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Role of the pro-apoptotic pyrin-interacting protein Siva in the pathogenesis of familial Mediterranean fever
  1. D Vassou1,
  2. E Petraki1,
  3. E Eliopoulos2,
  4. D Iliopoulos3,
  5. I Aksentijevich4,
  6. P Sidiropoulos5,
  7. D Kardassis6,
  8. D T Boumpas1,5,
  9. G N Goulielmos1
  1. 1Laboratory of Molecular Medicine and Human Genetics, Department of Medicine, University of Crete, Heraklion, Greece
  2. 2Laboratory of Genetics, Agricultural University of Athens, Greece
  3. 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA
  4. 4Genetics Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, USA
  5. 5Department of Rheumatology, Clinical Immunology and Allergy, University Hospital of Heraklion, Greece
  6. 6Department of Biochemistry, University of Crete Medical School, Heraklion, Crete

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Familial Mediterranean fever (FMF) is an autosomal recessive disease attributed to mutations in the MEFV gene encoding pyrin, which is characterised by recurrent, acute and self-limiting attacks of fever. Most disease-associated mutations in the MEFV gene reside on the C-terminal PRYSPRY (B30.2) domain of pyrin, an area found to interact with the pro-apoptotic protein Siva.


(1) To define the topology of the (full-length) Siva-1 with respect to its interaction with pyrin and other interacting proteins; (2) to investigate the ability of mutant forms of pyrin to interact with Siva-1; and (3) to explore the potential increase of GITR/Siva-induced apoptosis in the presence of the mutant forms of pyrin.

Materials and Methods

A three-dimensional model of Siva-1 was created with MODELLER and a Swiss model automated protein structure homology-modelling server. HEK293 (human embryonic kidney) and THP-1 (human monocytic leukaemia) cell lines were used for transfection experiments using Lipofectamine 2000 or the Amaxa nucleofection technology, respectively. Apoptosis was measured by FACS-Annexin-PE staining using the Annexin-V kit (Pharmingen).


The authors constructed a three-dimensional model of Siva-1 and three distinct domains were defined, an N-terminal domain containing amphipathic helices with potential to interact with the PRYSPRY domain of pyrin, a B-box-like domain and a zinc finger-like C-terminal domain that the authors assume to bind the cytoplasmic tail domain of GITR protein. In immunoprecipitation experiments on transfected HEK293 cells, Siva bound both to wild-type pyrin and its mutant forms, but no differences were observed in the rates of apoptosis in THP-1 upon transfection with either wild-type pyrin or mutant forms of pyrin. Patients with FMF did not display any mutations in the Siva gene.


The three-dimensional model of Siva-1 constructed showed that GITR and pyrin do not antagonise for the same binding site(s). The data obtained by using various mutants of pyrin and Siva-1 for cotransfecting HEK293 cells suggest that the positions of the PRYSPRY domain, found mutated in FMF patients, are not required for binding to Siva-1. The authors were not able to demonstrate an increase in GITR/Siva-induced apoptosis in the presence of the mutant forms of pyrin, thus suggesting that Siva is unlikely to contribute to the pathogenesis of FMF through the postulated molecular mechanism.

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