Proteasome regulation by O-glycosylation

通过 O-糖基化调节蛋白酶体

• 批准号: 7097359
• 负责人: Jeffrey E Kudlow
• 金额: $ 28.35万
• 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/7097359
• 关键词: N acetylglucosamine; acylation; adenosinetriphosphatase; apoptosis; enzyme mechanism; epithelium; genetically modified animals; glycosyltransferase; laboratory mouse; matrix assisted laser desorption ionization; peptidases; proteasome; protein structure; yeast two hybrid system

DESCRIPTION (provided by applicant): 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 proteasomeassociated 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.

描述（由申请人提供）： 实验室表明，O-连接的 N-乙酰氨基葡萄糖 (O-GlcNAc) 的蛋白质修饰在转录激活子和阻遏子的功能中起着直接作用。这种由葡萄糖代谢引起的修饰也调节蛋白酶体的功能，蛋白酶体是参与细胞内蛋白质降解的主要细胞器。通过将蛋白酶体与 O-GlcNAc 转移酶 (OGT) 一起孵育，可阻断 26S 蛋白酶体（而非 20S 蛋白酶体）针对 4 个氨基酸肽 (LLVY) 的胰凝乳蛋白酶活性。此外，完整蛋白酶体的 ATP 酶活性被 OGT 阻断。用高葡萄糖或葡萄糖胺处理的 NRK 细胞中生理失活的蛋白酶体可以通过重组 O-GlcNAcase（消除这种修饰的酶）重新激活。用 [3H]-GlcNAc 对纯化的蛋白酶体进行标记研究表明，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。由于蛋白酶体会降解 p53 等促凋亡因子及其许多下游靶标，因此抑制蛋白酶体功能可能会导致这些因子的积累，从而诱导细胞凋亡。化疗剂和 GlcNAc 类似物链脲佐菌素也通过其作为 O-GlcNAcase 非竞争性抑制剂的特性诱导细胞凋亡。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 阻断蛋白酶体功能的能力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。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. 使用这些酶在体外可逆地修饰蛋白酶体中的蛋白质，确定 O-GlcNAc 转移酶 (OGT) 和 O-GlcNAcase 对体外蛋白酶体功能的影响。 2. 鉴定含有 O-GlcNAc 修饰并以可逆方式调节蛋白酶体功能的蛋白酶体相关蛋白。 3. 确定蛋白酶体 19S 调节亚基的 O-GlcNAc 酰化如何改变蛋白酶体肽酶和 ATP 酶的功能。 4. 使用转基因小鼠，确定体内蛋白酶体阻断对上皮细胞凋亡和肌肉蛋白消耗的影响。