Title of project

Metabolite regulators of disease-relevant signaling proteins

Abstract

Small molecule metabolites are conventionally viewed as the elementary constituents of life, functioning as cellular building blocks, energy currency, and waste products. Beyond anabolic and catabolic metabolism, it is increasingly evident that metabolites serve as signalling molecules, directly regulating protein function to provide a rapid mechanism for the metabolic state to coordinate cellular processes that are energetically or biosynthetically demanding. Furthermore, metabolism internally balances influx and efflux through evolved metabolic surveillance, more specifically, metabolite-mediated orthosteric and allosteric regulation of enzyme activity. Although regulatory protein-metabolite interactions (PMIs) have been observed, the true scale of the protein-metabolite interactome is almost completely unknown and represents a major void in our understanding of cellular biology. Furthermore, extrapolations from multiple investigations suggest that the protein-metabolite interactome is immense (1-5). Towards this goal, we developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify PMIs (1, 6). The MIDAS platform is uniquely situated to systemically identify low millimolar and stronger binding between any soluble protein and any metabolite from a multiplexed metabolite library representative of the human metabolome. As such, the MIDAS platform is fully capable of revealing the set of interactions between the metabolome and proteome. We propose to leverage the power of MIDAS to reveal protein-metabolite interactions in two main areas. First, we propose to discover new metabolite interactions for the AMP-activated protein kinase (AMPK) trimeric complex. This protein has enormously important roles in controlling cellular and organismal energy homeostasis. It has primarily been believed to be a sensor of metabolic status via sensing adenine nucleotides (ATP, AMP). We propose, and this is supported by much recent data, that AMPK integrates other metabolic signals. We propose to discover the metabolite interactions underlying this signalling and determine how these interactions impact AMPK function. Second, we propose to leverage our newly implemented MIDAS augmentation, wherein we can analyze complicated membrane proteins, to discover unexpected interactions with G-protein coupled receptors (GPCRs). These proteins are well-appreciated to serve as sensors of metabolites and small- and macro-molecular hormones, but many GPCRs remained orphaned without known ligands. We propose to discover ligands for these as well as cooperating regulators that may act allosterically at distinct sites. In both cases, the discovery of these regulator ligands will inform our basic understanding of how metabolic signals are conveyed and received, but it will also inform the development of novel therapies that mimic or prevent action at novel regulatory sites.