Mitochondrial fatty acid ?-oxidation is traditionally viewed as an energy-generating, catabolic pathway but intermediates of this pathway can serve as key substrates for synthesis of other complex lipids. The long range objective of this project is to define the role of fatty acid oxidaton proteins in intermediary metabolism and the clinical impact of their deficiency due to inborn errors. The goal of the first funding period was to characterize the functional roles of LCAD, VLCAD, and ACAD9 and to explore the ramifications of genetic deficiencies of these enzymes in humans and mouse models. Significant progress has been made on each of these aims. Our chief findings include the ground breaking identification and partial purification of a multifunctional fatty acid oxidation complex containing all of the activities of this pathway in association with the mitochondrial respiratory chain super complexes and identification of the first patient with LCAD deficiency, presenting as predicted with a surfactant deficiency. The goal of this renewal application is to examine the expanding role of long chain fatty acid oxidation in normal metabolism and disease. It has three specific aims.
Specific Aim 1 is to characterize the structure of the multifunctional fatty acid oxidation complex and its interaction with the mitochondrial respiratory chain. I hypothesize that this contact is critical to the channeling of reducing equivalents from fatty acid oxidation to the respiratory chain and that disruption of this process reduces the efficiency of mitochondrial energy generation.
Specific Aim 1 a is to elucidate the mechanism of electron transfer by first deducing the structure of the multifunctional FAOD complex using cryoelectron microscopy.
Specific Aim 1 b is to examine the role of patient identified mutations on VLCAD function, including their effect on the integrity of the FAOD multifunctional complex.
Specific Aim 1 c is to characterize the integrity of the fatty acid oxidatin complex in VLCAD and LCAD deficient mouse models.
Specific Aim 2 is to characterize the disparate effects of null and point mutations in the ACAD9 gene on protein structure and function. I hypothesize that ACAD9 serves a duel function in mitochondria as a metabolic enzyme and a respiratory chain assembly/stability factor.
Specific Aim 2 a is to examine the metabolic and moonlighting functions of ACAD9 in cells from deficient human patients.
Specific Aim 2 b is to characterize the range of biochemical and clinical effects in an ACAD9 knock out mouse model.
Specific Aim 3 is to further characterize LCAD deficiency in patients and a knock out mouse model, and to elucidate its function in surfactant metabolism.
Specific Aim 3 a is to examine surfactant metabolism in wild type and LCAD deficient primary pneumocytes.
Specific Aim 3 b is to characterize the incidence and spectrum of clinical symptoms in LCAD deficiency.

Public Health Relevance

The acyl-CoA dehydrogenases are important enzymes in maintaining normal chemical balance in the body. We have identified two new genetic disorder of one of these enzymes that leads to liver failure and lung disease. Studying these disorders is important to learn more about their clinical presentation and treatment.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Therapeutic Approaches to Genetic Diseases (TAG)
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Leschek, Ellen W
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University of Pittsburgh
Schools of Medicine
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Leipnitz, Guilhian; Mohsen, Al-Walid; Karunanidhi, Anuradha et al. (2018) Evaluation of mitochondrial bioenergetics, dynamics, endoplasmic reticulum-mitochondria crosstalk, and reactive oxygen species in fibroblasts from patients with complex I deficiency. Sci Rep 8:1165
Repp, Birgit M; Mastantuono, Elisa; Alston, Charlotte L et al. (2018) Clinical, biochemical and genetic spectrum of 70 patients with ACAD9 deficiency: is riboflavin supplementation effective? Orphanet J Rare Dis 13:120
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Schiff, Manuel; Haberberger, Birgit; Xia, Chuanwu et al. (2015) Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency. Hum Mol Genet 24:3238-47
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