Curcumin prevents proteins expression changes of oxidative phosphorylation, cellular stress response, and lipid metabolism proteins in liver of mice fed a high-fructose diet.

Increased fructose consumption has been associated with the development of metabolic diseases due to the modification in protein expression, altering metabolic and signaling pathways. Curcumin is a natural compound with a regulatory effect on genes and metabolic pathways. To identify the fructose-induced protein expression changes and the effect of curcumin on the change of protein expression in the liver of mice fed a standard diet and a high fructose diet, to elucidate the global role of curcumin. Four groups (n = 4/group) of male mice (C57BL6J) of six-weeks-old were formed. One group received a standard diet (C); another received curcumin at 0.75% w/w in the feed (C + C); one more received 30% w/v fructose in drinking water (F); and one group received 30% w/v fructose in drinking water and 0.75% w/w curcumin in food (F + C); for 15 weeks. Proteomic analysis was performed by LC-MS/MS, using the label-free technique with the MaxQuant programs for identification and Perseus for expression change analysis. Differentially expressed proteins (fold change ≥1.5 and p < 0.5) were analyzed by gene ontology and KEGG. A total of 1047 proteins were identified, of which 113 changed their expression in mice fed fructose, compared to the control group, and curcumin modified the expression of 64 proteins in mice fed fructose and curcumin compared to mice that only received fructose. Curcumin prevented the change of expression of 13 proteins involved in oxidative phosphorylation (NDUFB8, NDUFB3, and ATP5L) in the cellular response to stress (PSMA5, HIST1H1D) and lipid metabolism (THRSP, DGAT1, ECI1, and ACOT13). Curcumin in mice fed the standard diet increased the expression of proteins related to oxidative phosphorylation, ribosomes, and PPAR pathways. In addition to fructose, increased expression of proteins involved in oxidative phosphorylation, ribosomes, lipid metabolism, and carbon metabolism. However, curcumin prevented expression change in 13 hepatic proteins of fructose-fed mice involved in oxidative phosphorylation, cellular stress response, and lipid metabolism. SIGNIFICANCE: Curcumin is a natural compound with a regulatory effect on proteins and metabolic pathways. So, curcumin prevents the change of expression in 13 hepatic proteins of fructose-fed mice involved in oxidative phosphorylation, cellular stress response and lipid metabolism, as a supplement with protector activity on fructose-induced toxic effects.

GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.

Hepatocellular carcinoma is the most frequent primary liver cancer. Macroautophagy/autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two hepatocellular carcinoma in vivo models. We showed that due to its lysosomotropic properties, GNS561 could reach and specifically inhibited its enzyme target, PPT1 (palmitoyl-protein thioesterase 1), resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of MTOR (mechanistic target of rapamycin kinase), lysosomal membrane permeabilization, caspase activation and cell death. Accordingly, GNS561, for which a global phase 1b clinical trial in liver cancers was just successfully achieved, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment.Abbreviations: ANXA5:annexin A5; ATCC: American type culture collection; BafA1: bafilomycin A1; BSA: bovine serum albumin; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CCND1: cyclin D1; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; CQ: chloroquine; iCCA: intrahepatic cholangiocarcinoma; DEN: diethylnitrosamine; DMEM: Dulbelcco's modified Eagle medium; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HDSF: hexadecylsulfonylfluoride; IC50: mean half-maximal inhibitory concentration; LAMP: lysosomal associated membrane protein; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3; LMP: lysosomal membrane permeabilization; MALDI: matrix assisted laser desorption ionization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MRI: magnetic resonance imaging; NH4Cl: ammonium chloride; NtBuHA: N-tert-butylhydroxylamine; PARP: poly(ADP-ribose) polymerase; PBS: phosphate-buffered saline; PPT1: palmitoyl-protein thioesterase 1; SD: standard deviation; SEM: standard error mean; vs, versus; Zn2+: zinc ion; Z-Phe: Z-Phe-Tyt(tBu)-diazomethylketone; Z-VAD-FMK: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone.

