Search of Syndecan- 1 binders using quantitative proteomic approach

Abstract

Sindecans are a family of cell surface proteoglycans that are involved in numerous biological processes such as migration, proliferation and adhesion. Our group shows that the syndecan-1 has been found in soluble form in squamous cell carcinoma (SCC-9) when treated with phorbol ester (PMA). This increase in soluble form has been associated with reduction of syndecan-1 on the membrane. Interestingly, these cells had higher migration and reduced cell adhesion when compared to the untreated control. A new information found in this study was that the peptide derived from syndecan-1 was also found in greater abundance in the same cells, especially when treated with PMA. This peptide was in turn capable of inducing migration in HaCaT cells and SCC-9. Thus, in accordance with the literature data and the data shown in our study, it is believed that, among others, are two mechanisms regulating cell migration with respect to the syndecan-1 signaling pathway: the loss of syndecan-1 from the membrane, as weel as performed by the signaling peptide. Thus, this project aims to further the study of our group in order to evaluate the signaling mechanisms of syndecan-1 The strategy chosen to help explain this issue will be to identify partners ligands soluble syndecan-1 to evaluate the network of interactions syndecan-1. For this, HaCaT cells (not tumorigenic) and SCC-9 (tumorigenic) are metabolically labeled by Stable Isotope Labeling by-Amino Acids (SILAC), transiently transfected with syndecan-1, and then activated (PMA) . The complexes of syndecan-1 + binders soluble in the culture medium will be immunoprecipitated and the identification thereof is performed by quantitative proteomics. To validate the identity of these complexes will be performed Western blot with antibody specific for each ligand and experiments of co-localization. Once confirmed partners of syndecan-1, a few will be chosen to characterize the interaction by solid phase assay and chemical cross-linking followed by mass spectrometry. Next, the functional role of this interaction is studied using loss-of-function IRNA ligand of interest in migration and adhesion assays. It is hoped that this project will contribute to the knowledge of the signaling mechanisms of syndecan-1 by the knowledge of their partners binders.