4-Octyl itaconate inhibits aerobic glycolysis by targeting GAPDH to exert anti-inflammatory effects.

Activated macrophages switch from oxidative phosphorylation to aerobic glycolysis, similar to the Warburg effect, presenting a potential therapeutic target in inflammatory disease. The endogenous metabolite itaconate has been reported to regulate macrophage function, but its precise mechanism is not clear. Here, we show that 4-octyl itaconate (4-OI, a cell-permeable itaconate derivative) directly alkylates cysteine residue 22 on the glycolytic enzyme GAPDH and decreases its enzyme activity. Glycolytic flux analysis by U13C glucose tracing provides evidence that 4-OI blocks glycolytic flux at GAPDH. 4-OI thereby downregulates aerobic glycolysis in activated macrophages, which is required for its anti-inflammatory effects. The anti-inflammatory effects of 4-OI are replicated by heptelidic acid, 2-DG and reversed by increasing wild-type (but not C22A mutant) GAPDH expression. 4-OI protects against lipopolysaccharide-induced lethality in vivo and inhibits cytokine release. These findings show that 4-OI has anti-inflammatory effects by targeting GAPDH to decrease aerobic glycolysis in macrophages.

In vitro photodynamic therapy of endothelial cells using hematoporphyrin monomethyl ether (Hemoporfin): Relevance to treatment of port wine stains.

Hemoporfin (hematoporphyrin monomethyl ether, HMME) is a relatively new photosensitizer that has achieved success in mediating photodynamic therapy (PDT) of port wine stains in China. However, the exact mechanism of Hemoporfin PDT on endothelial cell proliferation and apoptosis is unclear. The present study investigated the mechanism of action of HMME-PDT on endothelial cells in vitro. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. HMME-PDT treated the cells and detected the phototoxicity by cell counting kit-8 (CCK-8) assay, apoptosis by Flow cytometry assay and quantification of the secreted VEGF-A levels using ELISA and different proteins expression by quantitative real-time PCR and Western blotting. Phototoxicity was caused in an HMME and light dose-dependent manner. Apoptosis was induced as shown by Annexin-V/propidium iodide staining and morphological changes. The Bax/Bcl-2 ratio was increased as shown by Western blot for protein and RT-qPCR for mRNA. VEGF-A expression was reduced and signaling molecules in the Akt/mTOR pathway were inhibited as shown by ELISA and immunofluorescence. Hemoporfin (hematoporphyrin monomethyl ether, HMME) has achieved success in mediating photodynamic therapy (PDT) of port wine stains. The clinical success of HMME-PDT with low recurrence rates can be explained by inhibition of endothelial cell proliferation through VEGF/Akt /mTOR pathway.

Salicylic Acid Inhibits the Replication of Tomato bushy stunt virus by Directly Targeting a Host Component in the Replication Complex.

Although the plant hormone salicylic acid (SA) plays a central role in signaling resistance to viral infection, the underlying mechanisms are only partially understood. Identification and characterization of SA's direct targets have been shown to be an effective strategy for dissecting the complex SA-mediated defense signaling network. In search of additional SA targets, we previously developed two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SA-binding proteins (SABPs) from Arabidopsis. Using these approaches, we have now identified several members of the Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein family, including two chloroplast-localized and two cytosolic isoforms, as SABPs. Cytosolic GAPDH is a well-known glycolytic enzyme; it also is an important host factor involved in the replication of Tomato bushy stunt virus (TBSV), a single-stranded RNA virus. Using a yeast cell-free extract, an in vivo yeast replication system, and plant protoplasts, we demonstrate that SA inhibits TBSV replication. SA does so by inhibiting the binding of cytosolic GAPDH to the negative (-)RNA strand of TBSV. Thus, this study reveals a novel molecular mechanism through which SA regulates virus replication.

Catalysis of nitrite generation from nitroglycerin by glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Vascular relaxation to nitroglycerin (glyceryl trinitrate; GTN) requires its bioactivation by mechanisms that remain controversial. We report here that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the release of nitrite from GTN. In assays containing dithiothreitol (DTT) and NAD(+), the GTN reductase activity of purified GAPDH produces nitrite and 1,2-GDN as the major products. A vmax of 2.6nmolmin(-)(1)mg(-)(1) was measured for nitrite production by GAPDH from rabbit muscle and a GTN KM of 1.2mM. Reductive denitration of GTN in the absence of DTT results in dose- and time-dependent inhibition of GAPDH dehydrogenase activity. Disulfiram, a thiol-modifying drug, inhibits both the dehydrogenase and GTN reductase activity of GAPDH, while DTT or tris(2-carboxyethyl)phosphine reverse the GTN-induced inhibition. Incubation of intact human erythrocytes or hemolysates with 2mM GTN for 60min results in 50% inhibition of GAPDH's dehydrogenase activity, indicating that GTN is taken up by these cells and that the dehydrogenase is a target of GTN. Thus, erythrocyte GAPDH may contribute to GTN bioactivation.

Behavioral effects of cocaine mediated by nitric oxide-GAPDH transcriptional signaling.

Cocaine's behavioral-stimulant effects derive from potentiation of synaptic signaling by dopamine and serotonin leading to transcriptional alterations in postsynaptic cells. We report that a signaling cascade involving nitric oxide (NO) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mediates cocaine's transcriptional and behavioral actions. Lower, behavioral-stimulant doses enhance the cAMP response element-binding (CREB) signaling system, while higher, neurotoxic doses stimulate the p53 cytotoxic system. The drug CGP3466B, which potently and selectively blocks GAPDH nitrosylation and GAPDH-Siah binding, prevents these actions as well as behavioral effects of cocaine providing a strategy for anticocaine therapy.

