. Our data are consistent with the hypothesis that nuclear PI-PLC generates DAG to activate nuclear ␤ II PKC, whose activity is required for mitosis.The PKC 1 family of enzymes is involved in the transduction of external signals from many growth factors, cytokines, and hormones (reviewed in Refs. 1-3). Many of the details of the receptor-mediated signaling pathways that lead to PKC activation have been elucidated (2, 3). A common feature of these pathways is receptor-mediated activation of lipid-metabolizing enzymes that generate DAG. DAG in turn activates PKC family members. The best characterized of these pathways involves the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) activity (4). Two major classes of cellular receptor utilize the PI-PLC/PKC activation pathway. Growth factor receptors containing intrinsic tyrosine kinase activity activate PI-PLC ␥ isoforms through direct tyrosine phosphorylation and activation of the enzyme (4). Many G-protein-coupled receptors activate PI-PLC␤ isoforms through direct interaction with heterotrimeric G-proteins of the G q class (4). Activation of PI-PLC enzymes leads to generation of inositol trisphosphate and DAG, two metabolites of phosphatidylinositol 4,5-bisphosphate that stimulate intracellular calcium release and activate PKC isozymes, respectively. Extracellular ligands can also stimulate activation of phosphatidylcholine-specific phospholipase C (PC-PLC) and/or phospholipase D (PLD) activities (5). These phospholipases can give rise to increased cellular DAG levels and PKC activation, including the novel, calcium-independent isozymes (3). More recently, it has been demonstrated that phosphatidylinositol 3,4,5-trisphosphate, a product of growth factor receptor-activated PI3 kinase, can directly and selectively activate the atypical PKC isozymes (6 -10). Elucidation of these pathways has enhanced our understanding of how lipid metabolism and PKC activation are coupled to acute growth factor and hormone actions, including mitogenesis.In addition to its well established role in acute mitogenic signaling, PKC has also been implicated in intrinsic signaling pathways, including those involved in cell cycle control (1). We recently demonstrated that activation of the ␤ II PKC isoform at the nucleus is both cell cycle-regulated and necessary for entry of cells into mitosis (11-16). At the nucleus, ␤ II PKC directly phosphorylates the nuclear envelope polypeptide lamin B at sites involved in mitotic nuclear lamina disassembly (12,16). Inhibition of nuclear PKC activity leads to cell cycle arrest in the G 2 phase, indicating the importance of nuclear PKC in mitotic events (16). Although it is clear that nuclear PKC activity is cell cycle-regulated, the basis for this regulation is not clear. In the present study, we report that nuclear DAG levels fluctuate during cell cycle and that changes in nuclear DAG levels correlate with cell cycle progression through the G 2 /M phase. Furthermore, a nuclear PI-PLC activity has been identified that is active durin...

Thermal analyses of epoxidized soybean oil (ESO) were conducted and showed that it was stable up to temperatures as high as 240–260°C in air‐free environment. The solubility and transport characteristics of ESO in poly(vinylchloride) (PVC) were also investigated under various conditions. The absorption study showed that the resin had a void volume of ∼0.3 cm3/g. Furthermore, ESO was found to be sufficiently soluble in the PVC matrix to function as an effective plasticizer, with equilibrium solubility of 72 g/100 g PVC at 80°C and 163 g/100 g PVC at 120°C. The absorption of ESO in PVC grains was a three step process comprising “induction,” “swelling,” and “saturation” periods. Torque rheometer studies showed that higher mixer temperature and/or speed facilitated uptake of plasticizer in PVC and ultimate fusion. Migration studies of plasticized and stabilized PVC compositions showed no change in mass at 40°C, but increasingly greater mass loss as the temperature was raised up to 120°C. POLYM. ENG. SCI., 2013. © 2012 Society of Plastics Engineers

Calcineurin (CaN) is a calcium-dependent phosphatase involved in numerous signaling pathways. Its activation is in part driven by the binding of calmodulin (CaM) to a CaM recognition region (CaMBR) within CaN’s regulatory domain (RD). However, secondary interactions between CaM and the CaN RD may be necessary to fully activate CaN. Specifically, it is established that the CaN RD folds upon CaM binding and a region C-terminal to CaMBR, the “distal helix”, assumes an α-helix fold and contributes to activation [Dunlap, T. B., et al. (2013) Biochemistry 52, 8643–8651]. We hypothesized in that previous study that this distal helix can bind CaM in a region distinct from the canonical CaMBR. To test this hypothesis, we utilized molecular simulations, including replica-exchange molecular dynamics, protein–protein docking, and computational mutagenesis, to determine potential distal helix-binding sites on CaM’s surface. We isolated a potential binding site on CaM (site D) that facilitates moderate-affinity interprotein interactions and predicted that mutation of site D residues K30 and G40 on CaM would weaken CaN distal helix binding. We experimentally confirmed that two variants (K30E and G40D) indicate weaker binding of a phosphate substrate p-nitrophenyl phosphate to the CaN catalytic site by a phosphatase assay. This weakened substrate affinity is consistent with competitive binding of the CaN autoinhibition domain to the catalytic site, which we suggest is due to the weakened distal helix–CaM interactions. This study therefore suggests a novel mechanism for CaM regulation of CaN that may extend to other CaM targets.

Significant advances in our understanding of the molecular mechanisms that cause congenital long QT syndrome (LQTS) have been made. A wide variety of experimental approaches, including heterologous expression of mutant ion channel proteins and the use of inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LQTS patients offer insights into etiology and new therapeutic strategies. This review briefly discusses the major molecular mechanisms underlying LQTS type 2 (LQT2), which is caused by loss-of-function (LOF) mutations in the KCNH2 gene (also known as the human ether-à-go-go-related gene or hERG). Almost half of suspected LQT2-causing mutations are missense mutations, and functional studies suggest that about 90% of these mutations disrupt the intracellular transport, or trafficking, of the KCNH2-encoded Kv11.1 channel protein to the cell surface membrane. In this review, we discuss emerging strategies that improve the trafficking and functional expression of trafficking-deficient LQT2 Kv11.1 channel proteins to the cell surface membrane and how new insights into the structure of the Kv11.1 channel protein will lead to computational approaches that identify which KCNH2 missense variants confer a high-risk for LQT2.

Calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in mammalian tissue. The calcineurin (CaN) regulatory domain (RD) is responsible for regulating the enzyme's phosphatase activity, and is believed to be highly-disordered when inhibiting CaN, but undergoes a disorder-to-order transition upon diffusion-limited binding with the regulatory protein calmodulin (CaM). The prevalence of polar and charged amino acids in the regulatory domain (RD) suggests electrostatic interactions are involved in mediating calmodulin (CaM) binding, yet the lack of atomistic-resolution data for the bound complex has stymied efforts to probe how the RD sequence controls its conformational ensemble and long-range attractions contribute to target protein binding. In the present study, we investigated via computational modeling the extent to which electrostatics and structural disorder facilitate CaM/CaN association kinetics. Specifically, we examined several RD constructs that contain the CaM binding region (CAMBR) to characterize the roles of electrostatics versus conformational diversity in controlling diffusion-limited association rates, via microsecond-scale molecular dynamics (MD) and Brownian dynamic (BD) simulations. Our results indicate that the RD amino acid composition and sequence length influence both the dynamic availability of conformations amenable to CaM binding, as well as long-range electrostatic interactions to steer association. These findings provide intriguing insight into the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging diffusion-limited processes regulated by intrinsically-disordered proteins.