Targeted eradication of gastric cancer stem cells by CD44 targeting USP22 small interfering RNA-loaded nanoliposomes.

AIM: USP22, a member of ubiquitin-specific proteases (USPs), is a well-defined protein that promotes poor prognosis, invasion and metastasis, and also participates in the maintenance of cancer stem cells. USP22 siRNA-loaded nanoliposomes conjugated with CD44 antibodies (USP22-NLs-CD44) were constructed to enhance the therapeutic effect of USP22 siRNA against gastric cancer stem cells. MATERIALS & METHODS: The targeting and therapeutic efficacies of USP22-NLs-CD44 against gastric cancer stem cells were evaluated. RESULTS & CONCLUSION: USP22-NLs-CD44 was demonstrated to be able to effectively deliver USP22 siRNA to CD44+ gastric cancer stem cells, achieving superior therapeutic effects against CD44+ gastric cancer stem cells than nontargeted nanoliposomes. USP22-NLs-CD44 may provide a novel approach to eradicate gastric cancer stem cells in the near future.

Thioesterase superfamily member 2 promotes hepatic insulin resistance in the setting of glycerol-3-phosphate acyltransferase 1-induced steatosis.

Hepatic insulin resistance in the setting of steatosis is attributable at least in part to the accumulation of bioactive lipids that suppress insulin signaling. The mitochondria-associated glycerol-3-phosphate acyltransferase 1 (GPAT1) catalyzes the first committed step in glycerolipid synthesis, and its activity diverts fatty acids from mitochondrial ß-oxidation. GPAT1 overexpression in mouse liver leads to hepatic steatosis even in the absence of overnutrition. The mice develop insulin resistance owing to the generation of saturated diacylglycerol and phosphatidic acid molecular species that reduce insulin signaling by activating PKCϵ and by suppressing mTORC2, respectively. Them2, a mitochondria-associated acyl-CoA thioesterase, also participates in the trafficking of fatty acids into oxidative versus glycerolipid biosynthetic pathways. Them2-/- mice are protected against diet-induced hepatic steatosis and insulin resistance. To determine whether Them2 contributes to hepatic insulin resistance due to hepatic overexpression of GPAT1, recombinant adenovirus was used to overexpress GPAT1 in livers of chow-fed Them2+/+ and Them2-/- mice. Hepatic GPAT1 overexpression led to steatosis in both genotypes. In the setting of GPAT1 overexpression, glucose tolerance was reduced in Them2+/+ but not Them2-/- mice, without influencing whole-body insulin sensitivity or basal hepatic glucose production. Improved glucose tolerance in Them2-/- mice was associated with reduced PKCϵ translocation. Preserved insulin receptor activity was supported by Thr-308 phosphorylation of Akt following GPAT1 overexpression in Them2-/- hepatocytes. These findings suggest a pathogenic role of Them2 in the biosynthesis of glycerolipid metabolites that promote hepatic insulin resistance.

Synergistic effects of treating the spinal cord and brain in CLN1 disease.

Infantile neuronal ceroid lipofuscinosis (INCL, or CLN1 disease) is an inherited neurodegenerative storage disorder caused by a deficiency of the lysosomal enzyme palmitoyl protein thioesterase 1 (PPT1). It was widely believed that the pathology associated with INCL was limited to the brain, but we have now found unexpectedly profound pathology in the human INCL spinal cord. Similar pathological changes also occur at every level of the spinal cord of PPT1-deficient (Ppt1-/- ) mice before the onset of neuropathology in the brain. Various forebrain-directed gene therapy approaches have only had limited success in Ppt1-/- mice. Targeting the spinal cord via intrathecal administration of an adeno-associated virus (AAV) gene transfer vector significantly prevented pathology and produced significant improvements in life span and motor function in Ppt1-/- mice. Surprisingly, forebrain-directed gene therapy resulted in essentially no PPT1 activity in the spinal cord, and vice versa. This leads to a reciprocal pattern of histological correction in the respective tissues when comparing intracranial with intrathecal injections. However, the characteristic pathological features of INCL were almost completely absent in both the brain and spinal cord when intracranial and intrathecal injections of the same AAV vector were combined. Targeting both the brain and spinal cord also produced dramatic and synergistic improvements in motor function with an unprecedented increase in life span. These data show that spinal cord pathology significantly contributes to the clinical progression of INCL and can be effectively targeted therapeutically. This has important implications for the delivery of therapies in INCL, and potentially in other similar disorders.

