Characterization of a gene encoding two isoforms of a mitochondrial protein up-regulated by cyclosporin A in activated T cells.
Cyclosporin A (CSA) is an immunosuppressor used in organ transplantation. A recent proteomic analysis has revealed that activation of T cells in the presence of CSA induces the synthesis of hundreds of new proteins. Here we used representational difference analysis to characterize some of the corresponding induced genes. After cDNA bank screening we focused on one of these genes, which we named CSA-conditional, T cell activation-dependent (CSTAD) gene. This gene produces two mRNAs resulting from alternative splicing events. They encode two proteins of 104 and 141 amino acids, CSTADp-S and CSTADp-L, for the short and long forms, respectively. FK506 had the same effect as CSA, whereas rapamycin did not affect the level of CSTAD gene expression, demonstrating that inhibition of the calcineurin activation pathway is involved in CSTAD gene up-regulation. CSA also led to overexpression of CSTAD in mice immunized in the presence of CSA, confirming the in vitro analysis. Microscopic and cytofluorimetric analysis of cells expressing green fluorescent protein-tagged CSTADp-L and CSTADp-S showed that both proteins colocalize with mitochondrial markers and depolarize the mitochondrial transmembrane potential without causing release of cytochrome c, apoptosis, or necrosis. Both CSTADp isoforms are sensitive to proteinase K, implying that they are located in the mitochondrial outer membrane. These data reveal a new mechanism of action for CSA, which involves up-regulation of a gene whose products are sorted to mitochondria and depolarize the mitochondrial membrane.
Mascarell
Laurent
L
Unité de Biologie des Populations Lymphocytaires, CNRS 2582, Institut Pasteur, Paris, France.
Auger
Rodolphe
R
Alcover
Andres
A
Ojcius
David M
DM
Jungas
Thomas
T
Cadet-Daniel
Véronique
V
Kanellopoulos
Jean M
JM
Truffa-Bachi
Paolo
P
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D019703:Calcineurin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019286:Endopeptidase K / Q000494:pharmacology; D005434:Flow Cytometry; D016678:Genome; D049452:Green Fluorescent Proteins; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D009336:Necrosis; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection; D015854:Up-Regulation
C484941:Cstad protein, mouse; D018076:DNA, Complementary; D007166:Immunosuppressive Agents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010455:Peptides; D020033:Protein Isoforms; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D016572:Cyclosporine; D045304:Cytochromes c; D019703:Calcineurin; D019286:Endopeptidase K