Primary Supervisor: Dr Yu-Chiang Lai, School of Sport, Exercise and Rehabilitation Sciences
Secondary supervisor: Gareth Wallis
PhD project title: Identification of ubiquitin modifying enzymes as new exercise signalling mediators
University of Registration: University of Birmingham
The overarching aim of this PhD project is to understand how exercise improves skeletal muscle health at a molecular level. Protein phosphorylation and ubiquitylation are key post-translational modifications that regulate almost all aspects of biological function. There are considerable research interests on ubiquitin-modifying enzymes, such as MuRF1, Atrogin1 and USP19, in muscle atrophy. However, few information is known about which E3s/DUBs account for the beneficial effects of exercise, what are their molecular partners and signalling pathways. This project will specifically aim to fill these knowledge gaps by investigating some of the potential candidates that have been identified from our previous works (e.g. RNAseq and proteomics)
Using unbiased screening approaches, our lab has identified a list of E3s/DUBs (more than 15 candidates), whose gene and/or protein expressions increased in exercised human skeletal muscle. To better understand their translational and functional roles, the student will first verify their gene and protein expression and study their localisation in exercised human skeletal muscles (Objective 1). The student will also aim to test whether genetic manipulation of these enzymes can replicate the beneficial effects of exercise in cells and mouse muscles (Objective 2 and 3). Findings are expected to open new research avenues and accelerate the discovery of pharmaceutical targets for improving metabolic health.
Exercise helps maintain muscle health and is known as a preventative/interventional medicine to ameliorate ageing and metabolic diseases. Molecular signalling in muscle have long been a topic of intense research. Research over the past 30 years focused on protein phosphorylation. Now we have considerable knowledge of protein kinases- and phosphatases-dependent signalling pathways. In contrast, protein ubiquitylation, a crucial and versatile signalling machinery in cells, remains poorly understood, especially in relation to exercise. An increasing number of E3s/DUBs are reported to regulate myogenesis, muscle development, function and atrophy but none was studied in an exercise model.
In order to facilitate the pending breakthrough in the field, my lab has taken a number of unbiased approaches and identified a list of E3 ligases/DUBs, whose gene and/or protein expressions are up-regulated in responding to an aerobic exercise (cycling, 70% VO2max for 45min). We hypothesise that some of these candidates will account for the beneficial effects of exercise from a ubiquitin signalling point of view.
Our lab has been developing and collating a range of highly effective experimental tools for gaining mechanistic insights on the role of ubiquitin-modifying enzymes. For example, we established and successfully validated a state-of the-art CRISPR/Cas9 technology for generating gene knockout and knock-in cells, which will be crucial for this project. We also plan to use electroporation, a highly innovative and specialised gene delivery/overexpression technique to test functional outputs in mouse muscle.
Objective 1. To validate gene and protein expression and localisation of the E3s/DUBs in human muscles.
Objective 2. Loss-of-function knockout in muscle cells to investigate the E3-/DUB-mediated metabolic functions.
Objective 3. Gain-of-function overexpression to investigate E3-/DUB-mediated phenotypes and functions in mouse muscles.
- Nishimura Y, Musa I, Holm L, Lai YC. (2021) Recent advances in measuring and understanding the regulation of exercise-mediated protein degradation in skeletal muscle. Am J Physiol Cell Physiol 321(2): C276-C287 (*corresponding author).
BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing & Diet and Health
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
- Scientific data analysis and bioinformatics
Contact: Dr Yu-Chiang Lai, University of Birmingham