PROJECT ABSTRACT Fatty acid oxidation (FAO) is a critical energy producing pathway in heart, muscle, and liver, among other organs. Inborn errors in genes of the FAO pathway are associated with dysfunction in these organs and a high rate of mortality. Additionally, disruptions in FAO are seen in polygenic diseases such as obesity, diabetes, and cancer. With advances in mass spectrometry profiling of blood metabolites, FAO defects can be readily diagnosed. However, despite 30 years of intensive study, treatment options for modulating FAO in human patients remain limited and ineffective. Knowledge gaps regarding the regulation of FAO enzymes and the functional organization of the FAO pathway within the greater landscape of mitochondrial energy metabolism have limited the development of new therapies. In the previous funding period of this grant, we established reversible lysine post-translational modifications (acetylation, succinylation) as regulators of FAO. We showed that sirtuin enzymes, which deacylate target lysines and restore them to the native state, are important players in maximizing function of the FAO pathway. In the present proposal we hypothesize that lysine acylation regulates FAO enzyme activity, localization to the inner mitochondrial membrane, and the assembly of higher- order metabolic complexes between FAO proteins and the respiratory chain. In Specific Aim 1, we will employ in vitro methods that we pioneered in the previous funding period to identify sirtuin-targeted lysines on the membrane-associated FAO enzymes carnitine palmitoyltransferase-2 (CPT2), mitochondrial trifunctional protein (TFP), and acyl-CoA dehydrogenase-9 (ACAD9). We will perform mutagenesis studies to determine the functional role of each of the sirtuin-targeted lysine residues. In Specific Aim 2 we will investigate physical and functional interactions between the three mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5) and the inner mitochondrial membrane. We hypothesize that the sirtuins police the inner mitochondrial membrane in order to facilitate assembly and operation of higher-order metabolic complexes such as those formed between FAO and the electron transport chain. Finally, Specific Aim 3 will evaluate the effects of lysine acylation on these higher-order complexes using a combination of mouse models and protein complexes assembled in vitro. Understanding the role of the sirtuins in regulating FAO and metabolic supercomplexes will lay the ground work for developing new therapies that manipulate mitochondrial function in human patients with inborn errors of metabolism, as well as those with chronic diseases such as obesity, diabetes, and cancer.