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关系[1-7]。最近，修饰的核，细胞质和线粒体蛋白质的O-连接-？- N-乙酰葡糖胺（O-GlcNAc）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难以捉摸[19，30]，妨碍了对O-GlcNAc在许多细胞过程中的作用的理解，包括线粒体蛋白功能、衰老和神经变性。为了弥补我们知识上的这一差距，我将研究O-GlcNAc调节蛋白质从而调节细胞功能的一种机制：O-GlcNAc结合蛋白的表征。该项目的长期目标是定义O-GlcNAc结合基序，为O-GlcNAc调节蛋白质-蛋白质相互作用和疾病过程的机制提供分子见解。具体来说，我将调查的假设，即细胞内的透明质酸结合蛋白p32相互作用，并调节其他细胞蛋白质的O-GlcNAc依赖性的方式。该假设基于我的初步数据，其证明：1）p32在体外优先于其他糖缀合物结合糖缀合物BSA-AP-GlcNAc; 2）p32富集类似于O-GlcNAc特异性抗体RL 2的O-GlcNAc修饰的蛋白;和3）当O-GlcNAc水平升高时，与突变生物素连接酶融合的p32的过表达使更多的p32相关蛋白生物素化。为了确定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学以及由此的衰老过程中的作用，我将追求两个具体目标：1）确定p32的O-GlcNAc依赖性相互作用组;以及2）表征p32中的O-GlcNAc结合基序及其在调节线粒体生理学中的作用。本论文的主要工作如下：1）利用BioID方法鉴定p32与O-GlcNAc依赖性结合的蛋白质; 2）在体外和体内验证这些相互作用; 3）利用丙氨酸扫描诱变和ELISA方法确定p32中的O-GlcNAc结合基序;和4）在p32缺失细胞中再表达p32的O-GlcNAc结合突变体，以探测鉴定的相互作用在 线粒体生理学总之，这些研究将为理解O-GlcNAc在调节线粒体生理学和衰老过程中的作用奠定基础。这将最终导致对O-GlcNAc如何有助于线粒体生理学受到干扰的疾病的病因学的更好理解。

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.