Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD369↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.

We thank Ties Koopmans for the generation of several of the PM/Scl-75 mutants, Dr J. Reed (Burnham Institute, La Jolla, CA, USA) for the Jurkat/Neo and Jurkat/Bcl-2 cell lines, Dr M. Robertson (Indiana University, Bloomington, IN, USA) for the anti-Fas mAb 7C11, Dr J. Blenis (Department of Cell Biology, Harvard Medical School, Boston, MA, USA) for the caspase-8-deficient Jurkat cells and Dr J. Wilusz and Dr D. Mukherjee (UMDNJ New Jersey Medical School, Newark, NJ, USA) for the anti-PM/Scl-75 polyclonal mouse and rabbit antibodies. This work was supported in part by the Netherlands Organization for Scientific Research (NWO-CW). The work of LVW, XS and PV is supported by the Interuniversitaire Attractiepolen (IUAP-V), the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (grants 31.5189.00 and 3G.0006.01) and the EC-RTD (grant QLRT-CT-1999-00739), the Ghent University cofinanciering European Union project (011C0300) and GOA project 12050502. The work of LVW was also supported by the Instituut voor aanmoediging van Innovatie door Wetenschap en Technologie-Vlaanderen.

We thank Ties Koopmans for the generation of several of the PM/Scl75 mutants, Dr J. Reed (Burnham Institute, La Jolla, CA, USA) for the Jurkat/Neo and Jurkat/Bcl2 cell lines, Dr M. Robertson (Indiana University, Bloomington, IN, USA) for the antiFas mAb 7C11, Dr J. Blenis (Department of Cell Biology, Harvard Medical School, Boston, MA, USA) for the caspase8deficient Jurkat cells and Dr J. Wilusz and Dr D. Mukherjee (UMDNJ New Jersey Medical School, Newark, NJ, USA) for the antiPM/Scl75 polyclonal mouse and rabbit antibodies. This work was supported in part by the Netherlands Organization for Scientific Research (NWOCW). The work of LVW, XS and PV is supported by the Interuniversitaire Attractiepolen (IUAPV), the Fonds voor Wetenschappelijk OnderzoekVlaanderen (grants 31.5189.00 and 3G.0006.01) and the ECRTD (grant QLRTCT199900739), the Ghent University cofinanciering European Union project (011C0300) and GOA project 12050502. The work of LVW was also supported by the Instituut voor aanmoediging van Innovatie door Wetenschap en TechnologieVlaanderen.