The long term goal of my research program is to understand how fatty acid oxidation is regulated in order to develop new therapies for diseases of energy metabolism. Preliminary studies have identified protein acetylation/deacetylation as a novel mechanism regulating mitochondrial fatty acid oxidation. The key fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) has eight lysine acetylation sites and is demonstrated to be a target of the mitochondrial NAD-dependent deacetylase sirtuin-3 (Sirt3). When co- expressed with Sirt3 in HEK-293 cells, LCAD shows reduced lysine acetylation which is associated with a doubling of enzymatic activity. Other members of the acyl-CoA dehydrogenase enzyme family are also acetylated on numerous lysines and it is hypothesized that they are targets for sirtuin deacetylases.
Specific Aim 1 will investigate interactions between the mitochondrial sirtuins Sirt3, Sirt4, and Sirt5 and the enzymes very long-chain acyl-CoA dehydrogenase (VLCAD), medium chain-acyl-CoA dehydrogenase (MCAD), and isovaleryl-CoA dehydrogenase (IVD). Acetylation/deacetylation of their redox partner electron transferring flavoprotein (ETF) will also be studied. It is hypothesized that, similar to LCAD, activity of these enzymes will be modulated by sirtuin deacetylation. Proteomics methods will be used to identify acetylation sites responsible for regulating enzyme function. These sites will be further investigated using site-directed mutagenesis and three-dimensional molecular modeling.
Specific Aim 2 will focus on the mechanism by which acetylation alters LCAD activity. Preliminary data suggest that the effect of Sirt3 on LCAD activity is mediated by deacetylation of residue K42. Experiments are proposed to test the hypothesis that acetylation at K42 reduces enzymatic activity by interfering with the binding and transfer of electrons to ETF. Based on a shared quaternary structure among the acyl-CoA dehydrogenases the mechanism is anticipated to extend to other enzymes.
Specific Aim 3 a will study regulation of LCAD by acetylation/deacetylation in vivo using transgenic mice that express Flag- tagged LCAD as a reporter enzyme that can be easily recovered from tissue extracts for analysis of acetylation levels and function. LCAD-Flag transgenic mice will be crossed with Sirt3 knockout mice and studied under normal versus perturbed metabolic states including fasting and high-fat diet-induced obesity. I hypothesize that acetylation of LCAD will change with metabolic state and that Sirt3 activity on residue K42 is important for maintaining LCAD function in vivo.
Specific Aim 3 b will use preparative isoelectric focusing to separate differentially acetylated VLCAD, MCAD, IVD and ETF isoforms from mouse liver. Acetylation and enzyme function will be evaluated in protein preparations from Sirt3-/- mice versus wildtype. In summary, it is expected that this project will fundamentally alter our understanding of how acyl-CoA dehydrogenases and fatty acid oxidation are regulated and will uncover important new targets for treating diseases of energy metabolism.

Public Health Relevance

The acyl-CoA dehydrogenases are an important family of fatty acid oxidation enzymes and mutations in these genes are among the most common inborn errors of metabolism. The proposed project seeks to elucidate the molecular events behind a reversible modification to the acyl-CoA dehydrogenase enzymes known as lysine acetylation. Understanding this novel form of regulating acyl-CoA dehydrogenase function is important for developing new therapies for inborn errors of metabolism as well as for polygenic diseases such as diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK090242-01A1
Application #
8113569
Study Section
Therapeutic Approaches to Genetic Diseases (TAG)
Program Officer
Laughlin, Maren R
Project Start
2011-06-15
Project End
2016-05-31
Budget Start
2011-06-15
Budget End
2012-05-31
Support Year
1
Fiscal Year
2011
Total Cost
$332,615
Indirect Cost
Name
University of Pittsburgh
Department
Pediatrics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Meyer, Jesse G; Mukkamalla, Sushanth; Steen, Hanno et al. (2017) PIQED: automated identification and quantification of protein modifications from DIA-MS data. Nat Methods 14:646-647
Uppala, Radha; Dudiak, Brianne; Beck, Megan E et al. (2017) Aspirin increases mitochondrial fatty acid oxidation. Biochem Biophys Res Commun 482:346-351
Zhang, Yuxun; Bharathi, Sivakama S; Rardin, Matthew J et al. (2017) Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain. J Biol Chem 292:10239-10249
Dolezal, James M; Wang, Huabo; Kulkarni, Sucheta et al. (2017) Sequential adaptive changes in a c-Myc-driven model of hepatocellular carcinoma. J Biol Chem 292:10068-10086
Goetzman, Eric S (2017) Advances in the Understanding and Treatment of Mitochondrial Fatty Acid Oxidation Disorders. Curr Genet Med Rep 5:132-142
Wang, Huabo; Lu, Jie; Edmunds, Lia R et al. (2016) Coordinated Activities of Multiple Myc-dependent and Myc-independent Biosynthetic Pathways in Hepatoblastoma. J Biol Chem 291:26241-26251
Guarani, Virginia; Jardel, Claude; Chr├ętien, Dominique et al. (2016) QIL1 mutation causes MICOS disassembly and early onset fatal mitochondrial encephalopathy with liver disease. Elife 5:
Edmunds, Lia R; Otero, P Anthony; Sharma, Lokendra et al. (2016) Abnormal lipid processing but normal long-term repopulation potential of myc-/- hepatocytes. Oncotarget 7:30379-95
Edmunds, Lia R; Sharma, Lokendra; Kang, Audry et al. (2015) c-Myc programs fatty acid metabolism and dictates acetyl-CoA abundance and fate. J Biol Chem 290:20100
Uppala, Radha; McKinney, Richard W; Brant, Kelly A et al. (2015) Nickel inhibits mitochondrial fatty acid oxidation. Biochem Biophys Res Commun 463:806-10

Showing the most recent 10 out of 24 publications