Characterization of the O-GlcNAc-Binding Protein p32

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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.