New therapeutic targets to develop molecules active in drug-resistant epilepsies.

We have shown that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase involved in the endogenous phosphorylation of the alpha1 subunit of the gamma-aminobutyric acid (GABA)(A) receptor (GABA(A)R), maintaining GABA(A)-R function. GABA(A)R endogenous phosphorylation is opposed by one or several atypical phosphatases. We have shown in addition, using cerebral tissue obtained during epilepsy surgery and control tissue from patients undergoing brain tumor surgery, that both endogenous phosphorylation and GABA(A)R function are significantly reduced in the "epileptogenic" cerebral cortex when compared to control. This dysfunction likely contributes to seizure generation and/or transition from the interictal to the ictal state. The therapeutic challenge is to alleviate the endogenous phosphorylation deficiency of GABA(A)R in the epileptogenic cortical tissue, either through activating the endogenous kinase activity, or inhibiting dephosphorylation of the alpha1 subunit. Following the first trail, we have shown that spermine (the most effective polyamine) increases the GAPDH kinase activity on GABA(A)R and that subsequently such modulation potentiates its function as assessed by rundown studies on isolated neurons. Following the second trail, we have developed methods to identify these atypical membrane-bound phosphatases. Their activities were detected using two synthetic phosphopeptides corresponding to the alpha1 regions of phosphorylation by GAPDH. After purification, the active fractions are submitted to proteomic analysis by nanoLC-Maldi-TOF/TOF for protein identification. Two candidate proteins have been identified, which will be used as targets for high-throughput screening in order to develop original antiepileptic molecules.

A peptide inhibitor of c-Jun N-terminal kinase for the treatment of endotoxin-induced uveitis.

PURPOSE: To evaluate the effect of XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide that selectively inhibits the c-Jun N-terminal kinase, in the treatment of endotoxin-induced uveitis (EIU). METHODS: EIU was induced in Lewis rats by LPS injection. XG-102 was administered at the time of LPS challenge. The ocular biodistribution of XG-102 was evaluated using immunodetection at 24 hours after either 20 microg/kg IV (IV) or 0.2 microg/injection intravitreous (IVT) administrations in healthy or uveitic eyes. The effect of XG-102 on EIU was evaluated using clinical scoring, infiltration cell quantification, inducible nitric oxide synthase (iNOS) expression and immunohistochemistry, and cytokines and chemokines kinetics at 6, 24, and 48 hours using multiplex analysis on ocular media. Control EIU eyes received vehicle injection IV or IVT. The effect of XG-102 on c-Jun phosphorylation in EIU was evaluated by Western blot in eye tissues. RESULTS: After IVT injection, XG-102 was internalized in epithelial cells from iris/ciliary body and retina and in glial and microglial cells in both healthy and uveitic eyes. After IV injection, XG-102 was concentrated primarily in inflammatory cells of uveitic eyes. Using both routes of administration, XG-102 significantly inhibited clinical signs of EIU, intraocular cell infiltration, and iNOS expression together with reduced phosphorylation of c-Jun. The anti-inflammatory effect of XG-102 was mediated by iNOS, IFN-gamma, IL-2, and IL-13. CONCLUSIONS: This is the first evidence that interfering with the JNK pathway can reduce intraocular inflammation. Local administration of XG-102, a clinically evaluated peptide, may have potential for treating uveitis.

Phosphoinositide 3-kinase is a novel target of piceatannol for inhibiting PDGF-BB-induced proliferation and migration in human aortic smooth muscle cells.

AIMS: Abnormal migration and proliferation of human aortic smooth muscle cells (HASMCs) to the intima causes intimal thickening of the aorta, which is strongly related to the development of atherosclerosis. Previous studies have suggested that red wine polyphenols, particularly resveratrol, have great protective effects against cardiovascular diseases. Here, we compared the anti-atherosclerotic effect of piceatannol, a metabolite of resveratrol, and its underlying mechanisms. METHODS AND RESULTS: We demonstrated that piceatannol inhibited platelet-derived growth factor (PDGF)-BB-induced cell migration using a modified Boyden chamber assay and wound healing assay. Western blot analysis showed that PDGF-BB-induced phosphorylation of Akt, p70S6K, and p38 was inhibited by piceatannol, but not resveratrol. In vitro and ex vivo phosphoinositide 3-kinase (PI3K) assays demonstrated that piceatannol suppressed PI3K activity more effectively than resveratrol. PDGF-BB-induced migration and proliferation of HASMCs were inhibited by treatment with a commercial PI3K inhibitor, LY294002. Both in vitro and ex vivo pull-down assays revealed that piceatannol directly binds with sepharose 4B-PI3K beads in an ATP-competitive manner. CONCLUSION: The results of the present study demonstrate that piceatannol directly binds with PI3K in an ATP-competitive manner and suppresses PI3K activity with anti-atherosclerotic effects.

Glycolaldehyde induces apoptosis in a human breast cancer cell line.

Activated phagocytes employ myeloperoxidase to generate glycolaldehyde, 2-hydroxypropanal, and acrolein. Because alpha-hydroxy and alpha,beta-unsaturated aldehydes are highly reactive, phagocyte-mediated formation of these products may play a role in killing bacteria and tumor cells. Using breast cancer cells, we demonstrate that glycolaldehyde inactivates glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and Cu,Zn superoxide dismutase, suppresses cell growth, and induces apoptosis. These results suggest that glycolaldehyde might be an important mediator of neutrophil anti-tumor activity.