Proteolytic ectodomain shedding of muscle-specific tyrosine kinase in myasthenia gravis.

Autoantibodies to muscle-specific tyrosine kinase (MuSK) proteins at the neuromuscular junction (NMJ) cause refractory generalized myasthenia gravis (MG) with dyspnea more frequently than other MG subtypes. However, the mechanisms via which MuSK, a membrane protein locally expressed on the NMJ of skeletal muscle, is supplied to the immune system as an autoantigen remains unknown. Here, we identified MuSK in both mouse and human serum, with the amount of MuSK dramatically increasing in mice with motor nerve denervation and in MG model mice. Peptide analysis by liquid chromatography-tandem-mass spectrometry (LC-MS/MS) confirmed the presence of MuSK in both human and mouse serum. Furthermore, some patients with MG have significantly higher amounts of MuSK in serum than healthy controls. Our results indicated that the secretion of MuSK proteins from muscles into the bloodstream was induced by ectodomain shedding triggered by neuromuscular junction failure. The results may explain why MuSK-MG is refractory to treatments and causes rapid muscle atrophy in some patients due to the denervation associated with Ab-induced disruption of neuromuscular transmission at the NMJ. Such discoveries pave the way for new MG treatments, and MuSK may be used as a biomarker for other neuromuscular diseases in preclinical studies, clinical diagnostics, therapeutics, and drug discovery.

PCYT2 synthesizes CDP-glycerol in mammals and reduced PCYT2 enhances the expression of functionally glycosylated α-dystroglycan.

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface protein. Defective O-mannosyl glycan on α-DG is associated with muscular dystrophies and cancer. In the biosynthetic pathway of the O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP) transfer ribitol phosphate (RboP). Previously, we reported that FKTN and FKRP can also transfer glycerol phosphate (GroP) from CDP-glycerol (CDP-Gro) and showed the inhibitory effects of CDP-Gro on functional glycan synthesis by preventing glycan elongation in vitro. However, whether mammalian cells have CDP-Gro or associated synthetic machinery has not been elucidated. Therefore, the function of CDP-Gro in mammals is largely unknown. Here, we reveal that cultured human cells and mouse tissues contain CDP-Gro using liquid chromatography tandem-mass spectrometry (LC-MS/MS). By performing the enzyme activity assay of candidate recombinant proteins, we found that ethanolamine-phosphate cytidylyltransferase (PCYT2), the key enzyme in de novo phosphatidylethanolamine biosynthesis, has CDP-Gro synthetic activity from glycerol-3-phosphate (Gro3P) and CTP. In addition, knockdown of PCYT2 dramatically reduced cellular CDP-Gro. These results indicate that PCYT2 is a CDP-Gro synthase in mammals. Furthermore, we found that the expression of functionally glycosylated α-DG is increased by reducing PCYT2 expression. Our results suggest an important role for CDP-Gro in the regulation of α-DG function in mammals.

Diagnostic potential of serum extracellular vesicles expressing prostate-specific membrane antigen in urologic malignancies.

We aimed to develop a sandwich ELISA to detect prostate-specific membrane antigen (PSMA) on small extracellular vesicles (EVs) using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) as a capture molecule for EVs and to evaluate its diagnostic potential in urologic malignancies. First, we optimized the conditions for sandwich ELISA measuring the PSMA level on EVs captured from serum by Tim4 and found that the use of highly-purified EVs released from Tim4 that had captured EVs in serum reduced the background. Second, we confirmed its validity by studying mouse xenograft model for prostate cancer (PC). Lastly, we measured PSMA-EVs in serum of patients with urologic malignancies. The PSMA-EV levels were significantly higher in metastatic PC and castration-resistant PC (CRPC) patients than in therapy-naïve PC patients. In renal cell carcinoma (RCC) patients, PSMA-EVs were elevated in those with metastasis compared with those without metastasis, which may reflect the development of the neovasculature positive for PSMA in tumors. In conclusion, we developed a sandwich ELISA for detection of PSMA-EVs using highly-purified EVs isolated from serum by Tim4. Our results suggest that PSMA-EVs may be useful to diagnose and monitor not only PC but also RCC and possibly other hypervascular solid tumors.

DJ-1 is indispensable for the S-nitrosylation of Parkin, which maintains function of mitochondria.

