Proteasome regulation by O-glycosylation

实验室已经表明，用O-连接的N-乙酰葡糖胺（O-GlcNAc）修饰蛋白质， 在转录激活因子和抑制因子的功能中的直接作用。这一修改是由 葡萄糖代谢，也调节蛋白酶体的功能，蛋白酶体是参与葡萄糖代谢的主要细胞器。 蛋白质的细胞内降解。26 S蛋白酶体的胰凝乳蛋白酶活性，而不是20 S蛋白酶体 通过将蛋白酶体与O-GlcNAc孵育来阻断针对4个氨基酸肽（LLVY）的 转移酶（OGT）。此外，完整蛋白酶体的ATP酶活性被OGT阻断。 来自用高糖或葡糖胺处理的NRK细胞的生理失活的蛋白酶体可以被抑制。 通过重组O-GlcNAc酶重新激活，该酶去除这种修饰。标签研究 用[3 H]-GlcNAc纯化的蛋白酶体表明修饰的蛋白质具有约10000的分子量。 45 kDa，并且该底物位于蛋白酶体的19 S调节帽中。由于蛋白酶体 降解促凋亡因子如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。的 化疗剂和GlcNAc类似物，链脲霉素，也通过其作为抗肿瘤药物的性质诱导细胞凋亡。 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物化学联系。O-GlcNAc阻断 蛋白酶体的功能也可以将葡萄糖代谢与肌肉萎缩的氨基酸释放偶联。的 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-GlcNAc酶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- 相关蛋白，其含有O-GlcNAc修饰并调节蛋白酶体功能， 可逆的方式。3.确定蛋白酶体19 S调节亚基的O-GlcNAc酰化如何修饰 蛋白酶体肽酶和ATP酶的功能。4.使用转基因小鼠，确定 体内蛋白酶体阻断对上皮细胞凋亡和肌肉蛋白质消耗的影响。

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.