Characterization of a Gene Encoding Two Isoforms of a Mitochondrial Protein Up-regulated by Cyclosporin A in Activated T Cells

• Published in: The Journal of biological chemistry 2004-03, Vol.279 (11), p.10556-10563
• Main Authors: Mascarell, Laurent, Auger, Rodolphe, Alcover, Andres, Ojcius, David M., Jungas, Thomas, Cadet-Daniel, Véronique, Kanellopoulos, Jean M., Truffa-Bachi, Paolo
• Format: Article
• Language: English
• Subjects: Alternative Splicing; Amino Acid Sequence; Animals; Apoptosis; Base Sequence; Blotting, Western; Calcineurin; Calcineurin - metabolism; Cell Line; Cell Membrane; Cell Membrane - metabolism; CSTAD gene; cyclosporin A; Cyclosporine; Cyclosporine - pharmacology; Cytochromes c; Cytochromes c - metabolism; DNA, Complementary; DNA, Complementary - metabolism; Endopeptidase K; Endopeptidase K - pharmacology; FK506 protein; Flow Cytometry; Genome; Green Fluorescent Proteins; Humans; Immunology; Immunosuppressive Agents; Immunosuppressive Agents - pharmacology; In Vitro Techniques; Intracellular Membranes; Intracellular Membranes - metabolism; Life Sciences; Luminescent Proteins; Luminescent Proteins - metabolism; Lymphocyte Activation; Membrane Proteins; Membrane Proteins - biosynthesis; Membrane Proteins - genetics; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Mitochondria; Mitochondria - metabolism; Mitochondrial Proteins; Mitochondrial Proteins - biosynthesis; Mitochondrial Proteins - genetics; Molecular Sequence Data; Necrosis; Peptides; Peptides - chemistry; Plasmids; Plasmids - metabolism; Protein Isoforms; rapamycin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Messenger - metabolism; Subcellular Fractions; T-Lymphocytes; T-Lymphocytes - metabolism; Time Factors; Tissue Distribution; Transfection; Up-Regulation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M313770200

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.