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Defining the role of the ubiquitin system in skeletal muscle atrophy

Principal Supervisor: Dr. Yu-Chiang Lai, School of Life Sciences, University of Dundee.

Co-supervisor: Prof. Gareth Lavery, Centre for Endocrinology, Diabetes and Metabolism (CEDAM), University of Birmingham

PhD project title: Defining the role of the ubiquitin system in skeletal muscle atrophy

University of Registration: University of Birmingham


Project outline:

Background: Muscle atrophy is a systematic shift of the balance between protein synthesis and degradation toward a catabolic state. While muscle protein synthesis is primarily dependent on activation of the phosphoinositide 3-kinase-Akt (also known as PKB) and mammalian target of rapamycin (mTOR) pathways, the molecular mechanisms controlling muscle protein degradation are less well defined (1). An important breakthrough in the field was the use of microarray technology identifying more than a hundred atrophy-related genes, termed ‘atrogenes’. Among the identified genes most were associated with the ubiquitin proteasome and lysosome autophagy pathways, suggesting that these processes are central to the atrophy response. Ubiquitylation was initially regarded as a defined means to degrade proteins, but is now considered the most versatile protein modification regulating almost all aspects of biology. The process of protein ubiquitylation is carried out by dedicated ATP dependent machinery consisting of E1 activating, E2 conjugating and E3 ligases. Unlike phosphorylation or other types of posttranslational modifications, ubiquitylation is much more complicated and can result in several possible topological appearances. Ubiquitin can be attached to substrate proteins at a single or multi lysines, termed monoubiquitylation. Ubiquitin can further modify itself at seven lysines or at the amino terminus to form poly-ubiquitin chains. Important to this proposal, the ubiquitylated proteins depending on its linkage-chain types are recognised by differential receptors that contain ubiquitin-binding domains or by specialised proteases, termed deubiquitylase, to remove ubiquitin modification. Each modification can function as a distinct signal to alter the biological outcomes.

Objectives and Methods: This project aims to understand how ubiquitin systems regulate muscle protein degradation under physiological and pathological conditions.

The first aim is to elucidate how two E3 ligases, MURF1 and MAFbx contribute to muscle atrophy. Initially we will generate stable muscle cell lines expressing MURF1 and MAFbx, respectively, and then applying affinity-mediated proteomics will identify downstream substrates and regulatory interactors. In parallel, we will deploy quantitative di-Gly capture proteomics in the generated stable cell lines to establish MuRF1 and MAFbx specific ubiquitylome database, a publishable resource for elucidating targets of the ubiquitin signalling under atrophy process. All potential substrates identified in these studies will then be validated in mouse models of muscle atrophy.

The second aim will be to identify novel E3 ligases and downstream effectors of ubiquitylation during muscle atrophy. In collaboration with Dr Satpal Virdee (University of Dundee) we have recently established an activity-based proteomics platform that allows us to identify active E3 ligases and deubiquitylase in cell lysates (2). The project will adopt this platform to identify specific E3 ligases in lysate from genetic mouse models of muscle atrophy or glucocorticoid treated mice.

 

References:

  • Cohen S, et al. (2015) Muscle wasting in disease: molecular mechanisms and promising therapies. Nature Reviews Drug Discovery 14(1):58-74.
  •  Pao KC, et al. (2016) Probes of ubiquitin E3 ligases enable systematic dissection of Parkin activation. Nature Chemical Biology 12(5):324-331.

 

BBSRC Strategic Research Priority: Molecules, Cells and Systems


Techniques that will be undertaken during the project:
  • Molecular cloning, gene transfection, and CRISPR/Cas9 knock out/in technology.
  • Protein expression, purification, characterisation, and chemical modification.
  • Biochemical and enzymatic assays
  • Manipulating genetic mouse model and muscle tissue isolation and incubation.
  • Scientific data analysis and bioinformatics

Contact: Dr Yu-Chiang Lai, School of Life Sciences, University of Dundee.