Characterization of the O-GlcNAc-Binding Protein p32

描述(由申请人提供)：越来越多的研究表明，衰老和线粒体功能障碍之间存在因果联系[1-7]。最近，O-连接-N-乙酰氨基葡萄糖(O-GlcNAc)对核蛋白、细胞质和线粒体蛋白质的修饰与线粒体生理学、寿命、衰老和年龄相关疾病有关[8-29]。然而，O-GlcNAc执行这些功能的分子机制仍然不清楚[19，30]，这使得人们无法理解O-GlcNAc在许多细胞过程中的作用，包括线粒体蛋白功能、衰老和神经退化。为了弥合我们知识上的这一差距，我将探讨O-GlcNAc调节蛋白质从而调节细胞功能的一种机制：O-GlcNAc结合蛋白的特征。该项目的长期目标是定义一个O-GlcNAc结合基序，为O-GlcNAc调节蛋白质-蛋白质相互作用从而调节疾病过程的机制提供分子洞察力。具体地说，我将研究一种假设，即细胞内的透明质酸结合蛋白p32以O-GlcNAc依赖的方式与其他细胞蛋白相互作用并调节。这一假说是基于我的初步数据，这些数据表明：1)p32在体外优先结合糖偶联物BSA-AP-GlcNAc；2)p32富含O-GlcNAc修饰的蛋白质，类似于O-GlcNAc特异性抗体RL2；以及3)当O-GlcNAc水平升高时，p32的过度表达与突变的生物素连接酶生物素化更多的p32相关蛋白。为了明确O-GlcNAc在调节蛋白质-蛋白质相互作用中的作用以及这些相互作用在调节线粒体生理从而衰老过程中的作用，我将追求两个特定的目标：1)定义依赖O-GlcNAc的p32相互作用组；2)表征p32中的O-GlcNAc结合基序及其在调节线粒体生理中的作用。我将通过以下方法实现这些目标：1)使用BioID方法鉴定与p32以O-GlcNAc依赖的方式结合的蛋白质；2)在体外和体内验证这些相互作用；3)使用丙氨酸扫描突变和ELISA确定p32中的O-GlcNAc结合基序；以及4)在p32缺失细胞中重新表达p32的O-GlcNAc结合突变体，以探讨已发现的相互作用在p32缺失细胞中的作用 线粒体生理学。总之，这些研究将为理解O-GlcNAc在调节线粒体生理从而调节衰老过程中的作用奠定基础。这最终将导致对O-GlcNAc如何在线粒体生理学受到干扰的疾病的病因中起到更大作用的更好的理解。

DESCRIPTION (provided by applicant): A growing body of research suggests that there is a causative link between aging and mitochondrial dysfunction [1-7]. Recently, the modification of nuclear, cytoplasmic, and mitochondrial proteins by O-linked-?-N-acetylglucosamine (O-GlcNAc) has been linked to mitochondrial physiology, lifespan, aging, and age-related disorders [8-29]. However, the molecular mechanisms by which O-GlcNAc carries out these functions remain elusive [19, 30], precluding an understanding of the role of O-GlcNAc in many cellular processes including mitochondrial protein function, aging, and neurodegeneration. In order to close this gap in our knowledge, I will pursue one mechanism by which O-GlcNAc regulates protein and thus cellular function: the characterization of an O-GlcNAc binding protein. The long-term goal of this project is to define an O-GlcNAc-binding motif, providing molecular insight into the mechanisms by which O-GlcNAc regulates protein-protein interactions and therefore disease processes. Specifically, I will investigate the hypothesis that the intracellular hyaluronan- binding protein p32 interacts with and regulates other cellular proteins in an O-GlcNAc-dependent manner. This hypothesis is based on my preliminary data which demonstrates that: 1) p32 binds the glycoconjugate BSA-AP-GlcNAc in vitro in preference to other glycoconjugates; 2) p32 enriches O-GlcNAc-modified proteins similar to the O-GlcNAc-specific antibody RL2; and 3) overexpression of p32 fused to a mutant biotin ligase biotinylates more p32-associated proteins when O-GlcNAc levels are elevated. In order to define the role of O- GlcNAc in regulating protein-protein interactions and the role of these interactions in regulating mitochondrial physiology and thus the aging process, I will pursue two specific aims: 1) Define the O-GlcNAc-dependent interactome of p32; and 2) Characterize the O-GlcNAc-binding motif in p32 and its role in regulating mitochondrial physiology. I will carry out these aims by: 1) identifying proteins bound by p32 in an O-GlcNAc- dependent manner using the BioID method; 2) validating these interactions in vitro and in vivo; 3) defining the O-GlcNAc-binding motif in p32 using alanine-scanning mutagenesis and ELISA; and 4) re-expressing O- GlcNAc-binding mutants of p32 in p32 null cells to probe the role of the identified interactions in mitochondrial physiology. Together, these studies will form the foundation for understanding the role of O-GlcNAc in regulating mitochondrial physiology and thus the aging process. This will ultimately lead to a greater understanding of how O-GlcNAc contributes to the etiology of diseases in which mitochondrial physiology is perturbed.