A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants. In this paper, we derive a new PtdIns4P biosensor using the PtdIns4P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila. PtdIns4P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7-positive late endosomes/lysosomes. PtdIns4P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. Therefore, P4M reports a wider cellular distribution of PtdIns4P than previous probes and therefore will be valuable for dissecting the biological functions of PtdIns4P in its assorted membrane compartments.     

Therapeutic Potential of Clove Essential Oil in Diabetes: Modulation of Pro-Inflammatory Mediators,Oxidative Stress and Metabolic Enzyme Activities

Type 1 diabetes is characterized by insulin deficiency due to the destruction of pancreatic β cells, leading to hyperglycemia, which in turn induces vascular complications. In the current study, we investigated the effect of intraperitoneal administration of clove essential oil (CEO: 20 mg/kg body weight) on certain oxidative stress and glucose metabolism enzymes, as well as the expression of proinflammatory mediators. Administration of CEO to diabetic rats showed a significant decline in blood glucose levels, total cholesterol, and xanthine oxidase, compared to the streptozotocin group. Furthermore, these treated rats elicited a notable attenuation in the levels of lipid peroxides, and thiols groups in both liver and brain tissues. The activities of antioxidant and metabolic enzymes were reverted to normality in diabetic upon CEO administration. In addition to its protective effects on red blood cell hemolysis, CEO is a potent α-amylase inhibitor with an IC₅₀=298.0±2.75 μg/mL. Also, treatment of diabetic rats with CEO significantly reduced the iNOS expression in the spleen. Our data showed that CEO has potential beneficial effects on diabetes, which can possibly prevent the pathogenesis of diabetic micro- and macrovascular complications.     

Caenorhabditis elegans as a model for intracellular pathogen infection

The genetically tractable nematode Caenorhabditis elegans is a convenient host for studies of pathogen infection. With the recent identification of two types of natural intracellular pathogens of C. elegans, this host now provides the opportunity to examine interactions and defence against intracellular pathogens in a whole‐animal model for infection. C. elegans is the natural host for a genus of microsporidia, which comprise a phylum of fungal‐related pathogens of widespread importance for agriculture and medicine. More recently, C. elegans has been shown to be a natural host for viruses related to the Nodaviridae family. Both microsporidian and viral pathogens infect the C. elegans intestine, which is composed of cells that share striking similarities to human intestinal epithelial cells. Because C. elegans nematodes are transparent, these infections provide a unique opportunity to visualize differentiated intestinal cells in vivo during the course of intracellular infection. Together, these two natural pathogens of C. elegans provide powerful systems in which to study microbial pathogenesis and host responses to intracellular infection.     

Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β₂ and β₃ strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.     

In silico 3D structure modeling and inhibitor binding studies of human male germ cell-associated kinase

Human male germ cell-associated kinase (hMAK) is an androgen-inducible gene in prostate epithelial cells, and it acts as a coactivator of androgen receptor signaling in prostate cancer. The 3D structure of the hMAK kinase was modeled based on the crystal structure of CDK2 kinase using comparative modeling methods, and the ATP-binding site was characterized. We have collected five inhibitors of hMAK from the literature and docked into the ATP-binding site of the kinase domain. Solvated interaction energies (SIE) of inhibitor binding are calculated from the molecular dynamics simulations trajectories of protein–inhibitor complexes. The contribution from each active site residue in hMAK toward inhibitor binding revealed the nature and extent of interactions between inhibitors and individual residues. The main chain atoms of Met79 invariably form hydrogen bonds with all five inhibitors. The amino acids Leu7, Val15, and Leu129 stabilize the inhibitors via CH–pi interactions. The Asp140 in the active site and Glu77 in hinge region show characteristic hydrogen bonding interactions with inhibitors. From SIE, the residue-wise interactions revealed the nature of non-bonding contacts and modifications required to increase the inhibitor activity. Our work provides 3D model structure of hMAK and molecular basis for the mechanisms of hMAK inhibition at atomic level that aid in designing new potent inhibitors.     

