X-linked IAP is a direct inhibitor of cell-death proteases

We show that human X-chromosome-linked IAP directly inhibits at least two members of the caspase family of celldeath proteases, caspase-3 and caspase-7

Quinn L. Deveraux; Ryosuke Takahashi; Guy S. Salvesen; John C. Reed

2002

Scholarcy highlights

  • To exclude contamination of genomic DNA, control cDNA reactions in which reverse transcriptase was omitted were prepared in parallel
  • For our initial investigation of human X-chromosome-linked IAP, we used recombinant XIAP protein and a cell-free system in which cytochrome c was added to cytosolic extracts
  • The partially processed caspase-3 seen in extracts treated with the combination of caspase-8 and Recombinant XIAP bound efficiently to immobilized glutathione S-transferase–XIAP, consistent with the idea that cleavage between the large and small subunit but not removal of the prodomain is required for protease activation
  • A XIAP-encoding cDNA was obtained by RT-PCR using a first-strand cDNA derived from Jurkat cells as the template and specific primers based upon Genbank accession number U32974
  • We found that the Mothers against dpp protein MH1 plus linker, expressed as a glutathione S-transferase fusion protein, bound DNA and protected a single interval within the quadrant enhancer in a DNase I footprinting experiment; GSTand the GST–MAD linker plus MH2 fail to bind DNA
  • To our knowledge for the first time, a mechanism of action for an IAP-family protein: namely, inhibition of active cell-death proteases

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