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Identification and Characterization of the Biochemical And IDENTIFICATION AND CHARACTERIZATION OF THE BIOCHEMICAL AND PHYSIOLOGICAL FUNCTIONS OF ACYL-COA DEHYDROGENASE 10 by Kaitlyn Nicole Kormanik BS Biology, Wilkes University, 2009 Submitted to the Graduate Faculty of Graduate School of Public Health in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2014 UNIVERSITY OF PITTSBURGH GRADUATE SCHOOL OF PUBLIC HEALTH This dissertation was presented by Kaitlyn Nicole Kormanik It was defended on April 17, 2014 and approved by Dissertation Director: Jerry Vockley, M.D. Ph.D., Professor Pediatrics, School of Medicine, Professor, Graduate School of Public Health, University of Pittsburgh Al-Walid Mohsen, Ph.D., Associate Professor, Pediatrics School of Medicine, University of Pittsburgh Candace Kammerer, Ph.D., Professor, Human Genetics Graduate School of Public Health, University of Pittsburgh Zsolt Urban, Ph.D., Associate Professor, Human Genetics Graduate School of Public Health, University of Pittsburgh David Finegold, Ph.D., Professor, Pediatrics School of Medicine, University of Pittsburgh ii Copyright © by Kaitlyn N. Kormanik 2014 iii IDENTIFICATION AND CHARACTERIZATION OF THE BIOCHEMICAL AND PHYSIOLOGICAL FUNCTIONS OF ACYL-COA DEHYDROGENASE 10 Kaitlyn Nicole Kormanik, PhD University of Pittsburgh, 2014 ABSTRACT The acyl-CoA dehydrogenase, ACAD, genes encode enzymes that are essential to cellular metabolism as evidenced by the identification in the past 20 years of genetic defects associated with nine members of this gene family. In total, these defects constitute the most common inborn errors of metabolism identified through newborn screening and so are concerns to public health. Recently, two new ACADs, ACAD10 and 11, of unknown function have been identified. This discovery raises the specter of additional unrecognized disorders since prior studies on these two ACADs suggested a novel role in metabolism. Recently, ACAD10 has been linked to type 2 diabetes in Pima Indians, which draws additional attention to its role in metabolism and public health importance. Both ACAD10 and ACAD11 are highly conserved but with unique additional large protein domains compared to other ACADs, likely encoded from multiple coding domains. ACAD10 antigen in mice is present in lung, muscle, kidney, and pancreas, and localized to mitochondria, while a weak signal is also observed in peroxisomes of mouse lung. Human tissues including lung, kidney, liver, muscle, and pancreas reveal antigen present in mitochondria, and a weak antigen signal in peroxisomes in kidney and pancreas. To investigate the role of ACAD10 in metabolism further, I have generated an Acad10 deficient mouse and have characterized it through pathological, biochemical, and molecular studies. Deficient animals are viable and fertile, but become obese. Pathological studies reveal iv inflammatory liver disease and secondary splenic extramedullary hematopoiesis. Skeletal muscle findings were abnormal, consistent with deficient mice have elevated creatine kinase when fasting, indicative of rhabdomyolysis. Metabolomics analysis identified elevated levels of a variety of acylcarnitine species in deficient mouse samples consistent with mild, global energy dysfunction. Most dramatically, animals develop a syndrome consistent with insulin insensitivity characteristic of type 2 diabetes mellitus in humans. A better understanding of the biochemical pathways and physiological role of ACAD10, as well as the pathophysiology of disorders occurring among this new ACAD family member will allow identification and treatment of patients with ACAD10 deficiency, as well as its role in obesity and type 2 diabetes. v TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................................. XVII ABBREVIATIONS .................................................................................................................. XIX 1.0 INTRODUCTION ........................................................................................................ 1 1.1 MITOCHONDRIAL FATTY ACID OXIDATION ......................................... 1 1.1.1 Structural aspects of ACADs .......................................................................... 4 1.1.2 Mouse Models of ACAD Deficiencies ............................................................ 5 1.2 ACAD 10 AND 11 ................................................................................................. 6 1.2.1 Multiple transcripts from ACAD10 and ACAD11 genes .............................. 6 1.2.2 Tissue distribution of ACAD10 and ACAD11 transcripts .......................... 9 1.2.3 Subcellular location and mitochondrial fractionation of ACAD11 protein isoforms ....................................................................................................................... 