Proteomic screening for the identification of membrane proteins undergoing TMEM165-dependent endocytosis alteration

Details

Supervisors

Morsomme, Pierre

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

TMEM165 is a transmembrane protein localized in the Golgi apparatus of mammalian cells. This protein was discovered through mutations which were associated with type II congenital disorder of glycosylation, a rare genetic disease. The subsequent protein glycosylation defects in these patients were notably explained by the role of TMEM165 in the entry of Mn2+ into the Golgi apparatus, Mn2+ which is a cofactor for enzymes involved in the glycosylation process. Indeed, TMEM165 is a member of the GDT1 protein family and seems to act as a Ca2+/Mn2+:H+ antiporter. In addition, TMEM165 has been recently linked to cancer and more precisely, to migration. Its knockout was shown to significantly reduce the migratory and invasive properties of different cancer cell lines. Our current hypothesis is that the loss of TMEM165 leads to underglycosylation of plasma membrane proteins impacting their endocytosis and, as a result, their abundance at the cell surface. The latter would lead to alterations in cell adhesion and cell migration. This master thesis project was created to identify new cell surface proteins which are unproperly endocytosed due to glycosylation defects caused by TMEM165 knockout. As a result, these new proteins could be studied more thoroughly in the proposed mechanism linking TMEM165 and cancer cell migration. To identify endocytosis changes, cell surface proteins abundance was studied using mass spectrometry, as a disrupted endocytosis is indicated by significant differences in surface levels. To do so, cell surface proteins have been isolated via affinity chromatography in wild type, TMEM165 knockout and a rescue cell line expressing TMEM165 fused to mCherry in HeLa and HEK293T cells. Then, a reliable quantification method (to ensure that the same amount of samples was sent to the mass spectrometer) was used: silver staining gels. These samples were analyzed by mass spectrometry to determine which proteins are statistically more (or less) abundant on the cell surface in the three different conditions (wild type, TMEM165 knockout or rescue). Finally, the most interesting candidates have been validated using Western blotting (to detect glycosylation defects and validate the difference in abundance) and endocytosis assays (to check for a deficient abundance at the cell surface, correlated to endocytosis). The protocol of cell surface proteins isolation was validated and optimized notably using CD166 protein, showing glycosylation defects and enrichment in terms of abundance in the condition TMEM165 knockout of cell surface isolation samples. These observations could demonstrate a measurable impact of TMEM165 knockout on glycosylation, endocytosis and surface abundance of cell surface glycoproteins in HeLa and HEK293T cells. However, verification of cell lines could be essential to ensure the relevance and accuracy of these findings.