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Structure and function of novel tetraspanin complexes involved in autophagy and biogenesis of exosomes

Principal Supervisor: Dr Fedor Berditchevski, Institute of Cancer and Genomic Sciences

Co-supervisor: Dr Fiyaz Mohammed

PhD project title: Structure and function of novel tetraspanin complexes involved in autophagy and biogenesis of exosomes

University of Registration: University of Birmingham

Project outline:

This project offers the opportunity to elucidate the structure and molecular interactions of a novel tetraspanin complex which may link autophagic pathway with intercellular communications in normal tissues and in various pathological conditions. The tetraspanin superfamily includes 33 human proteins, and mediates the development of nervous and immune systems, infectious disease, fertilization and cell adhesion and motility. Tetraspanin proteins are known to facilitate assembly of various membrane complexes that play a critical role in intracellular signalling, endocytic trafficking and in defining a composition of cell-derived vesicles that function as signalling organelles supporting communication between various cell types.

We discovered that tetraspanin proteins form novel complexes which include proteins highly relevant to autophagy and production of exosomes. These complexes consist of four types of proteins: tetraspanins that are directly linked to either a cytoplasmic adaptor protein called syntenin-1 or to the proteins of the LC3 family. The syntenin-1 facilitates multiple interactions within tetraspanin-containing complexes thus serving as a critical molecular hub which regulates various cellular processes involving the endocytic vesicular trafficking and production of exosomes. These pathways rely on ubiquitin-binding cellular proteins, which interact with syntenin-1 in a unique manner involving both C- and N-termini of the protein. However, our understanding of the mechanisms that control assembly of the tetraspanin-syntenin-1 complexes remains in its infancy, with no detailed structural information available which would allow us to intervene with their function in tetraspanin-driven pathological conditions.

We have already established that syntenin-1 is phosphorylated by Src and Ulk1 kinases and that the phosphorylation of syntenin-1 plays an important role in regulation of the assembly of the tetraspanin-syntenin-ubiquitin complex. These kinases also control the assembly of a new class of tetraspanin complexes involving several members of the LC3 family. Since LC3 proteins are known to have a critical role in autophagy, these novel observations identify tetraspanins and syntenin-1 as key decision making molecular switches that control a previously unsuspected link between autophagy and production of exosomes.

Thus, the proposed project give an excellent opportunity to examine the structural-functional relationship between the newly identified molecular complexes and their phosphorylation-controlled dynamics and autophagy and exosome secretion, two key physiological processes. Detailed structural characterisation of the tetraspanin-based molecular complexes will involved small-angle scattering of X-rays (SAXS), X-ray crystallography and nuclear magnetic resonance (NMR). Complementary to this, a mass spectrometry based approach will be employed to identify cellular proteins that bind both phosphorylated and non-phosphorylated forms of syntenin-1. We predict that phosphorylation of syntenin-1 will affect the ability of the protein to recruit ubiquitylated cellular partners (see above). The role of ubiquitination in the functional assembly of the tetraspanin-syntenin-1 complexes in protein trafficking and methods to disrupt this key interaction will be explored using confocal microscopy and life cell imaging.

BBSRC Strategic Research Priority: Understanding the rules of life: Structural Biology

Techniques that will be undertaken during the project:

Eukaryotic cell biology, Flow cytometry, Recombinant protein expression and purification, SDS-PAGE, pull-down assays, western blotting, immunocytochemistry, mass spectrometry, Surface Plasmon resonance, NMR spectroscopy, X-ray crystallography, small angle X-ray scattering, culturing human cells, confocal microscopy, live cell imaging.

Contact: Dr Fedor Berditchevski, University of Birmingham