Glucose Sensing by AMPK

AMP-activated protein kinase (AMPK) is an energy-sensing protein that originated in single-celled eukaryotes to promote survival during starvation and evolved in multicellular organisms to regulate cellular metabolism and energy status (Hardie et al., 2012). The aberrant regulation of AMPK has been associated with chronic disease states, including cancer, type 2 diabetes and inflammation, and, as such, AMPK has become a key drug target (Hardie and Ashford, 2014). Activation of AMPK occurs both allosterically by AMP and by phosphorylation of Thr172, located within the alpha-catalytic domain. Interestingly, recent evidence has demonstrated non-canonical activation of AMPK, where short term glucose deprivation activates AMPK at the lysosome, independently of changes in nucleotide ratios (Zhang et al., 2017). Glucose deprivation is sensed by the enzyme aldolase via a decrease in its substrate, the glycolytic intermediate F-1,6-BP, which relays this signal to the lysosomal v-ATPase, resulting in interaction between Lamtor1 and AXIN1-LKB1 on the lysosome surface and recruitment and activation of AMPK (Zhang et al. 2013, 2014, 2017). At present, the precise mechanisms of this regulation remain unknown and require further investigation as their elucidation may have implications in designing new drug therapies targeting type 2 diabetes. The PhD project involves investigating the mechanisms by which AMPK localises to the lysosome upon myristoylation of its beta-subunit during glucose starvation, interacts with AXIN-LKB1, and dissociates from the lysosome, in addition to investigating potential cross-talk between AMPK and mTORC1. This will be studied using techniques of CRISPR/nickase genome editing, tissue culture, fluorescence confocal microscopy, kinase assays, nucleotide analysis, cloning, mass spectrometry, and protein biochemistry.
Key Words and Skills:
AMPK, glucose sensing, fructose-1,6-bisphosphate, aldolase, lysosome, Lamtor1, AXIN, LKB1
CRISPR/Nickase genome editing, tissue culture, fluorescence confocal microscopy, kinase assays, nucleotide analysis, cloning, mass spectrometry, and protein biochemistry.
1. Explain interdisciplinary interface:
Project involves advanced cell therapies, including gene editing and cloning, protein biochemistry techniques, including kinase assays to measure enzyme activity, size exclusion chromatography and immunoprecipitation to investigate protein-protein interactions, immunoblotting to investigate the effect of treatments on the behaviour of proteins, protein purification techniques, capillary electrop electrophoresis for nucleotide analysis, and fluorescent confocal microscopy to investigate AMPK localisation. Project and AMPK related science communicated at conferences, seminars and symposiums via poster and oral presentations. Public health engagement participation to educate the general public (including adults and children) on the positive benefits and impact of science within society. Collaboration with Xiamen University, requiring inter-lab discussions, presentations and training to perform technical methods. Opportunity to attend specific skill training courses and to be taught on specific techniques by other departments/institutes.
2. Does project require significant amount of quantitative skills?
Substantial use of bioinformatics to analyse and interpret proteomic and genetic data. Use of analytical systems to perform and analyse data for kinase assays, immunoblotting, mass spectrometry, immunocytochemistry, protein purifications, gene sequencing, cell sorting, and capillary electrophoresis. Compulsory statistical analysis modules, detailing appropriate use of statistical tests, handling of data, and analysis of data using different programmes, including R-Studio
3. Does project require significant amount of whole organism physphysiology skills? NO