（如疟疾）到酵母和酵母