The DJ-1 gene, a causative gene for familial Parkinson's disease (PD), has been reported to have various functions, including transcriptional regulation, antioxidant response, and chaperone and protease functions; however, the molecular mechanism associated with the pathogenesis of PD remains elusive. To further explore the molecular function of DJ-1 in the pathogenesis of PD, we compared protein expression profiles in brain tissues from wild-type and DJ-1-deficient mice. Two-dimensional difference gel electrophoresis analysis and subsequent analysis using data mining methods revealed alterations in the expression of molecules associated with energy production. We demonstrated that DJ-1 deletion inhibited S-nitrosylation of endogenous Parkin as well as overexpressed Parkin in neuroblastoma cells and mouse brain tissues. Thus, we used genome editing to generate neuroblastoma cells with DJ-1 deletion or S-nitrosylated cysteine mutation in Parkin and demonstrated that these cells exhibited similar phenotypes characterized by enhancement of cell death under mitochondrial depolarization and dysfunction of mitochondria. Our data indicate that DJ-1 is required for the S-nitrosylation of Parkin, which positively affects mitochondrial function, and suggest that the denitrosylation of Parkin via DJ-1 inactivation might contribute to PD pathogenesis and act as a therapeutic target.

Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation.

Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-family implicated in the regulation of cell division and morphogenesis. However, the molecular mechanisms underlying STK38 stability remain largely unknown. Here, we show that treatment of cells with either heat or the calcium ionophore A23187 induced STK38 degradation. The calpain inhibitor calpeptin suppressed hyperthermia-induced degradation or the appearance of A23187-induced cleaved form of STK38. An in vitro cleavage assay was then used to demonstrate that calpain I directly cleaves STK38 at the proximal N-terminal region. Deletion of the N-terminal region of STK38 increased its stability against hyperthermia. We further demonstrated that the MAPKK kinase (MAP3K) MEKK2 prevented both heat- and calpain-induced cleavage of STK38. MEKK2 knockdown enhanced hyperthermia-induced degradation of STK38. We performed an in vitro MEKK2 assay and identified the key regulatory site in STK38 phosphorylated by MEKK2. Experiments with a phosphorylation-defective mutant demonstrated that phosphorylation of Ser 91 is important for STK38 stability, as the enzyme is susceptible to degradation by the calpain pathway unless this residue is phosphorylated. In summary, we demonstrated that STK38 is a calpain substrate and revealed a novel role of MEKK2 in the process of STK38 degradation by calpain.

Age-associated proteomic alterations in human aortic media.

AIM: Human vascular senescence, which mainly occurs in media, is not completely understood. Here, we used proteomic approaches to investigate age-associated changes in human aortic media with the goal of understanding the molecular mechanisms underlying vascular senescence. METHOD: Cryopreserved autopsy samples of aortic media from older-aged (aged 70-100 years, n = 25), middle-aged (aged 49-68 years, n = 24), and young (aged 21-39 years, n = 12) subjects were collected. We used two proteomic techniques, two-dimensional differential gel electrophoresis and isobaric tags for relative and absolute quantitation, and we subjected differentially-expressed proteins among age groups to immunohistochemical analyses. RESULTS: Proteomic analyses showed that the expression of lactadherin, which produces medin, was elevated in aortic media of older-aged individuals. Immunohistochemical and Congo red staining showed that lactadherin and apolipoprotein E were deposited, and that amyloidosis was enhanced in older-aged aortic media. Furthermore, the markers of oxidative damage (8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal) were significantly elevated in aortic media of middle-aged or older-aged individuals. The immunohistochemical expression of anti-oxidant proteins (thioredoxin and extracellular superoxide dismutase) was also high in middle-aged and older-aged groups. Oxidative damage might induce the disruption of smooth muscle cells, resulting in the decrement of α-actin, a highly-expressed protein in smooth muscle cells, and matrix remodeling, in which several proteins associated with extracellular matrix were altered with aging. CONCLUSIONS: Proteomic approaches showed that the elevated expression of lactadherin might contribute to amyloid deposition, enhancement of oxidative stress, induction of antioxidant proteins and matrix remodeling in older-aged aortic media. Geriatr Gerontol Int 2019; 19: 1054-1062.

Identification of Diketopiperazine-Containing 2-Anilinobenzamides as Potent Sirtuin 2 (SIRT2)-Selective Inhibitors Targeting the "Selectivity Pocket", Substrate-Binding Site, and NAD+-Binding Site.

