Long-chain fatty acid oxidation disorders (LC-FAODs) are a heterogenous group of disorders characterized by the inability to break down long-chain fatty acids in the mitochondria for energy. Peroxisomal fatty acid oxidation (FAO) is a parallel pathway to mitochondrial FAO that could be leveraged to alleviate fatty acid accumulation in patients with LC-FAODs. However, there is currently no pharmacological means of stimulating peroxisomal FAO in humans. The ability to develop new peroxisome-stimulating therapies is limited by knowledge gaps regarding the factors that regulate activity of peroxisomal FAO enzymes. Here, it is proposed that sirtuin-5 (Sirt5) and lysine succinylation—a post-translational modification reversed by Sirt5—represent a new mechanism for manipulating peroxisomal function. When mice are fed a class of fatty acids called dicarboxylic acids (DCAs), lysine succinylation accumulates on peroxisomal proteins. Further preliminary data suggest that lysine succinylation increases peroxisomal function. The capacity of Sirt5 to reverse these effects remains unclear. The central hypothesis of this grant is that feeding DCAs can improve disease pathology in mouse models of mitochondrial LC-FAOD by driving protein succinylation and peroxisomal activation. This is supported by preliminary data in which seven days of DCA feeding improved muscle function in an LC-FAOD mouse model. The central hypothesis will be fully explored in three Specific Aims. 1) Aim 1 will quantify the effects of DCA feeding and Sirt5 ablation on the peroxisomal acylome. Sirt5 partially localizes to the peroxisome but its activity there has not been characterized. A quantitative, site-level lysine “acylome” ± DCA feeding will be compiled for liver, muscle, and heart—the key tissues affected in LC-FAODs—and all Sirt5 target sites identified. 2) Aim 2 is to delineate the effects of DCA feeding ± Sirt5 ablation on the function of peroxisomal enzymes and pathway fluxes. This will be done using purified recombinant proteins, cultured cells with manipulated Sirt5 levels in the peroxisome, and Sirt5-deficient mice. Metabolomics, 14C-substrate flux studies, and enzyme stability/function testing will be used to determine how reversible lysine PTMs affect the peroxisome. 3) Aim 3 will be to test DCA feeding as a therapeutic strategy in LC-FAOD mouse models. It is proposed that DCAs will distribute beyond the liver to the peripheral organs, serving as a source of energy via partial chain shortening and peroxisomal gain-of-function. Mild and severe LC-FAOD mouse models ± long- term DCA feeding will be evaluated for liver, heart, and muscle functioning as well as the response to fasting stress. Ablation of Sirt5 in this context may further enhance peroxisomal function. To test this, the LC-FAOD mouse models will be crossed onto a Sirt5-/- background. Together, completion of these Specific Aims will form critical new knowledge for manipulating peroxisomal function to treat LC-FAODs. ...