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The Golgi membrane protein GOLM1/GP73/GOLPH2 has been found to impact cytokine production in both infectious disease and cancer. In viral infections, GOLM1 levels are increased, and this lowers the production of type I interferons and other inflammatory cytokines. However, elevated GOLM1 expression levels due to mutations are linked to a higher production of interleukin (IL)‐6 during Candida infections, potentially explaining an increased susceptibility to candidemia in individuals carrying these mutations. In cancer, the protease Furin produces a soluble form of GOLM1 that has oncogenic properties by promoting the production of the chemokine CCL2 and suppressing the production of inflammatory cytokines such as IL‐12 and interferon gamma. This review will focus on the role of GOLM1 in cytokine production, highlighting how it can both promote and inhibit cytokine production. It is crucial to understand this in order to effectively target GOLM1 for therapeutic purposes in diseases associated with abnormal cytokine production, including cancer and infectious disease. [ABSTRACT FROM AUTHOR]

Aim: Defects in hepatic glycogen synthesis contribute to post‐prandial hyperglycaemia in type 2 diabetic patients. Chromogranin A (CgA) peptide Catestatin (CST: hCgA352‐372) improves glucose tolerance in insulin‐resistant mice. Here, we seek to determine whether CST induces hepatic glycogen synthesis. Methods: We determined liver glycogen, glucose‐6‐phosphate (G6P), uridine diphosphate glucose (UDPG) and glycogen synthase (GYS2) activities; plasma insulin, glucagon, noradrenaline and adrenaline levels in wild‐type (WT) as well as in CST knockout (CST‐KO) mice; glycogen synthesis and glycogenolysis in primary hepatocytes. We also analysed phosphorylation signals of insulin receptor (IR), insulin receptor substrate‐1 (IRS‐1), phosphatidylinositol‐dependent kinase‐1 (PDK‐1), GYS2, glycogen synthase kinase‐3β (GSK‐3β), AKT (a kinase in AKR mouse that produces Thymoma)/PKB (protein kinase B) and mammalian/mechanistic target of rapamycin (mTOR) by immunoblotting. Results: CST stimulated glycogen accumulation in fed and fasted liver and in primary hepatocytes. CST reduced plasma noradrenaline and adrenaline levels. CST also directly stimulated glycogenesis and inhibited noradrenaline and adrenaline‐induced glycogenolysis in hepatocytes. In addition, CST elevated the levels of UDPG and increased GYS2 activity. CST‐KO mice had decreased liver glycogen that was restored by treatment with CST, reinforcing the crucial role of CST in hepatic glycogenesis. CST improved insulin signals downstream of IR and IRS‐1 by enhancing phospho‐AKT signals through the stimulation of PDK‐1 and mTORC2 (mTOR Complex 2, rapamycin‐insensitive complex) activities. Conclusions: CST directly promotes the glycogenic pathway by (a) reducing glucose production, (b) increasing glycogen synthesis from UDPG, (c) reducing glycogenolysis and (d) enhancing downstream insulin signalling. [ABSTRACT FROM AUTHOR]

In autophagy, LC3‐positive autophagophores fuse and encapsulate the autophagic cargo in a double‐membrane structure. In contrast, lipidated LC3 (LC3‐II) is directly formed at the phagosomal membrane in LC3‐associated phagocytosis (LAP). In this study, we dissected the effects of autophagy inhibitors on LAP. SAR405, an inhibitor of VPS34, reduced levels of LC3‐II and inhibited LAP. In contrast, the inhibitors of endosomal acidification bafilomycin A1 and chloroquine increased levels of LC3‐II, due to reduced degradation in acidic lysosomes. However, while bafilomycin A1 inhibited LAP, chloroquine did not. Finally, EACC, which inhibits the fusion of autophagosomes with lysosomes, promoted LC3 degradation possibly by the proteasome. Targeting LAP with small molecule inhibitors is important given its emerging role in infectious and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Aim: A "leaky" gut barrier has been implicated in the initiation and progression of a multitude of diseases, for example, inflammatory bowel disease (IBD), irritable bowel syndrome and celiac disease. Here we show how pro‐hormone Chromogranin A (CgA), produced by the enteroendocrine cells, and Catestatin (CST: hCgA352‐372), the most abundant CgA‐derived proteolytic peptide, affect the gut barrier. Methods: Colon tissues from region‐specific CST‐knockout (CST‐KO) mice, CgA‐knockout (CgA‐KO) and WT mice were analysed by immunohistochemistry, western blot, ultrastructural and flowcytometry studies. FITC‐dextran assays were used to measure intestinal barrier function. Mice were supplemented with CST or CgA fragment pancreastatin (PST: CgA250‐301). The microbial composition of cecum was determined. CgA and CST levels were measured in blood of IBD patients. Results: Plasma levels of CST were elevated in IBD patients. CST‐KO mice displayed (a) elongated tight, adherens junctions and desmosomes similar to IBD patients, (b) elevated expression of Claudin 2, and (c) gut inflammation. Plasma FITC‐dextran measurements showed increased intestinal paracellular permeability in the CST‐KO mice. This correlated with a higher ratio of Firmicutes to Bacteroidetes, a dysbiotic pattern commonly encountered in various diseases. Supplementation of CST‐KO mice with recombinant CST restored paracellular permeability and reversed inflammation, whereas CgA‐KO mice supplementation with CST and/or PST in CgA‐KO mice showed that intestinal paracellular permeability is regulated by the antagonistic roles of these two peptides: CST reduces and PST increases permeability. Conclusion: The pro‐hormone CgA regulates the intestinal paracellular permeability. CST is both necessary and sufficient to reduce permeability and primarily acts by antagonizing PST. [ABSTRACT FROM AUTHOR]