The NAD+-dependent deacetylase SIRT2 represents an attractive target for drug development. Here, we designed and synthesized drug-like SIRT2-selective inhibitors based on an analysis of the putative binding modes of recently reported SIRT2-selective inhibitors and evaluated their SIRT2-inhibitory activity. This led us to develop a more drug-like diketopiperazine structure as a "hydrogen bond (H-bond) hunter" to target the substrate-binding site of SIRT2. Thioamide 53, a conjugate of diketopiperazine and 2-anilinobenzamide which is expected to occupy the "selectivity pocket" of SIRT2, exhibited potent SIRT2-selective inhibition. Inhibition of SIRT2 by 53 was mediated by the formation of a 53-ADP-ribose conjugate, suggesting that 53 is a mechanism-based inhibitor targeting the "selectivity pocket", substrate-binding site, and NAD+-binding site. Furthermore, 53 showed potent antiproliferative activity toward breast cancer cells and promoted neurite outgrowth of Neuro-2a cells. These findings should pave the way for the discovery of novel therapeutic agents for cancer and neurological disorders.

Proteomic study on neurite responses to oxidative stress: search for differentially expressed proteins in isolated neurites of N1E-115 cells.

Reactive oxygen species attack several living organs and induce cell death. Previously, we found axonal/dendrite degeneration before the induction of cell death in hydrogen peroxide-treated neuroblastoma: N1E-115 cells and primary neurons. This phenomenon may be connected with membrane oxidation, microtubule destabilization and disruption of intracellular calcium homeostasis. However, its detailed mechanisms are not fully understood. Here, we identified proteins after treatment with hydrogen peroxide using isolated neurites by liquid chromatography-matrix-assisted laser desorption/ionization-time of flight/time of flight analysis. Twenty-one proteins were increased after treatment with hydrogen peroxide. Specifically, 5 proteins which were secretogranin-1, heat shock protein family D member 1, Brain acid soluble protein 1, heat shock 70-kDa protein 5 and superoxide dismutase 1, were identified of all experiments and increased in isolated neurites of hydrogen peroxide-treated cells compared to the controls. Furthermore, secretogranin-1 and heat shock protein family D member 1 protein expressions were significantly increased in normal aged and Alzheimer's transgenic mice brains. These results indicate that secretogranin-1 and heat shock protein family D member 1 might contribute to reactive oxygen species-induced neurite degeneration. Both proteins have been related to neurodegenerative disorders, so their study may shed light on neurite dysfunction.

Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation.

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.

Characteristic glycopeptides associated with extreme human longevity identified through plasma glycoproteomics.

BACKGROUND: Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105 years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity. METHODS: Plasma proteins from Japanese semi-supercentenarians (SSCs, 106-109 years), aged controls (70-88 years), and young controls (20-38 years) were analysed by using lectin microarrays and liquid chromatography/mass spectrometry (LC/MS). Peak area ratios of glycopeptides to corresponding normalising peptides were subjected to orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, plasma levels of clinical biomarkers were measured. RESULTS: We found two lectins such as Phaseolus vulgaris, and Erythrina cristagalli (ECA), of which protein binding were characteristically increased in SSCs. Peak area ratios of ECA-enriched glycopeptides were successfully discriminated between SSCs and controls using OPLS-DA, and indicated that tri-antennary and sialylated N-glycans of haptoglobin at Asn207 and Asn211 sites were characterized in SSCs. Sialylated glycans of haptoglobin are a potential biomarker of several diseases, such as hepatocellular carcinoma, liver cirrhosis, and IgA-nephritis. However, the SSCs analysed here did not suffer from these diseases. CONCLUSIONS: Tri-antennary and sialylated N-glycans on haptoglobin at the Asn207 and Asn211 sites were abundant in SSCs and characteristic of extreme human longevity. GENERAL SIGNIFICANCE: We found abundant glycans in SSCs, which may be associated with human longevity.

Quantitative analysis of O-GlcNAcylation in combination with isobaric tag labeling and chemoenzymatic enrichment.

