A complete Rab screening reveals novel insights in Weibel-Palade body exocytosis.

Weibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

CD39 reveals novel insights into the role of transmembrane domains in protein processing, apical targeting and activity.

Cargo proteins of the biosynthetic secretory pathway are folded in the endoplasmic reticulum (ER) and proceed to the trans Golgi network for sorting and targeting to the apical or basolateral sides of the membrane, where they exert their function. These processes depend on diverse protein domains. Here, we used CD39 (NTPdase1), a modulator of thrombosis and inflammation, which contains an extracellular and two transmembrane domains (TMDs), as a model protein to address comprehensively the role of native TMDs in folding, polarized transport and biological activity. In MDCK cells, CD39 exits Golgi dynamin-dependently and is targeted to the apical side of the membrane. Although the N-terminal TMD possesses an apical targeting signal, the N- and C-terminal TMDs are not required for apical targeting of CD39. Folding and transport to the plasma membrane relies only on the C-terminal TMD, while the N-terminal one is redundant. Nevertheless, both N- and C-terminal anchoring as well as genuine TMDs are critical for optimal enzymatic activity and activation by cholesterol. We conclude therefore that TMDs are not just mechanical linkers between proteins and membranes but are also able to control folding and sorting, as well as biological activity via sensing components of lipid bilayers.

The orchestrated signaling by PI3Kα and PTEN at the membrane interface.

The oncogene PI3Kα and the tumor suppressor PTEN represent two antagonistic enzymatic activities that regulate the interconversion of the phosphoinositide lipids PI(4,5)P2 and PI(3,4,5)P3 in membranes. As such, they are defining components of phosphoinositide-based cellular signaling and membrane trafficking pathways that regulate cell survival, growth, and proliferation, and are often deregulated in cancer. In this review, we highlight aspects of PI3Kα and PTEN interplay at the intersection of signaling and membrane trafficking. We also discuss the mechanisms of PI3Kα- and PTEN- membrane interaction and catalytic activation, which are fundamental for our understanding of the structural and allosteric implications on signaling at the membrane interface and may aid current efforts in pharmacological targeting of these proteins.

Design and synthesis of novel 7-aminosubstituted pyrido[2,3-b]pyrazines exhibiting anti-breast cancer activity.

Breast cancer (BrCa) remains an unmet medical need despite the revolutionary development of antibody treatments and protein kinase inhibitors. In the current study, a series of novel substituted pyridopyrazine derivatives have been rationally designed and evaluated as multi-kinase inhibitors in the PI3K pathway. The target compounds were prepared from 6-amino-2-picoline, which upon nitration and selective reduction was converted to suitably substituted 6-methyl-7-aminopyrido[2,3-b]pyrazines. Suitable manipulation of the former amines provided the designed analogues, which were then assessed in vitro against several BrCa cell lines using the MTT cytotoxicity assay. The most potent compounds underwent evaluation in a broad spectrum of protein kinases, while their pharmacokinetic parameters were measured by LC-MS/MS. In vivo evaluation of a hit compound (14a) was performed in a HER2 amplified BrCa xenograft model (HCC1954) and efficacy was determined using Western blot based phosphokinase assays and immunohistochemistry. This derivative showed low micromolar cytotoxic potency in all BrCa cell lines, a mild inhibition of the PI3Kα wild type and H1047R mutated enzyme and excellent pharmacokinetic parameters following oral and intraperitoneal administration at the designed dose of 10 mg/kg, with absence of in vivo phenotypic toxicity. Interestingly, compound 14a inhibited the growth of xenografted tumors. Analysis of excised tumors from the treated animals showed a significantly reduced population of Ki-67 positive cells, as well as downregulated levels of phosphorylated AKT, ERK1/2 and SRC compared to vehicle treated animals. Finally, the specificity of 14a was assessed in a panel of 31 kinases where a mild, but direct, inhibition of the MET receptor tyrosine kinase was observed.

