PROJECT ABSTRACT Methylglyoxal (MG) is a potent intracellular glycating agent that forms advanced glycation endproducts. Formed spontaneously from 3-carbon glycolytic intermediates, MG rapidly glycates proteins and nucleotides, damages mitochondria and directly increases reactive oxygen species production; thus inducing a pro-oxidant state and senescent-like condition. MG and the related glyoxalase enzymatic defense system are emerging as critical players in aging and age-related disease processes. Under physiologic conditions MG is rapidly detoxified by glyoxalase 1 (GLO1). However, when GLO1 is attenuated, MG flux is increased and MG-modified proteins accumulate (termed dicarbonyl stress), both within and outside the cell. Dicarbonyl stress promotes glucose intolerance, oxidative stress and inflammation. The mechanisms regulating GLO1 protein stability and enzymatic activity in skeletal muscle tissue, a tissue critical to glucose metabolism, are not well studied and there is a critical need to understand the functional consequences of reduced GLO1 in the context of obesity, aging and age- related disease. GLO1 is critical to cellular function and subject to numerous posttranslational modifications (PTMs) that regulate GLO1 protein stability and activity. Our objective is to establish robust translational models to delineate the mechanisms by which GLO1 is regulated to better understand the functional consequences of attenuated GLO1. The generation of new, state-of-the-art translational models will help to accelerate the understanding of GLO1 attenuation and dicarbonyl stress and the implications for skeletal muscle health across both the life-span and health-span. We aim to establish the functional relevance of both GLO1 loss and the impact of PTMs of GLO1 in human myotubes. Our approach is to attenuate GLO1 and mutate critical amino acid residues using CRISPR gene editing technology, coupled with measures of dicarbonyl stress. We expect to identify a novel, muscle specific mechanism of GLO1 dysregulation and methylglyoxal-mediated damage. The successful completion of this work will have an important positive impact on advancing the understanding, and provide potential therapeutic targets, to maintain skeletal muscle function with aging and age-related disease.

PROJECT NARRATIVE Skeletal muscle is a critical organ towards whole body metabolic regulation. We have identified novel mechanisms by which cellular metabolism maintains the abundance of critical antioxidant defense enzymes in skeletal muscle. This proposal will explore the regulation of these critical cell proteins in skeletal muscle tissues and the applicability of our finding in age-related diseases.