Protein O-GlcNAcylation regulates various biological processes, and is associated with several diseases. Therefore, the development of quantitative proteomics is important for understanding the mechanisms of O-GlcNAc-related diseases. We previously reported selective enrichment of O-GlcNAcylated peptides, which provided high-selectivity and effective release by a novel thiol-alkyne and thiol-disulfide exchange. Here, we describe a new approach using initial isobaric tag labeling for relative quantification followed by enrichment and ß-elimination/Michael addition with dithiothreitol for identification of both proteins and modification sites. The approach was validated using model proteins and peptides. This novel strategy could be used for quantitative O-GlcNAcome of biological samples.

Potent mechanism-based sirtuin-2-selective inhibition by an in situ-generated occupant of the substrate-binding site, "selectivity pocket" and NAD+-binding site.

Sirtuin 2 (SIRT2), a member of the NAD+-dependent histone deacetylase family, has recently received increasing attention due to its potential involvement in neurodegenerative diseases and the progression of cancer. Potent and selective SIRT2 inhibitors thus represent desirable biological probes. Based on the X-ray crystal structure of SIRT2 in complex with a previously reported weak inhibitor (6), we identified in this study the potent mechanism-based inactivator KPM-2 (36), which is selective toward SIRT2. Compound 36 engages in a nucleophilic attack toward NAD+ at the active site of SIRT2, which affords a stable 36-ADP-ribose conjugate that simultaneously occupies the substrate-binding site, the "selectivity pocket" and the NAD+-binding site. Moreover, 36 exhibits antiproliferative activity in cancer cells and remarkable neurite outgrowth activity. This strategy for the selective inhibition of SIRT2 should allow further probing of the biology of SIRT2, and promote the development of new disease treatment strategies.

Excess APP O-glycosylation by GalNAc-T6 decreases Aß production.

Alterations of the structure and/or amount of glycans present on proteins are associated with many diseases. We previously demonstrated that changes in N-glycans alter Aß production. In the present study, we focused on the relationship between Alzheimer's disease (AD) and O-glycan, another type of glycan. The UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) family functions in the first step of mucin-type O-glycan synthesis. Analysis of the expression of GalNAc-Ts in the human brain using real-time PCR revealed that the expression of several GalNAc-Ts was altered with sporadic AD progression. Three of these GalNAc-Ts (GalNAc-T1, GalNAc-T4 and GalNAc-T6) were transfected into HEK293T cells to examine their impact on Aß production. Transfection of GalNAc-T6 significantly reduced both Aß1-40 and Aß1-42 generation, but GalNAc-T1 and GalNAc-T4 only reduced Aß1-40 generation. Although these three GalNAc-Ts exhibited enzymatic activities on soluble amyloid precursor protein (APP), the GalNAc transferase activity of GalNAc-T6 to APP was most prominent. The expression of α-secretase and ß-secretase was slightly altered in the transfected cells, but the activities of α-secretase and ß-secretase were not significantly altered. These data suggest that excess O-glycosylation on APP by GalNAc-T6 inhibits Aß production.

Identification of SNAIL1 Peptide-Based Irreversible Lysine-Specific Demethylase 1-Selective Inactivators.

Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide 3, bearing hydrazine, displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3 is a mechanism-based LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cell-membrane-permeable oligoarginine conjugated with peptide 3, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide 37 decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1.

POMGNT1 Is Glycosylated by Mucin-Type O-Glycans.

Protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is a Golgi glycosyltransferase that catalyzes the formation of the N-acetylglucosamine (GlcNAc) ß1→2Man linkage of O-mannosyl glycan. POMGNT1 is not modified by N-glycans because there are no potential N-glycosylation sites; however, it is not clear whether POMGNT1 is modified by O-glycans. To determine whether POMGNT1 is O-glycosylated, we prepared recombinant human POMGNT1 from HEK293T cells. The recombinant POMGNT1 was recognized by Sambucus sieboldiana lectin (SSA), and sialidase digestion of POMGNT1 decreased SSA reactivity and enhanced the reactivity of Arachis hypogaea lectin (PNA). These results suggest that POMGNT1 is modified by a sialylated core-1 O-glycan. Next, we analyzed the structures of the O-glycans on POMGNT1 by ß-elimination and pyrazolone-labeling methods in combination with mass spectrometry. We identified several mucin-type O-glycans containing (NeuAc)1(Hex)1(HexNAc)1, (NeuAc)2(Hex)1(HexNAc)1, and (NeuAc)2(Hex)2(HexNAc)2. To examine whether the O-glycans affect the functions and properties of POMGNT1, we compared glycosylated and non-glycosylated forms of recombinant sPOMGNT1 for their activity and surface hydrophobicity using the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS). POMGNT1 activity and surface hydrophobicity were not affected by the presence or absence of O-glycans.