Immune cells communicate by releasing large quantities of cytokines. Although the mechanisms of cytokine secretion are increasingly understood, quantitative knowledge of the number of cytokines per vesicle is still lacking. Here, we measured with quantitative microscopy the release rate of vesicles potentially carrying interleukin‐6 (IL‐6) in human dendritic cells. By comparing this to the total secreted IL‐6, we estimate that secretory vesicles contain about 0.5–3 IL‐6 molecules, but with a large spread among cells/donors. Moreover, IL‐6 did not accumulate within most cells, indicating that synthesis and not trafficking is the bottleneck for IL‐6 production. IL‐6 accumulated in the Golgi apparatus only in ~ 10% of the cells. Understanding how immune cells produce cytokines is important for designing new immunomodulatory drugs. [ABSTRACT FROM AUTHOR]

We determined the diffusion coefficients ( D) of (macro)molecules of different sizes (from ∼0.5 to 600 kDa) in the cytoplasm of live Escherichia coli cells under normal osmotic conditions and osmotic upshift. D values decreased with increasing molecular weight of the molecules. Upon osmotic upshift, the decrease in D of NBD-glucose was much smaller than that of macromolecules. Barriers for diffusion were found in osmotically challenged cells only for GFP and larger proteins. These barriers are likely formed by the nucleoid and crowding of the cytoplasm. The cytoplasm of E. coli appears as a meshwork allowing the free passage of small molecules while restricting the diffusion of bigger ones. [ABSTRACT FROM AUTHOR]

The effect of osmotic stress on the intracellular diffusion of proteins in Escherichia coli was studied, using a pulsed version of fluorescence recovery after photo-bleaching, pulsed-FRAP. This method employs sequences of laser pulses which only partly bleach the fluorophores in a cell. Because the cell size and geometry are taken into account, pulsed-FRAP enables to measure diffusion in very small cells of different shapes. We found that upon an osmotic upshock from 0.15 to 0.6 Osm, imposed by NaCl or sorbitol, the apparent intracellular diffusion ( D) of mobile green fluorescent protein (GFP) decreased from 3.2 to 0.4 μm2 s−1, whereas the membrane permeable glycerol had no effect. Exposing E. coli cells to higher osmolalities (> 0.6 Osm) led to compartmentalization of the GFP into discrete pools, from where the GFP could not escape. Although free diffusion through the cell was hindered, the mobility of GFP in these pools was still relatively high ( D∼0.4 μm2 s−1). The presence of osmoprotectants restored the effect of osmotic stress on the protein mobility and apparent compartmentalization. Also, lowering the osmolality from 0.6 Osm back to 0.15 Osm restored the mobility of GFP. The implications of these findings in terms of heterogeneities and diffusive barriers inside the cell are discussed. [ABSTRACT FROM AUTHOR]

Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeIImtl, is a member of the phosphoenolpyruvate-dependent phosphotransferase system. During the transport cycle, mannitol is phosphorylated and released into the cytoplasm as mannitol-1-phosphate. Several studies have shown that EIImtl functions as an oligomeric species. However, the oligomerization number and stability of the oligomeric complex during different steps of the catalytic cycle, e.g., substrate binding and/or phosphorylation of the carrier, is still under discussion. In this paper, we have addressed the oligomeric state and stability of EIImtl using fluorescence correlation spectroscopy. A functional double-cysteine mutant was site-specifically labeled with either Alexa Fluor 488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins was followed in time during different steps of the catalytic cycle. The most important conclusions are that (1) in a detergent-solubilized state, EIImtl is functional as a very stable dimer; (2) the stability of the complex can be manipulated by changing the intermicellar attractive forces between PEG-based detergent micelles; (3) substrate binding destabilizes the complex whereas phosphorylation increases the stability; and (4) substrate binding to the phosphorylated species partly antagonizes the stabilizing effect. [ABSTRACT FROM AUTHOR]