Overexpressed human mitochondrial thioredoxin confers resistance to oxidant-induced apoptosis in human osteosarcoma cells.
Oxidative damage to mitochondria is a central mechanism of apoptosis induced by many toxic chemicals. Thioredoxin family proteins share a conserved Cys-X-X-Cys motif at their active center and play important roles in control of cellular redox state and protection against oxidative damage. In addition to the well studied cytosolic and extracellular form (Trx1), rat and avian mitochondrial forms of thioredoxin (mtTrx) have been reported. In this study, we cloned the full-length human mtTrx cDNA and performed localization and functional studies in 143B human osteosarcoma cells. The coding sequence of human mtTrx consists of a region with homology to Trx1 as well as a putative mitochondrial localization signal (MLS) at its N terminus. In stably transfected cell lines, mtTrx had a mitochondrial localization as measured by subcellular fractionation studies and by confocal fluorescence microscopy. Deletion of the MLS rendered mtTrx to be solely expressed in the cytosolic fraction. On SDS-PAGE, transfected mtTrx had the same apparent molecular weight as the MLS truncated form, indicating that the leader sequence is cleaved during or after mitochondrial import. Treatment with the oxidant tert-butylhydroperoxide induced apoptosis in 143B cells. This oxidant-induced apoptosis was inhibited by overexpressing the full-length mtTrx in 143B cells. Thus, human mtTrx is a member of the thioredoxin family of proteins localized to mitochondria and may play important roles in protection against oxidant-induced apoptosis.
Chen
Yan
Y
Program of Biochemistry, Cell, and Developmental Biology, Emory University, Atlanta, Georgia 30322, USA.
Cai
Jiyang
J
Murphy
T J
TJ
Jones
Dean P
DP
D000595:Amino Acid Sequence; D017209:Apoptosis; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D016923:Cell Death; D002470:Cell Survival; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D016877:Oxidants / Q000037:antagonists & inhibitors*; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013053:Spectrophotometry; D013347:Subcellular Fractions; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D000939:Epitopes; D016877:Oxidants; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins