University of Dundee

Applying quantitative proteomics to discover new components of glycosylphosphatidylinositol (GPI) biosynthesis.

This project will provide excellent training in parasite (trypanosome) cultivation and genetic modification, and state-of-the-art quantitative proteomics methodology.

Glycosylphosphatidylinositol (GPI) membrane anchors attach numerous eukaryotic cell surface proteins to the outer leaflet of the plasma membrane. The GPI-anchored protective variant surface glycoprotein (VSG) of Trypanosoma brucei (causative agent of human African trypanosomiasis) is essential for parasite survival and proliferation within the mammalian host, and GPI biosynthesis has been validated as a potential drug target. Despite conservation in the core structure of almost all GPI anchors across eukaryotic evolution, notable differences occur between T. brucei and mammalian GPI biosynthetic pathways, including fatty-acid remodelling of T. brucei anchors and the roles of inositol-acylation and de-acylation [1].

The aim of this project is to take new approaches to identify the proteins that catalyse key steps of GPI anchor biosynthesis in T. brucei. The basic idea is to epitope tag known components of the GPI biosynthetic pathway and perform pull-down experiments with antibodies to the tags and identify those proteins that are physically and specifically associated with the known components by SILAC proteomics [2]. Another approach will use the GPI-active cell-free system of washed trypanosome membranes. In this case we will perform chemical protein cross-linking followed by high-resolution gel-filtration in the presence of SDS to identify membrane-bound protein complexes [3]. This can be augmented by using the aforementioned epitope-tag pull-out methodology to enrich for GPI biosynthetic complexes. Some of the proteins we identify in these experiments are likely to be the unknown GPI pathway components because proteins involved in membrane-associated biosynthetic pathways tend to be present in complexes. Candidate genes that we identify using these proteomic approaches will be validated by the construction of conditional null mutants of these genes (see [10] for an example) to assess (i) whether they are essential to the parasite in vitro and/or in vivo, and therefore potential drug targets and (ii) to prepare cell-free systems from parasites where the proteins are depleted to provide biochemical evidence for the activity of the gene products using cell-free system radiolabelling/HPTLC methodology. Finally, recombinant protein will be made to test for the enzymatic activity using appropriate radiolabelled substrates generated in the cell-free system.

  1. Güther, ML and Ferguson, MAJ (1995) The role of inositol-acylation and inositol-deacylation in GPI biosynthesis in Trypanosoma brucei. EMBO J. 14, 3080-3093.
  2. Güther ML, Urbaniak MD, Tavendale A, Prescott A, Ferguson MA (2014) High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics. J Proteome Res 13, 2796-2806.
  3. Larance, M, Kirkwood MJ, Tinti, M, Alejandro Brenes Murillo, A, Ferguson, MAJ and Lamond, AI (2016) Global Membrane Protein Interactome Analysis using In vivo Crosslinking and MS-based Protein Correlation Profiling. Mol Cell Proteomics 15, 2476-2490.