Is vacuole the richest store of IP3-mobilizable calcium in plant cells?

Inositol trisphosphate is known to mobilize calcium from internal stores in plant cells. However, with the exception of the vacuole, the largest plant cell compartment, organelles responsive to inositol trisphosphate have not been extensively identified. In this way, we have separated membrane vesicles from the same carrot microsomal fraction and identified them, both by marker enzyme activities and electron microscopy. These correspond to pure plasma membrane, pure tonoplast and mixed mitochondria, endoplasmic reticulum, Golgi membrane fractions. All the fractions accumulated calcium in a ATP-dependent manner and were tightly sealed. Inositol trisphosphate-dependent calcium releases were accurately measured only in fractions corresponding functionally and structurally to tonoplast, the vacuolar membrane. The process was dose-dependent and fairly specific for inositol trisphosphate. While highly significant, approximately 40% of the mobile calcium only may be released from tonoplast vesicles by inositol trisphosphate which remained basically intact during the release experiments. From these results it is concluded that the vacuole is the richest store of calcium directly mobilizable by inositol trisphosphate in plant cells, but inositol trisphosphate is not able to release the overall mobile vacuolar calcium.     

The role of lipid second messengers in aldosterone synthesis and secretion

Second messengers are small rapidly diffusing molecules or ions that relay signals between receptors and effector proteins to produce a physiological effect. Lipid messengers constitute one of the four major classes of second messengers. The hydrolysis of two main classes of lipids, glycerophospholipids and sphingolipids, generate parallel profiles of lipid second messengers: phosphatidic acid (PA), diacylglycerol (DAG), and lysophosphatidic acid versus ceramide, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, respectively. In this review, we examine the mechanisms by which these lipid second messengers modulate aldosterone production at multiple levels. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis. Primary aldosteronism is a frequent endocrine cause of secondary hypertension. A thorough understanding of the signaling events regulating aldosterone biosynthesis may lead to the identification of novel therapeutic targets. The cumulative evidence in this literature emphasizes the critical roles of PA, DAG, and sphingolipid metabolites in aldosterone synthesis and secretion. However, it also highlights the gaps in our knowledge, such as the preference for phospholipase D-generated PA or DAG, as well as the need for further investigation to elucidate the precise mechanisms by which these lipid second messengers regulate optimal aldosterone production.     

Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.     

Denervation Supersensitivity of the Rat Pineal to Norepinephrine-Stimulated [3H]Inositide Turnover Revealed by Lithium and a Convenient Procedure

A convenient procedure for the assay of myo-[2-3H(N)]inositol ([3H]inositol) metabolites in cells or small amounts of tissue was developed. The procedure is a composite of modifications of published methods. After preincubation with [3H]inositol, rat pineal glands were disrupted in an acidified organic solvent mixture. Lipids were separated from the hydrophilic products and precursor using Sephadex G-25 columns and further analyzed by TLC. Hydrophilic products were further analyzed by anion-exchange column chromatography using Dowex AG1-X8 (formate form). In the presence of lithium, increases in inositol phosphates consequent to stimulation of the glands by norepinephrine were apparent within 10 min. The response in denervated glands was considerably greater than in intact pineals.     

Endoplasmic reticulum stress in insulin resistance and diabetes

The endoplasmic reticulum is the main intracellular Ca²⁺ store for Ca²⁺ release during cell signaling. There are different strategies to avoid ER Ca²⁺ depletion. Release channels utilize first Ca²⁺-bound to proteins and this minimizes the reduction of the free luminal [Ca²⁺]. However, if release channels stay open after exhaustion of Ca²⁺-bound to proteins, then the reduction of the free luminal ER [Ca²⁺] (via STIM proteins) activates Ca²⁺ entry at the plasma membrane to restore the ER Ca²⁺ load, which will work provided that SERCA pump is active. Nevertheless, there are several noxious conditions that result in decreased activity of the SERCA pump such as oxidative stress, inflammatory cytokines, and saturated fatty acids, among others. These conditions result in a deficient restoration of the ER [Ca²⁺] and lead to the ER stress response that should facilitate recovery of the ER. However, if the stressful condition persists then ER stress ends up triggering cell death and the ensuing degenerative process leads to diverse pathologies; particularly insulin resistance, diabetes and several of the complications associated with diabetes. This scenario suggests that limiting ER stress should decrease the incidence of diabetes and the mobility and mortality associated with this illness.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.     

Intraluminal hydrogen peroxide induces a permeability change of the endoplasmic reticulum membrane

Gulonolactone treatment of mice resulted in the elevation of hepatic ascorbate and hydrogen peroxide levels accompanied by transient liver swelling and reversible dilatation of endoplasmic reticulum cisternae. Although a decrease in glutathione (reduced form)/total glutathione ratio was observed in microsomes, the redox state of luminal foldases remained unchanged and the signs of endoplasmic reticulum stress were absent. Increased permeability of the microsomal membrane to various compounds of low molecular weight was substantiated. It is assumed that Gulonolactone-dependent luminal hydrogen peroxide formation in the endoplasmic reticulum provokes a temporary increase in non-selective membrane permeability, which results in the dilation of the organelle and in enhanced transmembrane fluxes of small molecules.     

高效合成葡萄糖二酸酿酒酵母工程菌的构建

葡萄糖二酸是天然存在的一种重要二元酸,其在医疗保健和化工工业等领域具有很高的实际应用价值,因此被称为“最具价值的生物炼制产品之一”。以酿酒酵母(Saccharomyces cerevisiae)为底盘微生物,文中考察了过量表达肌醇转运蛋白Itr1、融合表达肌醇加氧酶和葡萄糖醛酸脱氢酶以及弱化表达葡萄糖6-磷酸脱氢酶基因ZWF1三种策略对葡萄糖二酸产量的影响。研究结果显示,过量表达肌醇转运蛋白Itr1使葡萄糖二酸产量在摇瓶发酵条件下较出发菌株Bga-3提高了26%;MIOX4-Udh融合蛋白的表达使葡萄糖二酸的产量较Bga-3菌株提高了40%;在此基础上,弱化表达葡萄糖6-磷酸脱氢酶基因ZWF1后,葡萄糖二酸的产量达5.5 g/L,较相同发酵条件下Bga-3菌株提高了60%。在5 L发酵罐中,该菌株葡萄糖二酸的最高产量达10.85 g/L,较Bga-3菌株提高了80%。由此可见,上述代谢改造策略的应用在很大程度上提高了葡萄糖二酸的途径效率和产量,为通过代谢工程方法在酿酒酵母中合成其他化合物的研究提供了参考。     

Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay

The Orai1 Ca²⁺ permeable ion channel is an important component of store operated Ca²⁺ entry (SOCE) in cells. It’s over-expression in basal molecular subtype breast cancers has been linked with poor prognosis, making it a potential target for drug development. We pharmacologically characterised a number of reported inhibitors of SOCE in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader (FLIPR) assay, and show that the rank order of their potencies in this assay is the same as those reported in a wide range of published assays. The assay was also used in a screening project seeking novel inhibitors. Following a broad literature survey of classes of calcium channel inhibitors we used simplified ligand structures to query the ZINC on-line database, and following two iterations of refinement selected a novel Orai1-selective dichlorophenyltriazole hit compound. Analogues of this were synthesized and evaluated in the FLIPR assay to develop structure–activity relationships (SAR) for the three domains of the hit; triazole (head), dichlorophenyl (body) and substituted phenyl (tail). For this series, the results suggested the need for a lipophilic tail domain and an out-of-plane twist between the body and tail domains.