Platelets possess and require an active protein palmitoylation pathway for agonist-mediated activation and in vivo thrombus formation.

OBJECTIVE: Several platelet proteins are palmitoylated, but whether protein palmitoylation functions in platelet activation is unknown. We sought to determine the role of platelet protein palmitoylation in platelet activation and thrombus formation. METHODS AND RESULTS: Platelet proteins were depalmitoylated by infusing acyl-protein thioesterase 1 into permeabilized platelets. In intact platelets, platelet protein palmitoylation was blocked using the protein palmitoylation inhibitor cerulein. The effects of inhibiting platelet protein palmitoylation on platelet function and on thrombus formation in vivo were evaluated. When infused into permeabilized platelets, acyl-protein thioesterase 1 reduced total platelet protein palmitoylation and inhibited protease-activated receptor-1-mediated alpha-granule secretion with an IC50 of 175 nmol/L and maximal inhibition of > or = 90%. G(alpha q) and SNAP-23, membrane-associated proteins that are constitutively palmitoylated, translocated to the cytosol when permeabilized platelets were exposed to recombinant acyl-protein thioesterase 1. The protein palmitoylation inhibitor cerulein also inhibited platelet granule secretion and aggregation. Studies using intravital microscopy showed that incubation with cerulein decreased the rate of platelet accumulation into thrombi formed after laser-induced injury of mouse arterioles and inhibited maximal platelet accumulation by >60%. CONCLUSION: These studies show that platelets possess a protein palmitoylation machinery that is required for both platelet activation and platelet accumulation into thrombi. These studies show that inhibition of platelet protein palmitoylation blocks platelet aggregation and granule secretion. In a murine model of thrombus formation, inhibition of protein palmitoylation markedly inhibits platelet accumulation into thrombi at sites of vascular injury.

Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation.

The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.

Abundant expression of c-Jun in guinea pig sympathetic ganglia under basal conditions and allergen challenge.

Airway hyperresponsiveness, a keystone of allergic asthma, is mediated by the extrinsic airway innervation. As pathophysiological stimuli can induce the expression JUN proteins, which belong to the immediate early gene (IEG) family of transcription factors, the expression of c-Jun was examined under basal conditions and allergen challenge in guinea pig paravertebral and prevertebral sympathetic ganglia by quantitative double-labeling immunohistochemistry. C-Jun immunoreactivity was seen in 78.4 +/- 3.5% under normal and 82.6 +/- 4.6% under allergen-challenged conditions of protein-gene product (PGP) 9.5-positive sympathetic neurons of guinea pig superior cervical ganglia and 73.1 +/- 2.8% (normal) and 76.1 +/- 3.5% (allergen) of stellate ganglion neurons. In the coeliac-superior mesenteric ganglion, 59.5 +/- 5.0% (normal) and 57.5 +/- 4.4% (allergen) of the PGP 9.5-positive sympathetic neurons were labeled for c-Jun. The high basal levels of c-Jun expression indicate that the presence of c-Jun is not exclusively related to noxious stimulation such as allergic airway inflammation in the guinea pig.

A 29,000 M(r) protein derived from round spermatids regulates Sertoli cell secretion.

