Lysine acylation has emerged as a prominent post-translational modification (PTM) within mitochondrial proteins. Increased susceptibility of mitochondrial proteins to acylation is a function of the alkaline pH of the matrix, in conjunction with the relatively high concentration of acyl-CoA intermediates. Protein acylation varies as a function of the matrix acyl-CoA pool and is counterbalanced by the activity of various mitochondrial- localized deacylases (SIRT3, 4, 5). As such, metabolic conditions know to increase free acyl-CoAs (e.g., high- fat diet, caloric restriction), as well as genetic knockout of either SIRT3 or SIRT5 have revealed a wide range of acetyl and acyl (e.g., malonylation, succinylation, glutarylation) modifications throughout various mitochondrial proteins. Although in some cases lysine acylation has been linked to altered enzymatic flux (e.g., lysine acetylation inhibits is citrate dehydrogenase), the functional relevance of these modifications in the context of mitochondrial physiology and disease is largely unknown. Malonyl-CoA decarboxylase (MCD) is an enzyme responsible for the degradation of malonyl-CoA, and genetic ablation of the enzyme in MCD-/- mice results in elevated tissue levels of malonyl-CoA. Our preliminary studies show that MCD in muscle and liver is predominantly localized to the mitochondria. Moreover, Western blot assessment of global malonylation in MCD-/- mice revealed striking elevations in malonylated proteins, particularly in the mitochondrial compartment. Follow up experiments using LC mass spectrometry (MS)/MS revealed widespread hypermalonylation in skeletal muscle tissue of muscle-specific MCD deficient mice (MCDMCK-/-). Included among identified targets of malonylation were two of the three subunits of the pyruvate dehydrogenase complex (PDC), as well as enzymes involved in beta-oxidation, ATP synthesis, the TCA cycle and redox homeostasis. Thus, the current NRSA application will test the overarching hypothesis that MCD defends mitochondrial function in muscle and liver by protecting against hypermalonylation of matrix proteins.
In Aim 1 we will identify malonylated lysine residues in both skeletal muscle and liver of tissue specific MCD deficient mice under low fat and high fat fed conditions and then compare these sites with those previously reported for SIRT5-/- mice.
In Aim 2 we will focus on hits identified in preliminary studies and SA1 to determine if the hypermalonylation phenotype of the MCD deficient mice is accompanied by alternations in mitochondrial function (e.g., enzyme activities, respiratory kinetics and H2O2 emission/redox homeostasis). Lastly, in Aim 3 we will use a variety of pharmacological and genetic approaches to alter protein malonylation, coupled with targeted LC-MS(MS), to test the hypothesis that protein malonylation exists as a novel regulator of the PDC. Results could implicate lysine acylation as a cause of mitochondrial failure in the context of aging and/or chronic metabolic disorders.
Mitochondria serve as cellular engines that burn dietary fuels to produce energy. The current project aims to investigate a novel mechanism whereby mitochondria regulate energy metabolism, and to understand why nutrient surplus damages mitochondria. The project is relevant to public health because mitochondrial failure results in organ dysfunction and exercise intolerance, and alterations in mitochondrial metabolism have been linked to aging and prevalent chronic metabolic disorders such as obesity, type 2 diabetes and heart disease. The results of the proposed project could lay the groundwork for the development of novel therapeutic strategies to prevent and/or mitigate mitochondrial dysfunction in the context of aging and chronic metabolic disease.
|Fisher-Wellman, Kelsey H; Davidson, Michael T; Narowski, Tara M et al. (2018) Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes. Cell Rep 24:3593-3606.e10|
|Anderson, Kristin A; Huynh, Frank K; Fisher-Wellman, Kelsey et al. (2017) SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion. Cell Metab 25:838-855.e15|
|Krycer, James R; Fisher-Wellman, Kelsey H; Fazakerley, Daniel J et al. (2017) Bicarbonate alters cellular responses in respiration assays. Biochem Biophys Res Commun 489:399-403|