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Affinity - and chemical-based proximaty labelling method to study protein-protein interaction in vivo

Primary Supervisor: Dr Yu-Chiang Lai, School of Sport, Exercise and Rehabilitation Sciences

Secondary supervisor: Professor Gareth Lavery

PhD project title: Affinity- and chemical-based proximaty labelling method to study protein-protein interaction in vivo

University of Registration: University of Birmingham

Project outline:

Protein-protein interaction, a fundamental basis of cellular signalling events, regulates all aspects of biological functions, including growth, development, and metabolism. Therefore, defining specific protein-protein interactions will improve our understanding of how cellular and disease processes are regulated. However, obtaining those information is challenging mainly due to limited methodology developed. In this PhD research proposal, the student will apply cutting-edge biological techniques and advanced proteomics to develop new experimental procedures for exploring protein-protein interactions in living cells.

Affinity purification/mass spectrometry (AP/MS), e.g. immunoprecipitation or single/tandem-bait pulldown with subsequent mass spectrometery analysis, has been commonly used as a classic approach to study protein interactions and complexes. Depite its utility in identifying interactions, this approach is non-physiological and is limited to high-affinity interactions, often missing weak and transient interactions. Recent technological innovations in proximity labelling techniques, such as TurboID and mini-TurboID [1], overcome the deficiencies of AP/MS and provide the optimal sensitivity needed to identify protein interactions and complexes in a native, living cell environment. The TurboID and mini-TurboID techniques employ an engineered enzyme to covalently tag “biotin” to adjacent proteins, which allows the enrichment and identification of protein complexes using orbitrap mass spectrometory proteomics. 

The PhD research aims to develop new methologies for studying protein-protein interaction by merging affinity-based or chemical-inducible binding approaches with proximity labelling techniques. For the affinity-based binding approach, we will first fuse eGFP (enhanced Green Fluorescent Protein) to the protein of interest (bait protein) using CRISPR-Cas9 technology or virus-based overexpression systems. Secondly, we will generate an inducible cell lines that express TurboID fused with eGFP-nanobodies. The affinity-based binding between eGFP and eGFP nanobody will bring TurboID (the labelling system) and the bait protein together, facilitating TurboID’s labelling of the bait protein complex. Meanwhile, we will couple the proximity labelling system with chemical-inducible binding systems as a molecular switch to form induciable dimerization. For example, the bait protein will be fused with an FRB domain; the TurboID will be fused with FKBP. Both fusion proteins will be expressed in cells. Treating cells with rapamycin will induce binding of these two fusion proteins (FRB and FKBP) together before turboID executes its labelling function. These developments are expected to improve the temporal and special resolutions of identifying protein-protein interactions; this will facilitate the identification of dynamic changes in protein interactions and complexes over time or in response to cellular perturbation.

The prospective student will initially develop the methologies using muscle atrophy F-box (MAFbx, also known as FBXO32; a protein implicated in muscle atrophy) as a bait protein to produce a publishable  proof of concept study. This target was chosen because MAFbx is a ubiquitin E3 ligase and its ligase activity is known to function in complex with multiple proteins (such as Cullin1, RBX1, SKP1, CDC34 (E2) and NEDD8). We will then apply quantitative protemics and classic biochemical pulldown assays to confirm the candidate proteins presence in complex with MAFbx. In the second phase, the student will aim to validate and confirm newly identified, novel MAFbx interactors identified in the proteomic analysis. Finally, we will aim to understand the functional role of novel interactors that bind with MAFbx. While working on this project, the PhD candidate will develop an interdisciplinary skill set of molecular biology, chemical biology, biochemistry, quantitative proteomics, data analysis, and interpretations.

References:

  1. Branon et al. Efficient proximity labeling in living cells and organisms withTurboID. Nat Biotechnol. 2018, 36 (9):880-887

BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing

    Techniques that will be undertaken during the project:

    • Molecular cloning, gene transfection (such as virus-based overexpression system or CRISPR/Cas9-based knock-in technology.
    • Protein expression, purification, characterisation, and chemical modification in mammalian cells.
    • Biochemical and enzymatic assays
    • Scientific data analysis, bioinformatics, integrative thinking and interpretations

    Contact: Dr Yu-Chiang Lai, University of Birmingham