Programme 4: Identifying the prion interactome in prion-infected cells

Aims

Whilst the aggregation of disease-specific proteins is a hallmark of neurodegenerative diseases, the associated perturbation of cellular pathways is ill-defined. A major drawback to better understand the consequences of protein aggregation is the lack of knowledge on molecular interactions between disease-associated and cellular proteins. This project aims to identify the interactome of disease-associated prion protein (PrPd) to better define the affected protein networks.

Methods

We recently developed cell models in our laboratory that show unprecedentedly large build-ups of PrPd. These hyper-infected cells are a favourable tool to identify protein interaction partners as PrPd is abundantly and continuously produced. Preliminary results demonstrated interaction of PrPd with proteins in membrane microdomains, such as GM1 and CD81. We will use immuno-isolation, chemical crosslinking and mass spectrometry for a deep analysis of the prion interactome. Validation of interacting proteins will be conducted by gene deletion and aided by confocal or superresolution microscopy. Mass spectrometry will be conducted in the laboratory of our collaborator. Extensive training will be provided in biochemical methods to identify interacting proteins, which include, but are not limited to co-immunoisolation, Western blotting, confocal and Superresolution microscopy.

Rotation project

A three-month project will enable the student to independently immuno-isolate protein complexes between PrPd and interacting proteins and begin their characterisation. By using denaturing and native polyacrylamide gel electrophoresis the student will learn how to optimise and validate experimental parameters, like protein cross-linking and immuno-isolation. The student will also learn techniques in molecular imaging and acquire competence in working in microbiological containment level 3 laboratories.

PhD project

The subsequent PhD studies will focus on identifying the prion-interacting network and defining how cellular pathways are perturbed by rogue proteins. As the prion protein resides in membraneous structures of the cell, this PhD project will also include study of membrane microdomains, important signalling platforms in cells, to define how prion formation interferes with physiological signalling cues.

Whilst a number of studies characterised the interactome of cellular PrP (PrPc), proteins that interact with PrPd are unknown. Identifying such protein interactions may not only help to improve the diagnosis of prion diseases, but may also provide novel avenues for therapeutic intervention. A recent study which systematically identified cellular factors impacting viral propagation by use of a proteomic-interactomic approach in HIV-infected cells (Luo et al. 2016; reference below) provides guidance and underscores the feasibility of such approaches.

References

Marbiah MM, Harvey A, West BT, Louzolo A, Banerjee P, Alden J, Grigoriadis A, Hummerich H, Kan HM, Cai Y, Bloom GS, Jat P, Collinge J, Klöhn PC (2014) Identification of a gene regulatory network associated with prion replication. EMBO J. 14, 1527-47.

Luo Y, Jacobs EY, Greco TM, Mohammed KD, Tong T, Keegan S, Binley JM, Cristea JM, Fenyo D, Rout MP, Chait BT, Muesing MA (2016) HIV-host interactome revealed directly from infected cells. Nature Microbiol. doi:10.1038/nmicrobiol.2016.68.