26 research outputs found
Exploiting protein flexibility to predict the location of allosteric sites
Background: Allostery is one of the most powerful and common ways of regulation of protein activity. However, for most allosteric proteins identified to date the mechanistic details of allosteric modulation are not yet well understood. Uncovering common mechanistic patterns underlying allostery would allow not only a better academic understanding of the phenomena, but it would also streamline the design of novel therapeutic solutions. This relatively unexplored therapeutic potential and the putative advantages of allosteric drugs over classical active-site inhibitors fuel the attention allosteric-drug research is receiving at present. A first step to harness the regulatory potential and versatility of allosteric sites, in the context of drug-discovery and design, would be to detect or predict their presence and location. In this article, we describe a simple computational approach, based on the effect allosteric ligands exert on protein flexibility upon binding, to predict the existence and position of allosteric sites on a given protein structure. Results: By querying the literature and a recently available database of allosteric sites, we gathered 213 allosteric proteins with structural information that we further filtered into a non-redundant set of 91 proteins. We performed normal-mode analysis and observed significant changes in protein flexibility upon allosteric-ligand binding in 70% of the cases. These results agree with the current view that allosteric mechanisms are in many cases governed by changes in protein dynamics caused by ligand binding. Furthermore, we implemented an approach that achieves 65% positive predictive value in identifying allosteric sites within the set of predicted cavities of a protein (stricter parameters set, 0.22 sensitivity), by combining the current analysis on dynamics with previous results on structural conservation of allosteric sites. We also analyzed four biological examples in detail, revealing that this simple coarse-grained methodology is able to capture the effects triggered by allosteric ligands already described in the literature. Conclusions: We introduce a simple computational approach to predict the presence and position of allosteric sites in a protein based on the analysis of changes in protein normal modes upon the binding of a coarse-grained ligand at predicted cavities. Its performance has been demonstrated using a newly curated non-redundant set of 91 proteins with reported allosteric properties. The software developed in this work is available upon request from the authors
Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells
The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ĪĻM) in pancreatic Ī²-cells. This built on our previously introduced calculation of the absolute magnitude of ĪĻM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ĪĻP) indicator. ĪĻM is a central mediator of glucose-stimulated insulin secretion in pancreatic Ī²-cells. ĪĻM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ĪĻM and ĪĻP. Glucose addition caused hyperpolarization of ĪĻM and depolarization of ĪĻP. The hyperpolarization was a monophasic step increase, even in cells where the ĪĻP depolarization was biphasic. The biphasic response of ĪĻP was associated with a larger hyperpolarization of ĪĻM than the monophasic response. Analysis of the relationships between ĪĻP and ĪĻM revealed that primary dispersed Ī²-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with Ī²-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ĪĻM and ĪĻP. Finally, we demonstrate a significant problem with using an alternative ĪĻM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited Ī²-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusion
The role of energy metabolism in amino acid stimulated insulin release in pancreatic Ī²-HC9 cells
Fuel stimulation of insulin secretion from pancreatic p-cells is thought to be me-diated by metabolic coupling factors that are generated by energized mitochondria, including protons, adenine nucleotides and perhaps certain amino acids, as for instance aspartate, glutamate or glutamine. The goal of the present study was to evaluate the role of such factors when insulin release (IR) is stimulated by glucose or amino acids (AA), alone or combined, using 31P-, 23Na- and 1H-NMR-technology, respirometry and biochemical analysis to study the metabolic events that occur in continuously superfused mouse Ī²-HC9 cells contained in agarose beads and enhanced by the phosphodiesterase inhibitor IBMX. Exposing Ī²-HC9 cells to high glucose or 3.5 mM of a physiological mixture of 18 AA plus 2 mM glutamine (Q) caused a marked stimulation of insulin secretion associated with increased oxygen consumption (V02), c-AMP release and phosphorylation potential (P-potential) as evidenced by higher Phosphocreatine (PCr) and lower Pi peak areas of 31P NMR spectra. Diazoxide blocked stimulation of IR completely suggesting involvement of KATP channels in this process. However, levels of [MgATP] and [MgADP] which regulate channel activity changed only slowly and little while the rate of insulin release increased fast and very markedly. The involvement of other candidate coupling factors was therefore considered. High glucose or AAM + Q increased intracellular pH. The availability of temporal pH profiles allowed the precise computation of the phosphate potential (ATP/P Ć ADP) in fuel stimulated IR. Intracellular Na+ levels were greatly elevated by AAM + Q. However, glutamine alone or together with 2-amino-2-norbornane-carboxylic acid (BCH, which activates glutamate dehydrogenase) decreased Ī²-cell Na levels. Stimulation of Ī²-cells by glucose in the presence of AAM + Q (0.5 mM) was associated with rising cellular concentrations of glutamate, glutamine and strikingly lower aspartate levels. Methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, blocked the glucose enhancement of AMM + Q induced IR and associated changes in glutamine and aspartate but did not prevent the accumulation of glutamate. The results of this study demonstrate again that an increased phosphate potential and a functional KATP channel are essential for metabolic coupling during fuel stimulated insulin release but illustrate that determining the identity and relative importance of all participating coupling factors and second messengers remains a challenge largely unmet
Glucose metabolism and insulin release in mouse beta HC9 cells, as model for wild-type pancreatic beta-cells
Functional and Metabolomic Consequences of KATP Channel Inactivation in Human Islets
Loss-of-function mutations of Ī²-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+]i), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+]i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low Ī³-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI Ī²-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.</jats:p