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 哺乳动物。秘书蛋白是N-糖基化，O-和C-甘露糖基化和/或糖基磷脂酰肌醇 （GPI）进入内质网（ER）的管腔时被锚定。无法合成n-的酵母 糖基化是不可或缺的，并且具有与胚胎相同的缺陷的小鼠死亡。 在登革热和SARS-COV-2病毒感染中很重要，并且糖基化缺陷引起人类疾病。那， 防御性的O-ansnosylation dystroglycan是肌肉营养不良和GPI缺乏症的原因 造血人干细胞是溶血疾病阵发性夜间血红蛋白尿。 糖基化（CDG）的先天性疾病是患有神经系统症状的严重遗传疾病。 蛋白质糖基化反应需要糖基甘露蛋白甘露糖基和葡萄糖基 - 磷酸二甲醇（MPD，MPD， GPD）充当ER管腔中的糖供体。由于这些脂质是在细胞质侧合成的，因此 它们必须翻转过ER膜才能在管腔中起作用，这是需要特定的过程 尚未确定的转运蛋白，称为Scramblases。微粒体中两个串联酶的测定 并使用天然脂质和短链类似物作为记者进行重组的蔬菜，表明运输是 双向，非ATP独立且高度结构的特异性，区分结构异构体。 我们将使用化学蛋白质和生物信息学方法鉴定MPD和GPD串联酶。 部署由哈纳集团（伯尔尼大学）综合的新颖的照片可关注的问题，我们将确定 我们假设的MPD和GPD相互作用组将包括串联酶。我们的初步结果 验证这种方法：MPD探针在ER甘露糖基化中起作用并识别特定的酵母 微粒体蛋白。光成蛋白将通过定量蛋白质组学鉴定并测试 我们基于重建的测定法中的Scramblase活动。有希望的候选人将在体内证实 评估酵母突变体的表型。对于GPD拼写板，我们还将通过系统发育确定候选者 分析，一种用于分配蛋白质功能的生物信息学方法。这种方法符合照片 - 识别策略，并且已经产生了用于测试的GPD Scramblase候选列表。 这是发现ER蛋白质糖基化的关键参与者的结果建议。我们的广泛 研究串联酶的经验使我们处于解决这一目标的强大状态。我们发现了 A类GPCR的Scramblase活性，并且是第一个显示TMEM16离子通道脂质扰动的人。 现在，我们部署在硅，生化和生物物理方法中，以阐明其机制。我们将使用这个 未来工作的专业知识，以揭示结构特异性脂质的分子机制。 我们预测的MPD和GPD串联酶与当前已知的磷脂的不同 串联酶。在生物学层面，我们的发现将揭示与CDG相关的新遗传位置。