Discovery of New Aminosubstituted Pyrrolopyrimidines with Antiproliferative Activity Against Breast Cancer Cells and Investigation of their Effect Towards the PI3Kα Enzyme.

OBJECTIVE: A series of novel 2,4-diaminosubstituted pyrrolo[3,2-d]pyrimidines was synthesized together with their corresponding 7-phenyl or 7-isopropyl counterparts. RESULTS: Among the target derivatives, the 7-substituted analogues exhibited interesting cytotoxic activity against a panel of PI3Kα related human breast cancer cell lines, namely MCF7, T47D, MDA-MB-231 and HCC1954. Selected compounds were tested for potential PI3Kα inhibitory activity as well as for their cytotoxic effect in prostate cancer cell lines (DU145 and PC3). CONCLUSION: Derivatives bearing a specific substitution pattern consisting of 7-phenyl as well as a 2-(4- aminocyclohexylamino) moiety (16c, 16f) display kinase inhibitory activity, elucidated on the basis of molecular simulation studies, which revealed their interaction with the DFG motif of the kinase.

Exploring a non-ATP pocket for potential allosteric modulation of PI3Kα.

Allosteric modulators offer a novel approach for kinase inhibition because they target less conserved binding sites compared to the active site; thus, higher selectivity may be obtained. PIK-108, a known pan phosphoinositide 3-kinase (PI3K) inhibitor, was recently detected to occupy a non-ATP binding site in the PI3Kα C-lobe. This newly identified pocket is located close to residue 1047, which is frequently mutated in human cancers (H1047R). In order to assess the interactions, stability, and any possible allosteric effects of this inhibitor on PI3Kα, extensive molecular dynamics (MD) simulations in aqueous solution were performed for the wild type (WT) human, WT murine, and H1047R human mutant PI3Kα proteins with PIK-108 placed in both catalytic and non-ATP sites. We verify the existence of the second binding site in the vicinity of the hotspot H1047R PI3Kα mutation through binding site identification and MD simulations. PIK-108 remains stable in both sites in all three variants throughout the course of the simulations. We demonstrate that the pose and interactions of PIK-108 in the catalytic site are similar in the murine WT and human mutant forms, while they are significantly different in the case of human WT PI3Kα protein. PIK-108 binding in the non-ATP pocket also differs significantly among the three variants. Finally, we examine whether the non-ATP binding site is implicated in PI3Kα allostery in terms of its communication with the active site using principal component analysis and perform in vitro experiments to verify our hypotheses.

Cholesterol-dependent lipid assemblies regulate the activity of the ecto-nucleotidase CD39.

CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase1) is a plasma membrane ecto-enzyme that regulates purinergic receptor signaling by controlling the levels of extracellular nucleotides. In blood vessels this enzyme exhibits a thromboregulatory role through the control of platelet aggregation. CD39 is localized in caveolae, which are plasma membrane invaginations with distinct lipid composition, similar to dynamic lipid microdomains, called rafts. Cholesterol is enriched together with sphingolipids in both rafts and caveolae, as well as in other specialized domains of the membrane, and plays a key role in their function. Here, we examine the potential role of cholesterol-enriched domains in CD39 function. Using polarized Madin-Darby canine kidney (MDCK) cells and caveolin-1 gene-disrupted mice, we show that caveolae are not essential either for the enzymatic activity of CD39 or for its targeting to plasma membrane. On the other hand, flotation experiments using detergent-free or detergent-based approaches indicate that CD39 associates, at least in part, with distinct lipid assemblies. In the apical membrane of MDCK cells, which lacks caveolae, CD39 is localized in microvilli, which are also cholesterol and raft-dependent membrane domains. Interfering with cholesterol levels using drugs that either deplete or sequester membrane cholesterol results in a strong inhibition of the enzymatic and anti-platelet activity of CD39. The effects of cholesterol depletion are completely reversed by replenishment of membranes with pure cholesterol, but not by cholestenone. These data suggest a functional link between the localization of CD39 in cholesterol-rich domains of the membrane and its role in thromboregulation.