Research team

Mammalian thymus specific serine protease (TSSP): Development of tools to obtain structure-based insights into its biochemical functions. 01/10/2024 - 30/09/2028

Abstract

Thymus specific serine protease (TSSP) is a serine protease first identified in the late nineties. It is highly expressed in the thymus and barely detectable in other organs. TSSP is the third member of the S28 group of serine proteases, along with prolyl carboxypeptidase (PRCP) and dipeptidyl peptidase II (DPPII). It is named a serine protease because of its predicted enzymatic activity due to the presence of three amino acids (serine, aspartate and histidine) at positions highly similar to the ones forming the catalytic site of PRCP. PRCP cleaves off C-terminal amino acids adjacent to a proline, while DPPII only cleaves short peptides after proline at the second position starting from the N-terminus, hence the name 'dipeptidyl'-peptidase. This difference in substrate specificity makes it hard to predict the cleavage specificity of the third member, TSSP. Why should TSSP be studied further? In the thymus, TSSP is highly expressed by the cortical epithelial cells (cTEC's) and at lower levels by thymic dendritic cells. Since the functional T-cell repertoire is shaped in the thymus by positive and negative selection through interactions with Major Histocompatibility Complex (MHC)-peptide complexes expressed by cTECs and bone marrow derived antigen-presenting cells, TSSP could possibly be involved in this process. Peptides for loading on MHC class II molecules are generated by sequential proteolysis of endosomal proteins. Furthermore, during T-cell maturation in the thymus, massive cell death occurs in the cortical region as thymocytes that recognize self-antigens have to be deleted. The molecular mechanisms underlying this 'thymocyte cleanup campaign' and the high expression of TSSP in this region remain outstanding questions in the field. On the long term, a better knowledge on the structure-function relationship of TSSP may contribute to a better insight in the T-cell selection processes in the thymus. It is hypothesized that a primary function of TSSP is to somehow limit central tolerance to increase the diversity of the functional CD4+ T cell repertoire. Clearly, further characterization of TSSP's enzymatic activity is an essential next step in the search for its function in the shaping of the immune repertoire. In this basic science project we want to start the biochemical study of this hitherto uncharacterized protein by 1) structure-guided recombinant production, purification and quality control of recombinant mouse TSSP (rmTSSP) and human TSSP (rhTSSP); 2) characterization of TSSP substrate specificity and comparison with DPPII and PRCP and 3) selection and development of antibodies and inhibitors as further tools to study TSSP biology. To increase the chance of obtaining valuable antibodies we will use two approaches: i) selection of camelid single domain antibodies (sdAbs) from a library and ii) generation of classical monoclonal antibodies (mAbs). As a first step in the development of potent and selective inhibitors, we will screen a protease inhibitor library of small molecules to select a lead compound that can be used as a starting point for follow-up research. It is astonishing that this thymus specific enzyme has not yet been studied in more detail. One reason may be that the recombinant production of well-folded active TSSP could not be achieved to date. The Laboratory of Medical Biochemistry (LMB) has a longstanding expertise in studying the other members of the serine protease family S28. Therefore, there is a momentum to build on recent AlphaFold 2-based structural knowledge and experimental expertise on related proteins to definitely unravel the catalytic activity of TSS.

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  • Research Project