Anti-ACADS monoclonal antibody, clone FQS21973(C)

Members of the Acyl-CoA dehydrogenase (ACADs) family of enzymes play a crucial role in cholesterol and steroid catabolism and are widely studied in the oldest known human pathogen, Mycobacterium tuberculosis (Mtb). However, there is a paucity of information on ACADs involved in branched chain amino acid catabolism. Here we characterized one of the putative ACAD enzyme, fadE9, as "Isobutyryl CoA Dehydrogenase (IBDH)" using a combined computational and experimental approach, guided by homology modeled structural information, affirming its role in valine catabolism. Multiple sequence alignment and phylogenetic analysis place it in a separate cluster from a recently identified family of alpha(2)beta(2)-heterotetramer ACADs in Mtb, based on the position of the conserved Arg247 and catalytic Glu368 residues. The conserved Arg247 was predicted to play an essential role at the center of H-bonding network of reaction center and was confirmed by the reduced activity of R247K mutant Thus, in addition to the finding of an architecturally distinct alpha(2)beta(2)-heterotetramer among ACADs, these studies also highlight the differences between MtIBDH, fadE9 from the other ACADs that are involved in cholesterol and steroid catabolism of Mtb. (C) 2018 Published by Elsevier B.V.

Objective: To elucidate the pathophysiology of SCAD deficient patients who have a unique neurological phenotype, among fatty acid oxidation disorders, with early developmental delay, CNS malformations, intractable seizures, myopathy and clinical signs suggesting oxidative stress. Methods: We studied skin fibroblast cultures from patients homozygous for ACADS common variant c.625G > A (n = 10), compound heterozygous for c.625G > A/c.319C > T (n = 3) or homozygous for pathogenic c.319C > T (n = 2) and c.1138C > T (n = 2) mutations compared to fibroblasts from patients with carnitine palmitoyltransferase 2 (CPT2) (n = 5), mitochondrial trifunctional protein (MTP)/long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) (n = 7), and medium-chain acyl-CoA dehydrogenase (MCAD) deficiencies (n = 4) and normal controls (n = 9). All were exposed to 50 mu M menadione at 37 degrees C. Additonal conditions included exposure to 39 degrees C and/or hypoglycemia. Time to 100% cell death was confirmed with trypan blue dye exclusion. Experiments were repeated with antioxidants (Vitamins C and E or N-acetylcysteine), Bezafibrate or glucose and temperature rescue. Results: The most significant risk factor for vulnerability to menadione-induced oxidative stress was the presence of a FAO defect. SCADD fibroblasts were the most vulnerable compared to other FAO disorders and controls, and were similarly affected, independent of genotype. Cell death was exacerbated by hyperthermia and/or hypoglycemia. Hyperthermia was a more significant independent risk factor than hypoglycemia. Rescue significantly prolonged survival. Incubation with antioxidants and Bezafibrate significantly increased viability of SCADD fibroblasts. Interpretation: Vulnerability to oxidative stress likely contributes to neurotoxicity of SCADD regardless of ACADS genotype and is significantly exacerbated by hyperthermia. We recommend rigorous temperature control in SCADD patients during acute illness. Antioxidants and Bezafibrate may also prove instrumental in their management.