title: Mia40 is a facile oxidant of unfolded reduced proteins but shows minimal isomerase activity.
abstract: Mia40 participates in oxidative protein folding within the mitochondrial intermembrane space (IMS) by mediating the transfer of reducing equivalents from client proteins to FAD-linked oxidoreductases of the Erv1 family (lfALR in mammals). Here we investigate the specificity of the human Mia40/lfALR system towards non-cognate unfolded protein substrates to assess whether the efficient introduction of disulfides requires a particular amino acid sequence context or the presence of an IMS targeting signal. Reduced pancreatic ribonuclease A (rRNase), avian lysozyme, and riboflavin binding protein are all competent substrates of the Mia40/lfALR system, although they lack those sequence features previously thought to direct disulfide bond formation in cognate IMS substrates. The oxidation of rRNase by Mia40 does not limit overall turnover of unfolded substrate by the Mia40/lfALR system. Mia40 is an ineffective protein disulfide isomerase when its ability to restore enzymatic activity from scrambled RNase is compared to that of protein disulfide isomerase. Mia40's ability to bind amphipathic peptides is evident by avid binding to the isolated B-chain during the insulin reductase assay. In aggregate these data suggest that the Mia40/lfALR system has a broad sequence specificity and that potential substrates may be protected from adventitious oxidation by kinetic sequestration within the mitochondrial IMS.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States.
authors.forename: Devin A
authors.initials: DA
authors.lastname: Hudson
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States. Electronic address: cthorpe@udel.edu.
authors.forename: Colin
authors.initials: C
authors.lastname: Thorpe
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D003579:Cytochrome Reductases / Q000737:chemistry* / Q000648:ultrastructure*; D004789:Enzyme Activation; D006801:Humans; D007535:Isomerases / Q000737:chemistry* / Q000648:ultrastructure*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013329:Structure-Activity Relationship
chemical_list: C507282:CHCHD4 protein, human; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D016877:Oxidants; D003579:Cytochrome Reductases; C500897:GFER protein, human; D050862:Oxidoreductases Acting on Sulfur Group Donors; D007535:Isomerases
keywords: Augmenter of liver regeneration; Disulfide exchange; Mia40; Mitochondrial intermembrane space targeting sequence; Oxidative protein folding; Protein disulfide isomerase