Effect of long-term exposure of SH-SY5Y cells to morphine: a whole cell proteomic analysis

BACKGROUND: Opiate addiction reflects plastic changes that endurably alter synaptic transmission within relevant neuronal circuits. The biochemical mechanisms of these adaptations remain largely unknown and proteomics-based approaches could lead to a broad characterization of the molecular events underlying adaptations to chronic drug exposure. RESULTS: Thus, we have started proteomic analyses of the effects of chronic morphine exposure in a recombinant human neuroblastoma SH-SY5Y clone that stably overexpresses the μ-opioid receptor. Cells were treated with morphine for 6, 24 and 72 hours, the proteins were separated by 2-D gel electrophoresis and stained with Coomassie blue, and the protein map was compared with that obtained from untreated cells. Spots showing a statistically significant variation were selected for identification using mass spectrometric analyses. CONCLUSION: A total of 45 proteins were identified, including proteins involved in cellular metabolism, cytoskeleton organization, vesicular trafficking, transcriptional and translational regulation, and cell signaling

The Rab5 Effector Rabankyrin-5 Regulates and Coordinates Different Endocytic Mechanisms

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells

An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway

Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kβ, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinaseβ (PI3Kβ), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis

From toxic waste to beneficial nutrient: acetate boosts <i>Escherichia coli</i> growth at low glycolytic flux

Abstract Acetate is a major by-product of glycolytic metabolism in Escherichia coli and many other microorganisms. It has long been considered a toxic waste compound that inhibits microbial growth, but this counterproductive auto-inhibition, which represents a major problem in biotechnology, has puzzled the scientific community for decades. Recent studies have revealed that acetate is also a co-substrate of glycolytic nutrients and a global regulator of E. coli metabolism and physiology. However, most of these insights were obtained at high glycolytic flux and little is known about the role of acetate at lower glycolytic fluxes, conditions that are nevertheless frequently experienced by E. coli in natural, industrial and laboratory environments. Here, we used a systems biology strategy to investigate the mutual regulation of glycolytic and acetate metabolism. Computational and experimental results demonstrate that reducing the glycolytic flux enhances co-utilization of acetate and glucose through the Pta-AckA pathway. Enhanced acetate metabolism compensates for the reduction in glycolytic flux and eventually buffers carbon uptake so that acetate, far from being toxic, actually enhances E. coli growth under these conditions. The same mechanism of increased growth was also observed on glycerol and galactose, two nutrients with a natively low glycolytic flux. Therefore, acetate makes E. coli more robust to glycolytic perturbations and is a valuable nutrient, with a beneficial effect on microbial growth. Finally, we show that some evolutionarily conserved design principles of eukaryotic fermentative metabolism are also present in bacteria. Significance Statement Acetate, a by-product of glycolytic metabolism in many microorganisms including Escherichia coli , is traditionally viewed as a toxic waste compound. Here, we demonstrate that this is only the case at high glycolytic fluxes. At low glycolytic fluxes in contrast, acetate acts as a co-substrate of glycolytic nutrients and boosts E. coli growth. Acetate also improves E. coli ’s robustness to glycolytic perturbations. We clarify the functional relationship between glycolytic and acetate metabolisms, show that acetate is a beneficial co-substrate of glycolytic nutrients used by E. coli in bioprocesses and in the gut, and provide insights into the underlying biochemical and regulatory mechanisms

Acetate is a beneficial nutrient for E. coli at low glycolytic flux

International audienceAcetate, a major by-product of glycolytic metabolism in Escherichia coli and many other microorganisms, has long been considered a toxic waste compound that inhibits microbial growth. This counterproductive auto-inhibition represents a major problem in biotechnology and has puzzled the scientific community for decades. Recent studies have however revealed that acetate is also a co-substrate of glycolytic nutrients and a global regulator of E. coli metabolism and physiology. Here, we used a systems biology strategy to investigate the mutual regulation of glycolytic and acetate metabolism in E. coli. Computational and experimental analyses demonstrate that decreasing the glycolytic flux enhances co-utilization of acetate with glucose. Acetate metabolism thus compensates for the reduction in glycolytic flux and eventually buffers carbon uptake so that acetate, rather than being toxic, actually enhances E. coli growth under these conditions. We validated this mechanism using three orthogonal strategies: chemical inhibition of glucose uptake, glycolytic mutant strains, and alternative substrates with a natively low glycolytic flux. In summary, acetate makes E. coli more robust to glycolytic perturbations and is a valuable nutrient, with a beneficial effect on microbial growth