Potent inhibitors of CDK5 derived from roscovitine: Synthesis,biological evaluation and molecular modelling

Cyclin dependent kinase 5 (CDK5) is a serine/threonine kinase belonging to the cyclin dependent kinase (CDK) family. CDK5 is involved in numerous neuronal diseases (including Alzheimer’s or Parkinson’s diseases, stroke, traumatic brain injury), pain signaling and cell migration. In the present Letter, we describe syntheses and biological evaluations of new 2,6,9-trisubstituted purines, structurally related to roscovitine, a promising CDK inhibitor currently in clinical trials (CDK1/Cyclin B, IC₅₀ = 350 nM; CDK5/p25, IC₅₀ = 200 nM). These new molecules were synthesized using an original Buchwald–Hartwig catalytic procedure; several compounds (3j, 3k, 3l, 3e, 4k, 6b, 6c) displayed potent kinase inhibitory potencies against CDK5 (IC₅₀ values ranging from 17 to 50 nM) and showed significant cell death inducing activities (IC₅₀ values ranging from 2 to 9 μM on SH-SY5Y). The docking of the inhibitors into the ATP binding domain of the CDK5 catalytic site highlighted the discriminatory effect of a hydrogen bond involving the CDK5 Lys-89. In addition, the calculated final energy balances for complexation measured for several inhibitors is consistent with the ranking of the IC₅₀ values. Lastly, we observed that several compounds exhibit submicromolar activities against DYRK1A (dual specificity, tyrosine phosphorylation regulated kinase 1A), a kinase involved in Down syndrome and Alzheimer’s disease (3g, 3h, 4m; IC₅₀ values ranging from 300 to 400 nM).     

Chemical Polymorphism of Origanum compactum Grown in All Natural Habitats in Morocco

Origanum compactum L. (Lamiaceae) is one of the most important medicinal species in term of ethnobotany in Morocco. It is considered as a very threatened species as it is heavily exploited. Its domestication remains the most efficient way to safeguard it for future generations. For this purpose, wide evaluation of the existing variability in all over the Moroccan territory is required. The essential oils of 527 individual plants belonging to 88 populations collected from the whole distribution area of the species in Morocco were analyzed by GC/MS. The dominant constituents were carvacrol (0 – 96.3%), thymol (0 – 80.7%), p‐cymene (0.2 – 58.6%), γ‐terpinene (0 – 35.2%), carvacryl methyl ether (0 – 36.2%), and α‐terpineol (0 – 25.8%). While in the Middle Atlas region and the Central Morocco mainly carvacrol type samples were found, much higher chemotypic diversity was encountered within samples from the north part of Morocco (occidental and central Rif regions). The high chemical polymorphism of plants offers a wide range for selection of valuable chemotypes, as a part of breeding and domestication programs of this threatened species.     

Visualization and manipulation of plasma membrane-endoplasmic reticulum contact sites indicates the presence of additional molecular components within the STIM1-Orai1 Complex

STIM1, a recently identified endoplasmic reticulum (ER) protein, rapidly translocates to a plasma membrane-adjacent ER compartment upon depletion of the ER Ca(2+) stores. Here we use a novel means, namely a chemically inducible bridge formation between the plasma and ER membranes, to highlight the plasma membrane-adjacent ER compartment and show that this is the site where STIM1 and its Ca(2+) channel partner, Orai1, form a productive interaction upon store depletion. By changing the length of the linkers connecting the plasma and ER membranes, we show that Orai1 requires a larger space than STIM1 between the two membranes. This finding suggests that Orai1 is part of a larger macromolecular cluster with an estimated 11-14-nm protrusion to the cytoplasm, whereas the cytoplasmic domain of STIM1 fits in a space calculated to be less than 6 nm. We finally show that agonist-induced translocation of STIM1 is rapidly reversible and only partially affects STIM1 in the juxtanuclear ER compartment. These studies are the first to detect juxtaposed areas between the ER and the plasma membrane in live cells, revealing novel details of STIM1-Orai1 interactions.     

Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors caused a rapid (<3 min) translocation of the cytosolic fluorescence to ruffle-like membrane structures. This response was not observed in cells expressing GFP only and was completely inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004. Membrane-targeted PI 3-kinase also caused membrane localization of BtkPH-GFP that was slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain, which causes X-linked agammaglobulinemia, was introduced into the fluorescent construct, no translocation was observed after stimulation. In contrast, the E41K mutation, which confers transforming activity to native Btk, caused significant membrane localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type BtkPH-GFP, interfered with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of changes in this phospholipid in living cells.