For cells and organisms to survive and grow it is critical that energy supply matches energy demand. The demand for energy is a continuously varying parameter; we are interested in studying the processes involved in synchronizing metabolic pathways (those that produce energy vs those that consume it) to maintain the perfect balance.
A major focus of our research is investigating regulation of an enzyme called AMP-activated protein kinase (AMPK). Analogous to a car’s fuel gauge, AMPK detects when energy in the cell is low and co-ordinates multiple branches of metabolism (e.g. fat burning, protein synthesis, digestion of cellular components) to redress energy imbalance. AMPK also has body-wide effects, being a key regulator of appetite and responsible for adaption to exercise. These roles have elevated AMPK to its current standing as an attractive drug target for diseases such as type 2 diabetes, cardiovascular disease and cancer.
AMPK represents a nexus in a complicated network of signalling pathways that act as ‘detection systems’, allowing the cell to sense environmental stresses and availability of nutrients. Our aim is to uncover how these signalling pathways interact, the components involved and how we can use them to exploit the therapeutic potential of AMPK. Working closely with the Protein Chemistry & Metabolism Unit we use a combination of biochemical and cell-based techniques, protein crystallography, medicinal chemistry, mass spectrometry and animal models to provide insight into the regulatory control of this important enzyme.
AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase consisting of an α catalytic subunit and regulatory β and γ subunits. Multiple isoforms exist for each subunit (α1/2, β1/2 and γ1/2/3) with each displaying tissue-specific expression profiles. Given the wide range of cellular effects attributed to AMPK it is unsurprising that the enzyme is subject to complex regulation, not only by adenine nucleotides but also other signalling networks. Phosphoproteomic analyses have identified >100 phosphorylation sites on AMPK, however understanding of the biological consequences of these phosphorylation events is limited to just a handful. For example phosphorylation of Thr172 in the activation loop of the α-subunit kinase domain by LKB1 or CaMKKβ activates the enzyme and facilitates signalling in response to increases in AMP/ATP ratio. Alternatively, phosphorylation of Ser485 in the α-subunit C-terminus by Akt (or by autophosphorylation) leads to suppression of Thr172 phosphorylation and down-regulation of AMPK signalling. Other phosphorylation sites are predicted to localize AMPK to specific cellular components or membranes, thereby conferring temporospatial specificity to AMPK signalling. Expanding upon the known signaling networks that communicate cellular state to AMPK is vitally important given its central role in energy homeostasis.
The molecule adenosine triphosphate (ATP) is regarded as the molecular unit of currency of intracellular energy transfer. It provides the energy used to drive virtually every cellular process, from muscle contraction to DNA synthesis. Human adults make ~50kg ATP daily due to rapid turnover to the low energy monophosphate form AMP. AMPK is able to sense elevations in AMP/ATP ratio (indicative of energy shortfall) via 3 nucleotide binding sites within its γ regulatory subunit, triggering phosphorylation of AMPK by upstream kinase LKB1 and CaMKK2 and subsequent AMPK signalling. We are interested in examining the molecular mechanisms by which this occurs.
An estimated 380 million people worldwide have type 2 diabetes. The metabolic dimensions of this disease, along with cardiovascular disease, obesity and cancer have encouraged efforts to develop small compound, direct-acting AMPK regulators as novel therapeutics. Crystal structures of AMPK/drug complexes have shown two distinct drug sites exist in the AMPK complex; one at an interface formed between the β-subunit carbohydrate binding module (CBM) and α-subunit kinase domain (occupied by drugs such as A-769662), the other within the γ-subunit (compound C2). Our research aims to understand how drug-binding at these sites leads to regulation of AMPK signalling, thereby driving development of treatments for metabolic diseases.