Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate

Ca2+-activated Cl- channels are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P-4) (Xie, W. Haetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092-14097), a novel second messenger that is formed after stimulus-dependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P-3) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P-4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P-3 inhibited Ins(3,4,5,6)P-4 1-kinase activity that was either (i) in lysates of AR4-2J pancreatoma cells or (ii) purified 22,500-fold (yield = 13%) from bovine aorta. Next, we incubated [H-3]inositol-labeled AR4-2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P-3 2.7-fold, while [H-3]Ins(3,4,5,6)P-4 levels increased a-fold; there were no changes to levels of other H-3-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P-4 are regulated by Ins(1,3,4)P-3 in vivo, independently of Ins(1,3,4)P-3 being metabolized to Ins(3,4,5,6)P-4. In addition, we found that the Ins(1,3,4)P-3 metabolites, namely Ins(1,3)P-2 and Ins(3,4)P-2, were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P-3 itself (IC50 = 0.17 mu M). This result shows that dephosphorylation of Ins(1,3,4)P-3 in vivo is an efficient mechanism to "switch-off" the cellular regulation of Ins(3,4,5,6)P-4 levels that comes from Ins(1,3,4)P-3-mediated inhibition of the 1-kinase. We also found that Ins(1,3,6)P-3 and Ins(1,4,6)P-3 were poor inhibitors of the 1-kinase (IC50 = 17 and >30 mu M, respectively). The non-physiological trisphosphates, D/L-Ins(1,2,4)P-3, inhibited 1-kinase relatively potently (IC50 = 0.7 mu M), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P-3 acts in an important signaling cascade.X1140sciescopu

Crystal structure of a mammalian phosphoinositide-specific phospholipase Cδ\delta

Mammalian phosphoinositide-specific phospholipase C enzymes (PI-PLC) act as signal transducers that generate two second messengers, inositol-l,4,5-trisphosphate and diacylglycerol. The 2.4-Å structure of phospholipase Cδ1 reveals a multidomain protein incorporating modules shared by many signalling proteins. The structure suggests a mechanism for membrane attachment and Ca2+ -dependent hydrolysis of second-messenger precursors. The regulation and reversible membrane association of PI-PLC may serve as a model for understanding other multidomain enzymes involved in phospholipid signalling

Recent Progress in Density Functional Methodology for Biomolecular Modeling

International audienceDensity Functional Theory (DFT) has become the workhorse of applied computational chemistry. DFT has grown in a number of different directions depending on the applications concerned. In this chapter, we provide a broad review of a number of DFT and DFT-based methods, having in mind the accurate description of biological systems and processes. These range from pure "cluster" DFT studies of the structure, properties, and reactions of biochemical species (such as enzymatic catalysts) using either straight DFT or dispersion-corrected functionals (DFT-D), to Born-Oppenheimer-DFT dynamics of systems containing up to a hundred atoms or more (such as glycero-lipids), to hybrid DFT/Molecular Mechanical Molecular Dynamics methods which include protein and solvent environments (for enzymes or ion channels, for example), to constrained-DFT (working within the Marcus framework for electron-transfer reactions), to Interpretational-DFT (which provides the interpretational benefits of the Kohn-Sham DFT methodology)