Scramblases for protein glycosylation

用于蛋白质糖基化的 Scramblases

• 批准号: 10600063
• 负责人: ANANT K MENON
• 金额: $ 51.2万
• 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/10600063
• 关键词: 2019-nCoV; Alkynes; Antibodies; Azides; Benzophenones; Biochemical; Bioinformatics; Biological; Biological Assay; Carrier Proteins; Cell-Adhesion Molecule Receptors; Cells; Cellular biology; Characteristics; Complex; Congenital disorders of glycosylation; Cytoplasm; 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; Genetic Complementation Test; 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; Proliferating; Protein Glycosylation; Proteins; Proteomics; Reaction; Reporter; Side; Source; Specificity; Structure; Testing; Trypanosoma; Universities; Vesicle; Virus; Virus Diseases; Work; Yeasts; adduct; analog; biophysical techniques; candidate identification; chemoproteomics; 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，从引起疾病的原生生物（例如疟疾）到酵母和 哺乳动物。分泌蛋白是 N-糖基化、O-和 C-甘露糖基化和/或糖基磷脂酰肌醇 (GPI)-锚定当它们进入内质网(ER)的管腔时。不能合成N-的酵母 糖蛋白或 GPI 蛋白是无法存活的，具有相同缺陷的小鼠会像胚胎一样死亡。糖基化是 在登革热和 SARS-CoV-2 病毒感染中很重要，糖基化缺陷会导致人类疾病。因此， 肌营养不良症 (dystroglycan) 的 O-甘露糖基化缺陷是肌营养不良和 GPI 缺乏的原因 人类造血干细胞是溶血性疾病阵发性睡眠性血红蛋白尿的基础。 先天性糖基化障碍（CDG）是一种伴有神经系统症状的严重遗传性疾病。 蛋白质糖基化反应需要糖脂甘露糖基和葡萄糖基磷酰多乙醇 (MPD, GPD）作为 ER 腔内的糖供体。由于这些脂质是在细胞质侧合成的， 它们必须翻转穿过内质网膜才能在管腔中发挥作用，这一过程需要特定的 运输者，称为扰乱，尚未确定。微粒体中两种混杂酶的测定 和重构的囊泡，使用天然脂质和短链类似物作为报告基因，揭示了运输是 双向、不依赖 ATP、高度结构特异性、可区分结构异构体。 我们将使用化学蛋白质组学和生物信息学方法来鉴定 MPD 和 GPD 扰乱酶。 部署由 Häner 小组（伯尔尼大学）合成的新型可照片点击探针，我们将确定 MPD 和 GPD 相互作用组，我们假设将包括乱序。我们的初步结果 验证此方法：MPD 探针在 ER 甘露糖基化中发挥作用，并通过照片识别特定酵母 微粒体蛋白。光加合蛋白将通过定量蛋白质组学进行鉴定并进行测试 我们基于重构的测定中的扰乱酶活性。有前途的候选者将通过体内验证 评估酵母突变体的表型。对于 GPD 扰乱酶，我们还将通过系统发育来确定候选者 分析，一种用于分配蛋白质功能的生物信息学方法。这种方法补充了照片- 识别策略，并且已经产生了用于测试的 GPD 扰乱酶候选者列表。 这是发现 ER 蛋白糖基化中关键参与者的一项相应建议。我们广泛的 研究扰乱的经验使我们有能力实现这一目标。我们发现了 A 类 GPCR 的置乱酶活性，并且是第一个通过 TMEM16 离子通道显示脂质置乱的。 我们现在采用计算机、生物化学和生物物理方法来阐明其机制。我们将使用这个 未来工作中的专业知识揭示了结构特异性脂质扰乱的分子机制 我们预测 MPD 和 GPD 扰乱酶与目前已知的磷脂的扰乱酶不同 混乱。在生物学水平上，我们的发现将揭示与 CDG 相关的新遗传位点。