Differential regulation of T cell plasma membrane proteins by N-glycan branching and clathrin-mediated endocytosis

N-聚糖分支和网格蛋白介导的内吞作用对 T 细胞质膜蛋白的差异调节

• 批准号: 10646138
• 负责人: Haik Mkhikian
• 金额: $ 46.71万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646138
• 关键词: Adaptor Signaling Protein; Affect; Anti-Inflammatory Agents; Asparagine; CD4 Positive T Lymphocytes; CD8B1 gene; CTLA4 gene; Carbohydrates; Cell Surface Proteins; Cell Surface Receptors; Cell membrane; Cell physiology; Cell surface; Cellular biology; Clathrin; Complex; Coupled; Data; Diagnostic; Endocytosis; Endocytosis Pathway; Endoplasmic Reticulum; Enzymes; Epitopes; Face; Family; Galactose Binding Lectin; Glycoproteins; Goals; Immunity; Individual; Inflammation; Inflammatory; Knowledge; Lectin; Letters; Mannose; Mass Spectrum Analysis; Measures; Mediating; Membrane; Membrane Proteins; Mentors; Methodology; Methods; Modeling; Molecular; Monosaccharides; Mutagenesis; N-acetyllactosamine; Outcome; Pathway interactions; Physiological; Polysaccharides; Protein Secretion; Proteins; Proteome; Proteomics; Receptor Signaling; Recycling; Regulation; Rest; Role; Structure; Surface; T cell regulation; T-Cell Activation; T-Cell Development; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; carbohydrate binding protein; carbohydrate receptor; crosslink; extracellular; glycosylation; immunoregulation; insight; knowledgebase; novel strategies; protein degradation; protein expression; receptor; receptor expression; response; therapeutic target; therapeutically effective; virtual

PROJECT SUMMARY/ABSTRACT Nearly every aspect of T cell biology is determined in part by the relative expression of a multitude of cell-surface proteins. For example, the relative expression of co-stimulatory and co-inhibitory receptors impact pro- versus anti-inflammatory outcomes. N-glycosylation is a critical but poorly understood regulator of cell- surface protein turnover. Virtually every cell-surface and secreted protein is modified with covalently attached complex carbohydrates at asparagine (N) residues. These N-glycans are variably edited by a large number of glycosylation enzymes along the secretory pathway, producing many possible glycan structures. The N-glycan branching pathway serves as a key gateway between relatively simple high-mannose structures and more complex N-glycans that carry glyco-epitopes recognized by numerous families of carbohydrate-binding proteins, known as lectins. The branching pathway controls N-acetyllactosamine (LacNAc) incorporation into N-glycans, which are recognized by the galectin family of soluble lectins. At the cell surface, the interaction of multivalent soluble galectins with glycoproteins carrying LacNAc units, leads to the formation of a molecular lattice-like structure. This galectin-glycoprotein lattice affects cell surface organization, receptor mobility in the plane of the membrane, and endocytosis rates. In part by regulating cell surface expression of CD4, CD8, IL2Rα, CTLA-4, and the T cell receptor (TCR), N-glycan branching regulates T cell development, TCR signaling, T cell activation, T cell proliferation and pro-inflammatory versus anti-inflammatory differentiation. Based on studies of these receptors, a model has emerged of the lattice as a unidirectional regulator of cell- surface retention which opposes glycoprotein loss and promotes cell surface retention. However counter- examples to this model have recently emerged demonstrating that some receptors are regulated in the opposite manner. Thus a comprehensive and unbiased analysis of branching regulated changes is needed. Furthermore, an understanding of the mechanisms involved that promote expression of some receptors while hindering expression of others is also lacking. Without such detailed information, therapeutic targeting of the complex glycosylation pathways that regulate T cell function will be limited. We have developed an approach to examine branching mediated effects on cell-surface expression at the proteome scale. We provide proof-of- concept that this approach is reliable and informative and propose to use it to tackle this outstanding aspect of T cell biology. In Aim1, we will extend this approach to more physiologically relevant primary T cells and examine a range of N-glycan branching states to derive a complete and informative picture. We will also adapt our approach to globally determine endocytosis and recycling rates of branching regulated proteins. In Aim 2, we will test the hypothesis that cargo adapter proteins in the clathrin-mediated endocytosis pathway cooperate with N-glycan branching to differentially regulate receptor turnover and expression. Together these studies will dramatically increase our knowledge of branching mediated regulation of T cell biology.

项目摘要/摘要 T细胞生物学的几乎每个方面都部分取决于众多的相对表达 细胞表面蛋白。例如，共刺激和共抑制受体的相对表达影响 促和抗炎结果。 N-糖基化是一个关键但知之甚少的细胞调节剂 表面蛋白更新。实际上，每个细胞表面和分泌的蛋白质都通过共价附着修饰 在天冬酰胺（n）上保留的复杂碳氢化物。这些N-聚糖被大量编辑 沿秘密途径的糖基化酶，产生许多可能的聚糖结构。 N-聚糖 分支途径是相对简单的高甘露糖结构之间的关键网关 复杂的N-聚糖，携带众多碳水化合物家族识别的糖 - 发plipopes 蛋白质，称为讲座。分支途径控制着纳入N-环甲胺（LACNAC） N-聚糖，由固体讲座的半乳糖素家族认可。在细胞表面，相互作用 多价固体甘染素含有糖蛋白带有LACNAC单位，导致形成分子 晶格状结构。这种乳肠蛋白 - 糖蛋白晶格影响细胞表面组织，受体迁移率 膜的平面和内吞作用率。部分由CD4的调节细胞表面表达表达 IL2Rα，CTLA-4和T细胞受体（TCR），N-聚糖分支调节T细胞的发育，TCR 信号传导，T细胞活化，T细胞增殖和促炎与抗炎分化。 基于对这些受体的研究，该模型已成为晶格作为细胞的单向调节剂 表面保留率反对糖蛋白损失并促进细胞表面保留率。但是反对 该模型的示例最近出现了，证明了某些受体受到调节 相反的方式。需要对分支监管变化进行全面且公正的分析。 此外，对促进某些受体表达的机制的理解 缺乏阻碍他人的表达。没有这样详细的信息，可以治疗定位 调节T细胞功能的复杂糖基化途径将受到限制。我们已经开发了一种方法 检查蛋白质组量表对细胞表面表达的介导的影响。我们提供证明 这种方法是可靠且内容丰富的概念，并且建议使用它来解决这个杰出的方面 T细胞生物学。在AIM1中，我们将将这种方法扩展到更相关的原代T细胞，并将其扩展 检查一系列的N-聚会分支状态，以获取完整而有益的图片。我们也会适应 我们的全球方法确定了分支调节蛋白的内吞作用和回收率。在AIM 2中， 我们将检验以下假设，即网状蛋白介导的内吞作用途径中的货物适配器蛋白坐标 N-Glycan分支向差异调节受体周转和表达。这些研究将共同 动态地增加我们对分支介导的T细胞生物学调节的了解。