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，从致病的原生生物，如疟疾，到酵母和 哺乳动物。分泌蛋白是N-糖基化、O-和C-甘露糖化和/或糖基化磷脂酰肌醇 (GPI)-当它们进入内质网(ER)的管腔时被锚定。不能合成N-的酵母 糖蛋白或GPI蛋白是不能存活的，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， 肌营养不良症患者的肌营养不良和GPI缺陷的原因之一 造血人干细胞是溶血性疾病阵发性睡眠性血红蛋白尿的基础。 先天性糖基化异常(CDGs)是一种具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醇和葡糖基磷二醇(MPD， GPD)作为内质网管腔内的糖供体。由于这些脂质是在细胞质一侧合成的， 它们必须翻转到内质网膜上才能在管腔中发挥作用，这一过程需要特定的 被称为加扰物的转运体，目前尚未确定。微生物体中两种扰乱酶的检测 而重组囊泡，使用天然脂质和短链类似物作为报告，揭示了运输是 双向，不依赖于三磷酸腺苷，高度结构特异性，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)合成的新型可光点击探针，我们将确定 MPD和GPD的相互作用，我们假设将包括扰码。我们的初步结果 验证这种方法：MPD探针在ER甘露糖化和光识别特定酵母中起作用 微体蛋白。光加成蛋白将通过定量蛋白质组学进行鉴定和测试 在我们基于重组的检测中，扰乱酶的活性。有希望的候选人将通过体内验证 酵母菌突变体的表型鉴定。对于GPD加扰酶，我们还将通过系统发育来确定候选基因 蛋白质功能定位的生物信息学方法。这种方法是对照片的补充- 识别战略，并已经产生了一份GPD加扰酶候选名单供测试。 这是一个相应的建议，以发现ER蛋白糖基化的关键角色。我们广泛的 研究加扰的经验使我们在解决这一目标方面处于有利地位。我们发现了 A类GPCRs的扰乱酶活性，并首次显示TMEM16离子通道对脂质的扰乱。 我们现在利用硅胶、生化和生物物理方法来阐明它们的作用机制。我们将使用这个 在未来的工作中的专业知识，以揭示结构特异性的脂质扰乱的分子机制 我们预测的MPD和GPD扰乱酶与目前已知的磷脂不同 杂乱无章。在生物学层面上，我们的发现将揭示与CDGs相关的新的遗传位点。