Peptides that mimic the pseudosubstrate region of protein kinase C bind to acidic lipids in membranes

The cytoplasmic form of protein kinase C (PKC) is inactive, probably because the pseudosubstrate region in its regulatory domain blocks the substrate-binding site in its kinase domain. Calcium ions cause a translocation to the membrane: maximum activation requires a negative lipid such as phosphatidylserine (PS) and the neutral lipid diacylglycerol (DAG) but the mechanism by which PS and DAG activate PKC is unknown. Pseudosubstrate region 19–36 of PKC-beta has six basic and one acidic amino acids and region 19–29 has five basic and no acidic amino acids. Since any binding of basic residues in the pseudosubstrate region to acidic lipids in the membrane should stabilize the active form of PKC, we studied how peptides with amino acids equivalent to residues 19–36 and 19–29 of PKC-beta bound to phospholipid vesicles. We made equilibrium dialysis, filtration, and electrophoretic mobility measurements. The fraction of bound peptide is a steep sigmoidal function of the mol fraction of negative lipid in the membrane, as predicted from a simple theoretical model that assumes the basic residues provide identical independent binding sites. The proportionality constant between the number of bound peptides/area and the concentration of peptide in the bulk aqueous phase is 1 micron for a membrane with 25% negative lipid formed in 0.1 M KCl. Equivalently, the association constant of the peptide with the membrane is 10(4) M-1, or the net binding energy is 6 kcal/mol. Thus the interaction of basic residues in the pseudosubstrate region with acidic lipids in the membrane could provide 6 kcal/mol free energy towards stabilizing the active form of PKC

Open-Sourced CIViC Annotation Pipeline to identify and annotate clinically relevant variants using single-molecule molecular inversion probes

PURPOSE: Clinical targeted sequencing panels are important for identifying actionable variants for patients with cancer; however, existing approaches do not provide transparent and rationally designed clinical panels to accommodate the rapidly growing knowledge within oncology. MATERIALS AND METHODS: We used the Clinical Interpretations of Variants in Cancer (CIViC) database to develop an Open-Sourced CIViC Annotation Pipeline (OpenCAP). OpenCAP provides methods to identify variants within the CIViC database, build probes for variant capture, use probes on prospective samples, and link somatic variants to CIViC clinical relevance statements. OpenCAP was tested using a single-molecule molecular inversion probe (smMIP) capture design on 27 cancer samples from 5 tumor types. In total, 2,027 smMIPs were designed to target 111 eligible CIViC variants (61.5 kb of genomic space). RESULTS: When compared with orthogonal sequencing, CIViC smMIP sequencing demonstrated a 95% sensitivity for variant detection (n = 61 of 64 variants). Variant allele frequencies for variants identified on both sequencing platforms were highly concordant (Pearson\u27s CONCLUSION: The OpenCAP design paradigm demonstrates the utility of an open-source and open-access database built on attendant community contributions with peer-reviewed interpretations. Use of a public repository for variant identification, probe development, and variant interpretation provides a transparent approach to build dynamic next-generation sequencing-based oncology panels

Rare DEGS1 variant significantly alters de novo ceramide synthesis pathway

The de novo ceramide synthesis pathway is essential to human biology and health but genetic influences remain unexplored. The core function of this pathway is the generation of biologically active ceramide from its precursor, dihydroceramide. Dihydroceramides have diverse, often protective, biological roles; conversely, increased ceramide levels are biomarkers of complex disease. To explore the genetics of the ceramide synthesis pathway, we searched for deleterious nonsynonymous variants in the genomes of 1,020 Mexican Americans from extended pedigrees. We identified a Hispanic ancestry−specific rare functional variant, L175Q, in DEGS1, a key enzyme in the pathway that converts dihydroceramide to ceramide. This amino acid change was significantly associated with large increases in plasma dihydroceramides. Indexes of DEGS1 enzymatic activity were dramatically reduced in heterozygotes. CRISPR/Cas9 genome editing of HepG2 cells confirmed that the L175Q variant results in a partial loss of function for the DEGS1 enzyme. Understanding the biological role of DEGS1 variants, such as L175Q, in ceramide synthesis may improve the understanding of metabolic-related disorders, and spur ongoing research of drug targets along this pathway

G protein-coupled receptor-mediated calcium signaling in astrocytes

Astrocytes express a large variety of G~protein-coupled receptors (GPCRs) which mediate the transduction of extracellular signals into intracellular calcium responses. This transduction is provided by a complex network of biochemical reactions which mobilizes a wealth of possible calcium-mobilizing second messenger molecules. Inositol 1,4,5-trisphosphate is probably the best known of these molecules whose enzymes for its production and degradation are nonetheless calcium-dependent. We present a biophysical modeling approach based on the assumption of Michaelis-Menten enzyme kinetics, to effectively describe GPCR-mediated astrocytic calcium signals. Our model is then used to study different mechanisms at play in stimulus encoding by shape and frequency of calcium oscillations in astrocytes.Comment: 35 pages, 6 figures, 1 table, 3 appendices (book chapter

Group IV cytosolic phospholipase A2 binds with high affinity and specificity to phosphatidylinositol 4,5-bisphosphate resulting in dramatic increases in activity.

The group IV cytosolic phospholipase A2 (cPLA2) exhibits a potent and specific increase in affinity for lipid surfaces containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at physiologically relevant concentrations. Specifically, the presence of 1 mol% PtdIns(4,5)P2 in phosphatidylcholine vesicles results in a 20-fold increase in the binding affinity of cPLA2. This increased affinity is accompanied by an increase in substrate hydrolysis of a similar magnitude. The binding studies and kinetic analysis indicate that PtdIns(4,5)P2 binds to cPLA2 in a 1:1 stoichiometry. The magnitude of the effect of PtdIns(4,5)P2 is unique among anionic phospholipids and larger than that for other polyphosphate phosphatidylinositols. The effect of PtdIns(4,5)P2 on the activity of cPLA2 is at least an order of magnitude larger than the concomitant changes in the fraction of the enzyme associated with lipid membranes. Striking parallels between the interaction of cPLA2 with PtdIns(4,5)P2 and the interaction of the pleckstrin homology domain of phospholipase C delta 1 with PtdIns(4,5)2 combined with sequence analysis of cPLA2 lead us to propose the existence and location of a pleckstrin homology domain in cPLA2. We further show that the very nature of the interaction of proteins such as cPLA2 with multiple ligands incorporated into membranes follows a specific model which necessitates the use of an experimental methodology suitable for a membrane interface to allow for a meaningful analysis of the data

Binding of peptides with basic residues to membranes containing acidic phospholipids.

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane