MOONLIGHTING IN MITOCHONDRIA: ACAD9 PLAYS A DUAL ROLE IN ENERGY METABOLISM. M. Schiff1, B. M. Haberberger2, E. S. Goetzman1, A. W. Mohsen1, H. Prokisch2, J. Vockley1 1) Pediatrics, University of Pittsburgh, Pittsburgh, PA; 2) Human Genetics, Technical University and Helmholtz Zentrum, University of Munich, Germany.

   Background: Liver Acyl-CoA dehydrogenase-9 (ACAD9) deficiency was first described in 2 patients with liver failure. Subsequently, point mutations in ACAD9 were shown to cause respiratory chain complex I (CI) deficiency due to a moonlighting function of ACAD9 as a CI assembly factor. However, the impact of these mutations on fatty acid oxidation (FAO) activity and the physiologic contribution of ACAD9 in eukaryotes to FAO remain unknown. Methods: Prokaryotic expression mutagenesis studies were performed for 13 missense ACAD9 mutations. Oxidation of long chain fatty acids was analyzed in very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain acyl-CoA dehydrogenase (LCAD) double knockout mouse fibroblasts. ACAD9 gene knockout in HEK293 cells was performed using Transcription Activator-like Effector Nucleases (TALEN) technology. Results: All but two ACAD9 mutants (R127Q, A326P) had a stable ACAD9 antigen. Three (R469W, R518H, R518C) had mildly (75% of wild type) decreased ACAD9 enzymatic activity while one had normal activity (R532W); activity was partially (20% of wild type) decreased in 2 mutants (L98S and R433Q) and undetectable in 7 (I87N, R127Q, R266Q, A326P, E413K, R417C and D418G). ACAD9 protein stability in patient fibroblasts largely mirrored these findings though some discordance was apparent. Doubly deficient VLCAD/LCAD knockout mouse fibroblasts retained ACAD9 activity and supported significant capacity for long-chain FAO. TALEN-mediated knockout HEK293 cells exhibited a total absence of ACAD9 antigen as well as defective FAO. Conclusions: The moonlighting function of ACAD9 as a CI assembly factor is independent of its FAO enzymatic activity. Mutations in ACAD9 can affect either or both functions. We also demonstrated that ACAD9 plays a significant role in eukaryotic FAO. Further characterization of genotype/phenotype correlations in ACAD9 deficient patients will be complicated by this dual function.

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