Title: Protein Archaeology: Using Reconstructed Ancestral Proteins to Understand the Evolution of Glucokinase Regulation by Its Regulatory Protein
Human glucokinase (GCK), the body’s glucose sensor in the liver and pancreas, is regulated via inhibition and sequestration by glucokinase regulatory protein (GKRP). GKRP is only able to bind to GCK’s super-open conformation, which can only be sampled at low glucose concentrations. Additionally, the strength of this inhibition is modulated by fructose-1-phosphate (F1P) and fructose-6-phosphate (F6P), although this is not true in all organisms. It is currently understood that GKRP arose from a bacterial peptidoglycan recycling enzyme, MurQ, via a gene duplication and unequal fusion event and that the binding site for F1P and F6P on GKRP was the active site of MurQ. How, then, did GCK and GKRP evolve this inhibitory protein-protein interaction wherein GKRP can only bind when glucose is low? And what causes the GKRPs of some organisms, such as tree frogs, to be insensitive to F1P and F6P while the GKRPs of other organisms, such as humans and rats, are sensitive? In this talk, I will discuss how ancestral sequence reconstruction (ASR) and experimental techniques are being used in the Miller lab to answer these questions. By reconstructing and characterizing select ancestral protein sequences along the GCK and GKRP lineages, we hope to identify the sequence changes that led to functional shifts in these proteins to give rise to their extant features.
Title: Additional Regulatory Role of 6-Phosphofructo-2-kinase/ Fructose 2, 6-Bisphosphatase?
The bifunctional enzyme 6-Phosphofructo-2-kinase/ Fructose 2, 6-Bisphosphatase (PFK2/FBPase2) is a key allosteric regulator of glycolysis and gluconeogenesis. The enzyme generates and degrades Fructose-2, 6-bisphosphate, which is responsible for aiding the progression of glycolysis and inhibition of gluconeogenesis. Recent studies in mammalian cells demonstrated that PFK2/FBPase2 is a cytosolic binding partner for glucokinase (GCK), the primary regulator of glucose homeostasis in the body. This observation suggests that the GCK:PFK2-FBPase2 interaction could play an important, but yet-to-be determined role in regulating central metabolism. The goal of my project is to reconstitute the GCK:PFK2-FBPase2 complex from purified components to facilitate structural and functional studies of the complex. In this presentation, I will provide an initial report on these efforts using both rat and human proteins.