From toxic waste to beneficial nutrient: acetate boosts <i>Escherichia coli</i> growth at low glycolytic flux

Acetate, a major by-product of glycolytic metabolism in Escherichia coli and many other microorganisms, has long been considered a toxic waste compound that inhibits microbial growth. This counterproductive auto-inhibition represents a major problem in biotechnology and has puzzled the scientific community for decades. Recent studies have revealed that acetate is also a co-substrate of glycolytic nutrients and a global regulator of E. coli metabolism and physiology. Here, we used a systems biology strategy to investigate the mutual regulation of glycolytic and acetate metabolism. Computational and experimental results demonstrate that reducing the glycolytic flux enhances co-utilization of acetate with glucose. Acetate metabolism thus compensates for the reduction in glycolytic flux and eventually buffers carbon uptake so that acetate, far from being toxic, actually enhances E. coli growth under these conditions. We validated this mechanism using three orthogonal strategies: chemical inhibition of glucose uptake, glycolytic mutant strains, and alternative substrates with a natively low glycolytic flux. Acetate makes E. coli more robust to glycolytic perturbations and is a valuable nutrient, with a beneficial effect on microbial growth

Control and regulation of acetate overflow in Escherichia coli

International audienceOverflow metabolism refers to the production of seemingly wasteful by-products by cells during growth on glucose even when oxygen is abundant. Two theories have been proposed to explain acetate overflow in Escherichia coli – global control of the central metabolism and local control of the acetate pathway – but neither accounts for all observations. Here, we develop a kinetic model of E. coli metabolism that quantitatively accounts for observed behaviors and successfully predicts the response of E. coli to new perturbations. We reconcile these theories and clarify the origin, control and regulation of the acetate flux. We also find that, in turns, acetate regulates glucose metabolism by coordinating the expression of glycolytic and TCA genes. Acetate should not be considered a wasteful end-product since it is also a co-substrate and a global regulator of glucose metabolism in E. coli. This has broad implications for our understanding of overflow metabolism

Cullin 5-RING E3 ubiquitin ligases, new therapeutic targets?

International audienceUbiquitylation is a reversible post-translational modification of proteins that controls a myriad of functions and cellular processes. It occurs through the sequential action of three distinct enzymes. E3 ubiquitin ligases (E3s) play the role of conductors of the ubiquitylation pathway making them attractive therapeutic targets. This review is dedicated to the largest family of multimeric E3s, the Cullin-RING E3 (CRL) family and more specifically to cullin 5 based CRLs that remains poorly characterized

A Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Approach to Identify the Origin of the Glycan Heterogeneity of Diptericin, anO-Glycosylated Antibacterial Peptide from Insects

International audienceIn a previous study, electrospray ionization mass spectrometry was used to analyze the structure of theO-glycopeptide diptericin, an antibacterial peptide from the fleshflyPhormia terranovae.Several glycoforms of diptericin differing in the length of their oligosaccharide chains were present at the final stage of purification. In order to determine the origin of this glycan heterogeneity, we analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI–MS) the relative abundance of the different diptericin glycoforms in fractions obtained after each purification step, and directly in the hemolymph and in the fat body which produces diptericin. MALDI–MS clearly shows that the purification procedure had no effect on theO-linked oligosaccharide chains of diptericin, suggesting that diptericin is synthesized as a family of heterogeneous glycopeptides. In addition, in these experiments, differential mapping by MALDI–MS of the hemolymph and fat body tissue from bacteria-challenged and naive larvae allowed us to detect induced or repressed molecules which may be involved in the immune response ofP. terranovae