Within the last decade it has become accepted that germ cells can modulate Sertoli cell function in a paracrine interactive manner during the regulation of spermatogenesis. In this context, we undertook to identify a specific factor in round spermatid conditioned media that could stimulate Sertoli cell secretory function. Rat round spermatids isolated by centrifugal elutriation were cultured and the concentrated conditioned media were fractionated by Sephacryl S-200 gel filtration column chromatography. The biological activity of the fractionated round spermatid protein was assessed as stimulation of total protein and transferrin secretion from Sertoli cells that had been isolated from 18-day-old immature rat testes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the gel-filtration fractions showed two predominant proteins of 29,000 and 24,500 molecular weight which coexisted in the fractions containing the greatest biological activity. These two proteins were transferred to a nitrocellulose membrane and excised to raise polyclonal antibodies. Western blot analysis of the 29,000 M(r) protein demonstrated that it specifically occurred in round spermatid conditioned media, whereas no immunoreactive band was observed in either the conditioned media or cell lysates of other testicular cell types such as primary spermatocytes, Sertoli cells and peritubular myoid cells. Following subcellular fractionation of round spermatids by differential centrifugation, the 29,000 M(r) protein was detected by Western blots specifically in the cytosolic fraction of round spermatids, and was absent from the nuclear, mitochondrial, lysosomal and microsomal fractions. The antibody did recognize a few higher molecular bands in the cytosolic fraction which may represent precursor forms of the 29,000 M(r) protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of proliferation of human promyelocytic leukaemia HL60 cells by S-D-lactoylglutathione in vitro.

Human promyelocytic leukaemia HL60 cells were incubated with the glyoxalase intermediate S-D-lactoylglutathione in culture. The effects on cell proliferation, maturation, viability and cell cycle were investigated. When HL60 cells (5 x 10(4)/ml) were incubated with 50-500 microM S-D-lactoylglutathione for two days, the rate of cell proliferation was decreased. This effect was maximal at 500 microM S-D-lactoylglutathione where the cell proliferation rate was only 16% of control levels. There was a concomitant decrease in cell viability but little differentiation. During the first day of treatment, there was a significant decrease in the percentage of cells in the G2-M phase of the cell cycle with a concomitant increase in the G0-G1 phase. In contrast, when HL60 cells were incubated with 1.0-1.5 mM S-D-lactoylglutathione, the inhibition of cell proliferation was progressively lifted, with a concomitant increase in the percentage of differentiated cells (27% differentiation with 1.5 mM S-D-lactoylglutathione). The activities of glyoxalase II and gamma-glutamyl transpeptidase were increased in these cells. S-D-Lactoylglutathione slowly entered the HL60 cells and was consumed over the period when changes in cell cycle distribution, growth arrest and decrease in cell viability were observed. The mechanism of inhibition of proliferation of HL60 promyelocytes by S-D-lactoylglutathione is unknown but it may be related to the ability of S-D-lactoylglutathione to stimulate the assembly of microtubules.

Inhibition of protein and DNA synthesis in tissue culture cells by a derivative of methyl glyoxal and ascorbate.

The inhibitory effect of a methyl glyoxal-ascorbate (MGA) adduct (NFCR 278021) on protein and DNA synthesis in monolayer cultures of GPK epithelial cells has been compared with the inhibitory action of methyl glyoxal (MG). GPK cells exhibited an ID50 of 0.98 microM MG for both protein and DNA synthesis compared with an ID50 of 0.92 mM for the adduct. Hill plots demonstrate that the characteristics of the receptor saturation are the same for MG and MGA, suggesting that the action of the two agents is mediated through the MG moiety which is modified by the presence of the ascorbate portion of the molecule in MGA. It is shown that MGA undergoes spontaneous oxidation in solution and is a substrate for ascorbate oxidase, but that no additional MG activity is released by total enzymic oxidation of MGA, and oxidised MGA possesses the same inhibitory characteristics as MGA. Inhibition of protein synthesis by ascorbate or dehydroascorbate were not demonstrated in the dose range employed for MGA. The inhibitory effect of the adduct on protein synthesis was found to be diminished in the presence of glutathione and glyoxalase I (Glo I) and II( Glo II).