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Study of the TSSP protease and its role in the diabete

Heads Christine KELLENBERGER

Coordinatrice: C. Kellenberger

The functional T cell repertoire is shaped in the thymus by positive and negative selection through interactions withMHC/peptide complexes expressed by thymic epithelial cells (TEC) and bone marrow-derived antigen (Ag) presenting cells. The size of the CD4 T cell compartment and its diversity are determined by the complexity of the self-peptide repertoire presented by MHC class II molecules in the thymus. Several cysteine and aspartic proteases have been involved in the generation of MHC class II/ peptide complexes, but indirect evidence suggest that serine and metalloproteases may also participate to Ag processing. In 1999, our collaborator A. Carrier identified a novel serine protease named Thymus Specific Serine Protease (TSSP) that is highly expressed in the endosomal compartment of TECs [Carrier et al., 1999]. In addition, TSSP was also initially described as a gene linked to a diabetes susceptibility locus in Human [Lie et al., 1999]. Our collaborators A. Carrier and S. Guerder showed that TSSPP is necessary to the positive selection of some CD4 T cell specificities [Gommeaux et al., 2009].

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Figure 1

The limited impact on the development of CD4 T cells expressing polyclonal TCR suggests a role for TSSP in the edition of the T cell repertoire in the thymus (Figure 1). Since TSSP was linked to a diabetes susceptibility locus, the study was carried on with NOD mice (a model for spontaneous insulin-dependent diabete). Studies showed the role of TSSP in the development of a functional CD4 repertoire in these mice, in the case of the antigen HEL (Hen Egg Lysozyme) [Viret et al., 2011]. Another recent study of our collaborators showed that TSSP-deficient NOD mice (TSSP-/-) are completely protected from diabete and insulitis l’insulite [Viret et al., 2011b] (Figure 2). The goal of the current studies is the characterization of TSSP substrates, one of which is an epitope of Phogrin, a membrane protein of Langerhans islet of the pancreas. TSSP is therefore the first example of a serine protease that plays a role in establishing the functional CD4 repertoire through its implication in editing the peptide repertoire in the thymus.

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Figure 2

Characterization of the structure and of the molecular function of TSSP

TSSP is a serine protease belonging to the S28 family, which contains the lysosomal prolylcarboxypeptidase (PRCP), also called angiotensinase C and which is involved in the activation of pre-kallikrein in endothelial cells and DPPII/VII, a peptidase expressed in human lymphocytes and that is thought to regulate the activation of T cells and survival. These proteases are studied at the biochemical, enzymatic and structural levels [Sharia-Madar et al., 2005] [Leiting et al., 2003]. On the contrary, this is not the case for TSSP. The first step is the production of recombinant protein. For this purpose, we have set up the mammalian cell expression system. First results were recently obtained on the expression of TSSP in HEK293 and HEK293-EBNA cells.
Functional studies will be carried out in order to characterize the enzymatic activity of TSSP and to define its substrates. Our collaborators use mainly two approaches: double-hybrid strategy and characterization of the functional CD4 repertoire of TSSP deficient mice. Natural identified substrates will be tested with the recombinant protein. It may be necessary to produce the substrates in E. Coli or in eukaryotic cells. In parallel, commercial substrates will also be tested. Structural study of TSSP will be done using X ray crystallography.

Footnotes


[Carrier et al., 1999] Carrier A, Nguyen C, Victorero G, Granjeaud S, Rocha D, Bernard K, Miazek A, Ferrier P, Malissen M, Naquet P, Malissen B, Jordan BR (1999) Immunogenetics 50 255-70

[Lie et al., 1999] Lie BA, Todd JA, Pociot F, Nerup J, Akselsen HE, Joner G, Dahl-Jorgensen K, Ronningen KS, Thorsby E, Undlien DE (1999) Am J Hum Genet 64 793-800

[Gommeaux et al., 2009] Gommeaux J, Gregoire C, Nguessan P, Richelme M, Malissen M, Guerder S, Malissen B, Carrier A (2009) Eur J Immunol 39 956-64

[Viret et al., 2011] Viret C, Lamare C, Guiraud M, Fazilleau N, Bour A, Malissen B, Carrier A, Guerder S (2011) J Exp Med 208 3-11

[Viret et al., 2011b] Viret C, Leung-Theung-Long S, Serre L, Lamare C, Vignali DA, Malissen B, Carrier A, Guerder S (2011) J Clin Invest 121 1810-21

[Sharia-Madar et al., 2005] Shariat-Madar Z, Rahimy E, Mahdi F, Schmaier AH (2005) Am J Physiol Heart Circ Physiol 289 H2697-703

[Leiting et al., 2003] Leiting B, Pryor KD, Wu JK, Marsilio F, Patel RA, Craik CS, Ellman JA, Cummings RT, Thornberry NA (2003) Biochem J 371 525-32
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