Proteasome regulation by O-glycosylation

描述(由申请人提供)： 实验室已经证明，O-连接N-乙酰氨基葡萄糖(O-GlcNAc)的蛋白质修饰在转录激活因子和抑制因子的功能中起着直接的作用。这种由葡萄糖代谢引起的修饰，也调节蛋白酶体的功能，蛋白酶体是参与蛋白质细胞内降解的主要细胞器。与O-GlcNAc转移酶(OGT)孵育可阻断26S蛋白酶体对4种氨基酸肽(LLVY)的胰凝乳酶活性，而20S蛋白酶体对LLVY的抑制作用不明显。此外，完整蛋白酶体的ATPase活性被OGT阻断。用高葡萄糖或氨基葡萄糖处理的NRK细胞中生理失活的蛋白酶体可以通过重组O-GlcNAcase重新激活，O-GlcNAcase是一种去除这种修饰的酶。用[~3H]-GlcNAc对纯化的蛋白酶体进行标记研究表明，修饰蛋白(S)的分子质量约为45 kDa，该底物位于蛋白酶体的19S调节帽中。由于蛋白酶体降解P53等促凋亡因子及其下游靶点，抑制蛋白酶体功能可能导致这些因子在诱导细胞凋亡的过程中积聚。化疗药物和GlcNAc类似物，链脲佐菌素，也通过其作为O-GlcNAcase的非竞争性抑制物而诱导细胞凋亡。建议的研究旨在确定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萎缩所释放的氨基酸结合在一起。具体目标如下：总目标：确定O-GlcNAc在蛋白酶体功能中的作用。1.用O-GlcNAc转移酶(OGT)和O-GlcNAcase体外可逆修饰蛋白酶体中的蛋白质，研究它们对蛋白酶体功能的影响。2.鉴定含有O-GlcNAc修饰的蛋白酶体相关蛋白(S)，并以可逆的方式调节蛋白酶体功能。3.确定蛋白酶体19S调节亚基的O-GlcN酰化如何改变蛋白酶体多肽酶和ATPase的功能。4.利用转基因小鼠，检测体内蛋白酶体阻断对小鼠上皮细胞凋亡和肌肉蛋白消耗的影响。

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.