Design and use of peptide-based antibodies decreasing superoxide production by mitochondrial complex I and complex II.
Mitochondria are the major source of reactive oxygen species. Both complex I and complex II mediate O2*- production in mitochondria and host reactive protein thiols. To explore the functions of the specific domains involved in the redox modifications of complexes I and II, various peptide-based antibodies were generated against these complexes, and their inhibitory effects were subsequently measured. The redox domains involved in S-glutathionylation and nitration, as well as the binding 2011. motif of the iron-sulfur cluster (N1a) of the complexes I and II were utilized to design B-cell epitopes for generating antibodies. The effect of antibody binding on enzyme-mediated O2*- generation was measured by EPR spin trapping. Binding of either antibody AbGSCA206 or AbGSCB367 against glutathione (GS)-binding domain to complex I inhibit its O2*- generation, but does not affect electron transfer efficiency. Binding of antibody (Ab24N1a) against the binding motif of N1a to complex I modestly suppresses both O2*- generation and electron transfer efficiency. Binding of either antibody Ab75 or Ab24 against nonredox domain decreases electron leakage production. In complex II, binding of antibody AbGSC90 against GS-binding domain to complex II marginally decreases both O2*- generation and electron transfer activity. Binding of antibody AbY142 to complex II against the nitrated domain modestly inhibits electron leakage, but does not affect the electron transfer activity of complex II. In conclusion, mediation of O2*- generation by complexes I and II can be regulated by specific redox and nonredox domains.
Kang
Patrick T
PT
Department of Integrative Medical Sciences, Northeastern Ohio Universities Colleges of Medicine and Pharmacy, Rootstown, OH 44272 USA.
Yun
June
J
Kaumaya
Pravin P T
PP
Chen
Yeong-Renn
YR
D020816:Amino Acid Motifs; D000818:Animals; D002417:Cattle; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D042963:Electron Transport Complex II / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D005260:Female; D008929:Mitochondria, Heart / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits
D024101:Mitochondrial Proteins; D010455:Peptides; D042963:Electron Transport Complex II; D042967:Electron Transport Complex I