Design and synthesis of substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides as positive allosteric modulators of the metabotropic glutamate receptor subtype 5

Based on SAR in the alkyne class of mGlu5 receptor negative allosteric modulators and a set of amide-based positive allosteric modulators, optimized substitution of the aryl ‘b’ ring was used to create substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides. Results from an mGlu5 receptor functional assay, using calcium fluorescence, revealed varying efficacies and potencies that provide evidence that subtle changes in compounds within a close structural class can have marked effects on functional activity including switches in modes of efficacy (i.e., negative to positive allosteric modulation).     

MS4A15 drives ferroptosis resistance through calcium-restricted lipid remodeling

Ferroptosis is an iron-dependent form of cell death driven by biochemical processes that promote oxidation within the lipid compartment. Calcium (Ca²⁺) is a signaling molecule in diverse cellular processes such as migration, neurotransmission, and cell death. Here, we uncover a crucial link between ferroptosis and Ca²⁺ through the identification of the novel tetraspanin MS4A15. MS4A15 localizes to the endoplasmic reticulum, where it blocks ferroptosis by depleting luminal Ca²⁺ stores and reprogramming membrane phospholipids to ferroptosis-resistant species. Specifically, prolonged Ca²⁺ depletion inhibits lipid elongation and desaturation, driving lipid droplet dispersion and formation of shorter, more saturated ether lipids that protect phospholipids from ferroptotic reactive species. We further demonstrate that increasing luminal Ca²⁺ levels can preferentially sensitize refractory cancer cell lines. In summary, MS4A15 regulation of anti-ferroptotic lipid reservoirs provides a key resistance mechanism that is distinct from antioxidant and lipid detoxification pathways. Manipulating Ca²⁺ homeostasis offers a compelling strategy to balance cellular lipids and cell survival in ferroptosis-associated diseases.Subject terms: Lipidomics, Cancer genetics, Cell biology     

Asymptotic Efficiency of the Chi-Square Test for Heterogeneity of Proportions in Some Sparse-Data Cases

We consider the problem of testing for heterogeneity of K proportions when K is not small and the binomial sample sizes may not be large. We assume that the binomial proportions are normally distributed with variance σ². The asymptotic relative efficiency (ARE) of the usual chi-square test is found relative to the likelihood-based tests for σ²=0. The chi-square test is found to have ARE = 1 when the binomial sample sizes are all equal and high relative efficiency for other cases. The efficiency is low only in cases where there is insufficient data to use the chi-square test.     

The Epoxyeicosatrienoic Acid Pathway Enhances Hepatic Insulin Signaling and is Repressed in Insulin-Resistant Mouse Liver*

Although it is widely accepted that ectopic lipid accumulation in the liver is associated with hepatic insulin resistance, the underlying molecular mechanisms have not been well characterized.Here we employed time resolved quantitative proteomic profiling of mice fed a high fat diet to determine which pathways were affected during the transition of the liver to an insulin-resistant state. We identified several metabolic pathways underlying altered protein expression. In order to test the functional impact of a critical subset of these alterations, we focused on the epoxyeicosatrienoic acid (EET) eicosanoid pathway, whose deregulation coincided with the onset of hepatic insulin resistance. These results suggested that EETs may be positive modulators of hepatic insulin signaling. Analyzing EET activity in primary hepatocytes, we found that EETs enhance insulin signaling on the level of Akt. In contrast, EETs did not influence insulin receptor or insulin receptor substrate-1 phosphorylation. This effect was mediated through the eicosanoids, as overexpression of the deregulated enzymes in absence of arachidonic acid had no impact on insulin signaling. The stimulation of insulin signaling by EETs and depression of the pathway in insulin resistant liver suggest a likely role in hepatic insulin resistance. Our findings support therapeutic potential for inhibiting EET degradation.Hepatosteatosis has a strong association with hepatic insulin resistance, which plays a major role in the early stages of type 2 diabetes. Although the contribution of the liver to total energy consumption is not as high as other tissues, the liver is the main organ responsible for endogenous glucose production through gluconeogenesis and glycogenolysis (1). The liver''s pivotal role in type 2 diabetes is underscored by a strong correlation between fasting hyperglycemia and endogenous glucose production in patients (2). Studies on the early stages of hepatosteatosis and hepatic insulin resistance are complicated by the fact that patients are often unaware of their impaired insulin sensitivity. Therefore, the transition of the liver to an insulin resistant state is not as well studied as other aspects of the disease.To study early stage hepatic insulin resistance in an unbiased fashion, we analyzed the transition of the liver to an insulin-resistant state in a mouse model fed a high fat diet (HFD)¹, rich in safflower oil, on the proteome level. Phenotypic characterization in combination with proteomic profiling resulted in the identification of alterations in protein patterns, which were correlated with hepatic insulin resistance in a time-resolved manner. Protein expression was monitored using state of the art LC-MS/MS based proteomics, employing non-targeted discovery as well as targeted strategies.The comparison of expression profiles from HFD-fed mice with standard diet-fed controls directed us to a group of eicosanoid lipid mediators - epoxyeicosatrienoic acids (EET). Our proteomic approach uncovered a down-regulation of the EET pathway at the protein level through HFD feeding in insulin resistant mouse liver. In order to link expression patterns to signaling alterations and connect alterations on the level of signaling pathways to insulin sensitivity we proceeded to investigate the influence of these eicosanoids on insulin signaling in primary hepatocytes. Up to now, EETs have been extensively studied in the biology of blood vessels (3) and have been found to have profound influence on intracellular signaling (4–6) and ion channel activity (7) in endothelial as well as smooth muscle cell. Their vasodilating (7), anti-inflammatory (8) and proliferation inducing effects on endothelial cells (5, 6) have made inhibition of the EET degrading enzyme Ephx2 an attractive pharmacological strategy for the treatment of hypertension, with clinical trials already in progress (9).Recent studies using genetic mouse models have shown that knockdown or overexpression of EET pathway enzymes affect insulin secretion (10) and glucose homeostasis (11, 12) and point to as yet poorly understood effects of EETs on insulin sensitivity (10, 11). Moreover, EETs have been implicated in activating insulin signaling directly by increasing insulin receptor (IR) phosphorylation (11). In line with this model, medium supplementation, but not acute stimulation of the human hepatoma cell line HepG2 with high doses (30 μm) of EETs, has been shown to increase insulin mediated activation of Akt, the central protein kinase in insulin signaling (13).We show here that acute application of 4 μm of exogenous EETs but not overexpression of the EET pathway enzymes in absence of arachidonic acid had a strong positive effect on insulin mediated phosphorylation of Akt in primary mouse hepatocytes. The activation was not associated with changes in IR or insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation through EETs. These results indicate that EET influence insulin signaling downstream of IRS-1 and upstream of Akt rather than at the level of the IR.     

Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.

The gastric pathogen Helicobacter pylori uses a type IV secretion system to inject the bacterial CagA protein into gastric epithelial cells. Within the host cell, CagA becomes phosphorylated on tyrosine residues and initiates cytoskeletal rearrangements. We demonstrate here that Src-like protein-tyrosine kinases mediate CagA phosphorylation in vitro and in vivo. First, the Src-specific tyrosine kinase inhibitor PP2 specifically blocks CagA phosphorylation and cytoskeletal rearrangements thereby inhibiting the CagA-induced hummingbird phenotype of gastric epithelial cells. Second, CagA is in vivo phosphorylated by transiently expressed c-Src. Third, recombinant c-Src and lysates derived from c-Src-expressing fibroblasts but not lysates derived from Src-, Yes-, and Fyn-deficient cells phosphorylated CagA in vitro. Fourth, a transfected CagA-GFP fusion protein is phosphorylated in vivo in Src-positive fibroblasts but not in Src-, Yes-, and Fyn-deficient cells. Because a CagA-GFP fusion protein mutated in an EPIYA motif is not efficiently phosphorylated in any of these fibroblast cells, the CagA EPIYA motif appears to constitute the major c-Src phosphorylation site conserved among CagA-positive Helicobacter strains.     

The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation

The gastric pathogen Helicobacter pylori translocates the CagA protein into epithelial cells by a type IV secretion process. Translocated CagA is tyrosine phosphorylated (CagA(P-Tyr)) on specific EPIYA sequence repeats by Src family tyrosine kinases. Phos phorylation of CagA induces the dephosphorylation of as yet unidentified cellular proteins, rearrangements of the host cell actin cytoskeleton and cell scattering. We show here that CagA(P-Tyr) inhibits the catalytic activity of c-Src in vivo and in vitro. c-Src inactivation leads to tyrosine dephosphorylation of the actin binding protein cortactin. Concomitantly, cortactin is specifically redistributed to actin-rich cellular protrusions. c-Src inactivation and cortactin dephosphorylation are required for rearrangements of the actin cytoskeleton. Moreover, CagA(P-Tyr)-mediated c-Src inhibition downregulates further CagA phosphorylation through a negative feedback loop. This is the first report of a bacterial virulence factor that inhibits signalling of a eukaryotic tyrosine kinase and on a role of c-Src inactivation in host cell cytoskeletal rearrangements.     

Clustering protein sequences with a novel metric transformed from sequence similarity scores and sequence alignments with neural networks

Background  

The sequencing of the human genome has enabled us to access a comprehensive list of genes (both experimental and predicted) for further analysis. While a majority of the approximately 30000 known and predicted human coding genes are characterized and have been assigned at least one function, there remains a fair number of genes (about 12000) for which no annotation has been made. The recent sequencing of other genomes has provided us with a huge amount of auxiliary sequence data which could help in the characterization of the human genes. Clustering these sequences into families is one of the first steps to perform comparative studies across several genomes.