10 1.2.4 Computer modeling of ACAD10 and ACAD11 .......................................... 13 1.3 PLANT ACADS ................................................................................................. 14 1.4 ACAD LOCALIZATION ON GLUT4-CONTAINING VESICLES ........... 15 2.0 EVIDENCE FOR INVOLVEMENT OF MEDIUM CHAIN ACYL-COA DEHYDROGENASE IN THE METABOLISM OF PHENYLBUTYRATE ....................... 16 2.1 ABSTRACT........................................................................................................ 16 2.2 INTRODUCTION ............................................................................................. 17 vi 2.3 MATERIALS AND METHODS ...................................................................... 20 2.3.1 Purification of recombinant human MCAD ............................................... 20 2.3.2 The electron transfer flavoprotein (ETF) purification .............................. 21 2.3.3 Fibroblast cell culture and extract preparation .......................................... 21 2.3.4 ETF fluorescence reduction assay ................................................................ 22 2.3.5 Phenylbutyryl-CoA synthesis ....................................................................... 22 2.3.6 Monitoring the interaction of MCAD with substrates ............................... 23 2.3.7 Molecular modeling ....................................................................................... 24 2.4 RESULTS ........................................................................................................... 24 2.4.1 Interaction of MCAD with substrates, the reductive half-reaction .......... 24 2.4.2 Interaction of MCAD: Substrate ternary complex with ETF, the oxidative half reaction ................................................................................................................ 27 2.4.3 The ETF fluorescence reduction assay of cell extract ................................ 28 2.5 DISCUSSION ..................................................................................................... 28 2.6 ACKNOWLEDGEMENTS .............................................................................. 33 3.0 CLONING AND EXPRESSION OF ACAD10 IN E.COLI .................................... 34 3.1 ABSTRACT........................................................................................................ 34 3.2 INTRODUCTION ............................................................................................. 35 3.3 MATERIALS AND METHODS ...................................................................... 37 3.3.1 Cloning and Expression ................................................................................ 37 3.3.2 Transient transaction and overexpression in HEK293T cells with pcDNA ACAD10 ...................................................................................................................... 39 3.3.3 His-Tag Protein Purification ........................................................................ 41 vii 3.3.4 Production and Purification of Anti-ACAD10 Antiserum ........................ 41 3.3.5 Western Blot analysis .................................................................................... 42 3.3.6 Identification of Proteins by Tandem Mass Spectrometry (MS/MS) ....... 43 3.4 RESULTS ........................................................................................................... 44 3.4.1 ACAD10 cDNA amplification and expression ............................................ 44 3.4.2 First half reaction .......................................................................................... 48 3.5 DISCUSSION ..................................................................................................... 53 4.0 CHARACTERIZATION OF AN ACAD10 DEFICIENT MOUSE MODEL: PATHOLOGICAL AND BIOCHEMICAL ANALYSES ....................................................... 56 4.1 ABSTRACT........................................................................................................ 56 4.2 INTRODUCTION ............................................................................................. 57 4.2.1 Mitochondrial fatty acid oxidation and diabetes ........................................ 57 4.3 MATERIALS AND METHODS ...................................................................... 59 4.3.1 Subcellular Location of ACAD10 Protein in Human Tissues ................... 59 4.3.2 Subcellular Location of ACAD10 Protein in Mouse Tissues ..................... 60 4.3.3 Generation of Mutant Mice .......................................................................... 61 4.3.4 Clinical Characterization .............................................................................
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