My laboratory is interested in the reciprocal coupling of core metabolism and other cellular processes. Metabolic and nutrient status elicit substantial effects on a wide array of cellular biologies, including cell growth, cell division, protein synthesis and many others. Conversely, many cellular signaling pathways exert control on important metabolic decisions. Our broad goal is to begin to understand how this crosstalk occurs in normal situations and how its impairment is involved in disease states. Our present focus is on how the availability and quality of nutrients and energy effect cellular decisions, and how these cellular decisions then determine the use of available nutrients and energy (see Lindsley and Rutter, 2004). Our research currently centers in three areas: First, we are examining the regulation and function of PAS kinase, a protein we think participates in this system by sensing and signaling nutrient status. Second, we have started a new project to study the interplay between the mitochondria and the rest of the cell as it relates to metabolism and oxidative stress. Third, we are working to develop new technologies for the discovery of regulatory small molecule-protein interactions.
PAS kinase regulation
PAS kinase function in yeast
PAS kinase function in mammals
Mitochondria
Allostery
We have found that nutrient status regulates PAS kinase activity both in mammalian and yeast cells. Specifically, PAS kinase is activated in cultured b-cells grown in elevated glucose (daSilva Xavier, et al. 2004), likely as a result of increased mitochondrial metabolism. Similarly, growth of yeast under conditions that require mitochondrial respiration (growth on non-fermentable carbon sources) also substantially activates PAS kinase (Grose, et al. 2007). We have also found that PAS kinase is an integral component of a system that maintains cell structural integrity (see below), and as such is activated by cell membrane stress (Grose, et al. 2007 and unpublished). Using both yeast and mammalian cells as model systems, we are trying to identify the factors that regulate PAS kinase, both transcriptionally and post-translationally.
Cells must adapt flux through different metabolic pathways to meet demand for the different resultant products. In yeast, one example of such an adaptation is the differential partitioning of glucose, in the form of UDP-glucose, to either storage carbohydrate (primarily glycogen) or structural carbohydrate (primarily cell wall material). We have identified a system that, in response to demand for cell wall production, shifts glucose partitioning from glycogen into cell wall material. We have identified two components of this system. PAS kinase is activated under conditions of cell membrane stress (interpreted by the cell as cell wall insufficiency) and phosphorylates Ugp1 (Rutter, et al. 2002a), the enzyme that produces UDP-glucose from UTP and glucose-1-phosphate. Interestingly, phosphorylation does not affect the ability of Ugp1 to catalyze the production of UDP-glucose, but instead determines its fate (Smith and Rutter, 2007). Phosphorylated Ugp1 is required for cell wall maintenance while unphosphorylated Ugp1 elicits increased glycogen production. While we don’t yet understand how the phosphorylation state of Ugp1 mediates this differential glucose partitioning, we have observed that phospho- and dephospho-Ugp1 adopt significantly different conformations. These alternate conformations of Ugp1 are likely responsible for the alternative fates of the UDP-glucose it produces, probably by localizing Ugp1 to the sites of differentially compartmentalized metabolism (Smith and Rutter, 2007). We are currently working to understand how Ugp1 phosphorylation works in detail. We are also examining other functions of PAS kinase in yeast, both metabolic and in other signaling pathways.
Diabetes mellitus is rapidly becoming one of the predominant health concerns of the western world. Fundamentally, diabetes is a failure of the insulin signaling system which functions to maintain blood glucose concentrations within a narrow range. A key component of this system is the pancreatic beta-cell which singularly has responsibility for insulin production and secretion in response to elevated serum glucose. We have found that PAS kinase is required for the synthesis of insulin in response to elevated glucose, at least in cultured beta-cells. We have extended these finding using mice wherein PAS kinase has been deleted (Pask-/- mice). These mice are hypoinsulinemic and as a result have an impaired ability to clear glucose after an injected glucose challenge. Further, isolated beta-cells from Pask-/- mice have a profound defect in glucose-stimulated insulin secretion in vitro. However, PASK-/- mice were resistant to high-fat diet induced obesity, hepatic steatosis and insulin resistance. This phenotype appears to be due to hypermetabolism in PASK-/- mice in vivo as measured by indirect calorimetry and in isolated skeletal muscle. These findings suggest an important physiological role of PASK in regulating metabolism and controlling energy balance in mammals (Hao, et al. 2007).
A major focus of our lab is to understand the mechanisms by which PASK controls both cellular and organismal energy metabolism. Specifically, we are working to understand how PAS kinase regulates mitochondrial metabolism in skeletal muscle (and probably in many cell types). We are also working to understand how PAS kinase controls hepatic lipid metabolism (as described in Hao, et al. 2007).
It is increasingly apparent that mitochondria and their dysfunction lie at the heart of many human diseases. These include type 2 diabetes, a number of neurodegenerative disorders, cancer and aging itself. The understanding, prevention and treatment of these and other diseases requires a more complete understanding of how mitochondrial function, both in energy production and in cellular decisions, is regulated and maintained.
While much is known about mitochondria and their workings, much still awaits discovery. This is illustrated by proteomics experiments which show that more than 20% of mitochondrial proteins are essentially uncharacterized. This includes a large number that are highly conserved throughout eukarya, a strong indication that they perform a fundamental and important function. We have chosen to initiate studies to determine the genetic and biochemical function of eight of these conserved mitochondrial proteins. Each has been identified as mitochondrial, but has not been further studied. Of the eight, we have made substantial progress on understanding the function of two. The others are in various stages of progress. We are also working on parallel low resolution characterization of all yeast mitochondrial proteins.
This project is based on a hypothesis that we recently published as a Perspective in PNAS (Lindsley and Rutter, 2006). Briefly, we propose that small molecule-based allosteric regulation of protein function has been inappropriately de-emphasized over the past thirty years of biological research. Previously, regulation of this type, wherein the dynamic fluctuations in the level of an intracellular metabolite regulate the activity of a protein through direct binding, was assayed routinely and discovered routinely. Indeed, most proteins that were studied were found to be allosterically regulated. In subsequent years, however, as the discovery of new genes and new proteins has become easier, we rarely bother to look for small molecule allostery and consequently rarely find it. We suggest that if we looked we would find this form of regulation to be widespread and to touch all aspects of cell biology.
If important and common, why has this form of regulation languished while others like phosphorylation or transcriptional regulation have exploded? This is due primarily to the relative difficulty of detecting and characterizing interactions that are low affinity. Allosteric interactions, to be dynamic and physiologically relevant, must have an affinity that is near the physiological concentration of the metabolite (typically mid-micromolar or weaker) and therefore are difficult to detect using standard binding techniques. Recognizing the significance of the biological problem and the technical challenges, we developed a very simple but reliable system for systematically identifying low affinity small molecule-protein interactions. To date, we have analyzed five proteins with known allosteric regulators. For four of the five, we detected the known interactions. Somewhat surprisingly, we also detected substrates and/or products for four of the five. Having validated the system, we are now moving on to the analysis of naïve proteins.
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