Proteasome regulation by O-glycosylation

通过 O-糖基化调节蛋白酶体

• 批准号: 7499426
• 负责人: Jeffrey E Kudlow
• 金额: $ 11.48万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7499426
• 关键词: 26S proteasome; ATP phosphohydrolase; Acetylglucosamine; Amino Acids; Apoptosis; Apoptotic; Biochemical; Cells; Enzymes; Epithelial Cells; Glucosamine; Glucose; Goals; In Vitro; Induction of Apoptosis; Label; Laboratories; Lead; Link; Modification; Muscle; Muscle Proteins; O-GlcNAc transferase; Organelles; Pathway interactions; Peptide Hydrolases; Peptides; Play; Post-Translational Protein Processing; Property; Proteasome Inhibition; Proteins; Recombinants; Regulation; Research Design; Role; Streptozocin; TP53 gene; Transcription Coactivator; Transgenic Mice; analog; chemotherapeutic agent; glucose metabolism; glycosylation; in vivo; inhibitor/antagonist; molecular mass; multicatalytic endopeptidase complex; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; protein degradation; wasting

The laboratory has shown that protein modification with O-linked N-acetylglucosamine (O-GlcNAc) plays a direct role in the function of transcriptional activators and repressors. This modification, which results from glucose metabolism, also modulates the function of the proteasome, the major organelle involved in intracellular degradation of proteins. The chymotryptic activity of 26S proteasomes, but not 20S proteasomes against 4 amino acid peptides (LLVY) is blocked by incubation of the proteasome with O-GlcNAc transferase (OGT). In addition, the ATPase activity of intact proteasomes is blocked by OGT. Physiologically inactivated proteasomes from NRK cells treated with high glucose or glucosamine can be reactivated by recombinant O-GlcNAcase, the enzyme that removes this modification. Labeling studies on purified proteasomes with [3H]-GlcNAc indicate that the modified protein(s) have a molecular mass of about 45 kDa and that this substrate resides in the 19S regulatory cap of the proteasome. Since the proteasome degrades pro-apoptotic factors such as p53 and many of its downstream targets, inhibition of proteasome function might lead to the accumulation of these factors with the induction of apoptosis. The chemotherapeutic agent and GlcNAc analog, streptozotocin, also induces apoptosis through its property as a non-competitive inhibitor of the O-GlcNAcase. The proposed studies are designed to determine the biochemical linkage between the O-GlcNAc pathway and the proteasome. The ability of O-GlcNAc to block proteasomal function may also couple glucose metabolism to amino acid release from muscle wasting. The specific aims are as follows: General goal: Determine the role of O-GlcNAc in proteasomal function. 1. Determine the effect of O-GlcNAc transferase (OGT) and O-GlcNAcase on proteasome function in vitro using these enzymes to reversibly modify proteins in the proteasome in vitro. 2. Identify proteasome- associated protein(s) that contain the O-GlcNAc modification and regulate proteasome function in a reversible manner. 3. Determine how O-GlcNAcylation of the proteasome 19S regulatory subunit modifies the function of the proteasomal peptidase and ATPases. 4. Using transgenic mice, determine the effect of proteasome blockade in vivo on epithelial cell apoptosis and muscle protein wasting.

实验室表明，O-连接的 N-乙酰氨基葡萄糖 (O-GlcNAc) 的蛋白质修饰起到了 直接作用于转录激活子和阻遏子的功能。这一修改的结果是 葡萄糖代谢，还调节蛋白酶体的功能，蛋白酶体是参与糖代谢的主要细胞器 细胞内蛋白质的降解。 26S 蛋白酶体的胰凝乳蛋白酶活性，但 20S 蛋白酶体没有 蛋白酶体与 O-GlcNAc 一起孵育可阻断针对 4 个氨基酸肽 (LLVY) 的作用 转移酶（OGT）。此外，完整蛋白酶体的 ATP 酶活性被 OGT 阻断。 来自用高葡萄糖或葡萄糖胺处理的 NRK 细胞的生理失活的蛋白酶体可以 由重组 O-GlcNAcase 重新激活，该酶可消除这种修饰。标签研究 用 [3H]-GlcNAc 纯化的蛋白酶体表明修饰的蛋白质的分子量约为 45 kDa 并且该底物位于蛋白酶体的 19S 调节帽中。由于蛋白酶体 降解促凋亡因子，例如 p53 及其许多下游靶标，抑制蛋白酶体 功能可能导致这些因子的积累并诱导细胞凋亡。这 化疗剂和 GlcNAc 类似物链脲佐菌素也通过其作为细胞凋亡的特性诱导细胞凋亡 O-GlcNAcase 的非竞争性抑制剂。拟议的研究旨在确定 O-GlcNAc 途径和蛋白酶体之间的生化联系。 O-GlcNAc 的阻断能力 蛋白酶体功能也可能将葡萄糖代谢与肌肉消耗中的氨基酸释放联系起来。这 具体目标如下： 总体目标：确定O-GlcNAc在蛋白酶体功能中的作用。 1. 体外测定 O-GlcNAc 转移酶 (OGT) 和 O-GlcNAcase 对蛋白酶体功能的影响 使用这些酶在体外可逆地修饰蛋白酶体中的蛋白质。 2. 鉴定蛋白酶体- 含有 O-GlcNAc 修饰并调节蛋白酶体功能的相关蛋白 可逆方式。 3.确定蛋白酶体19S调节亚基的O-GlcNAcylation如何修饰 蛋白酶体肽酶和 ATP 酶的功能。 4. 使用转基因小鼠，确定效果 体内蛋白酶体阻断上皮细胞凋亡和肌肉蛋白消耗。