Integrin ß4 and vinculin contained in exosomes are potential markers for progression of prostate cancer associated with taxane-resistance.

Treatment with taxanes for castration-resistant prostate cancer often leads to the development of resistance. It has been recently demonstrated that exosomes present in the body fluids contain proteins and RNAs in the cells from which they are derived and could serve as a diagnostic marker for various diseases. In the present study, we aimed to identify proteins contained in exosomes that could be markers for progression and taxane-resistance of prostate cancer. Exosomes were isolated by differential centrifugation from the culture medium of taxane-resistant human prostate cancer PC-3 cells (PC-3R) and their parental PC-3 cells. Isolated exosomes were subjected to iTRAQ-based quantitative proteomic analysis. Exosomes were also isolated from the culture medium by using anti-CD9 antibody-conjugated magnetic beads. Protein expression was knocked down by siRNA transfection followed by analysis of the silencing effects. Proteomic analysis showed that integrin ß4 (ITGB4) and vinculin (VCL) were upregulated in exosomes derived from PC-3R cells compared to PC-3 cells. The elevation of ITGB4 and VCL was confirmed in exosomes captured by anti-CD9 antibody from the culture medium of PC-3R cells. Silencing of ITGB4 and VCL expression did not affect proliferation and taxane-resistance of PC-3R cells, but ITGB4 knockdown attenuated both cell migration and invasion and VCL knockdown reduced invasion. Our results suggest that ITGB4 and VCL in exosomes could be useful markers for progression of prostate cancer associated with taxane-resistance, providing the basis for development of an exosome-based diagnostic system.

S-nitrosylation regulates mitochondrial quality control via activation of parkin.

Parkin, a ubiquitin E3 ligase of the ring between ring fingers family, has been implicated in mitochondrial quality control. A series of recent reports have suggested that the recruitment of parkin is regulated by phosphorylation. However, the molecular mechanism that activates parkin to induce mitochondrial degradation is not well understood. Here, and in contrast to previous reports that S-nitrosylation of parkin is exclusively inhibitory, we identify a previously unrecognized site of S-nitrosylation in parkin (Cys323) that induces mitochondrial degradation. We demonstrate that endogenous S-nitrosylation of parkin is in fact responsible for activation of its E3 ligase activity to induce aggregation and degradation. We further demonstrate that mitochondrial uncoupling agents result in denitrosylation of parkin, and that prevention of denitrosylation restores mitochondrial degradation. Our data indicates that NO both positive effects on mitochondrial quality control, and suggest that targeted S-nitrosylation could provide a novel therapeutic strategy against Parkinson's disease.

Rapid discovery of highly potent and selective inhibitors of histone deacetylase 8 using click chemistry to generate candidate libraries.

To find HDAC8-selective inhibitors, we designed a library of HDAC inhibitor candidates, each containing a zinc-binding group that coordinates with the active-site zinc ion, linked via a triazole moiety to a capping structure that interacts with residues on the rim of the active site. These compounds were synthesized by using click chemistry. Screening identified HDAC8-selective inhibitors including C149 (IC(50) = 0.070 µM), which was more potent than PCI-34058 (6) (IC(50) = 0.31 µM), a known HDAC8 inhibitor. Molecular modeling suggested that the phenylthiomethyl group of C149 binds to a unique hydrophobic pocket of HDAC8, and the orientation of the phenylthiomethyl and hydroxamate moieties (fixed by the triazole moiety) is important for the potency and selectivity. The inhibitors caused selective acetylation of cohesin in cells and exerted growth-inhibitory effects on T-cell lymphoma and neuroblastoma cells (GI(50) = 3-80 µM). These findings suggest that HDAC8-selective inhibitors have potential as anticancer agents.

Proteomic analysis of the role of S-nitrosoglutathione reductase in lipopolysaccharide-challenged mice.

S-Nitrosoglutathione reductase (GSNOR) is a key regulator of protein S-nitrosylation, the covalent modification of cysteine residues by nitric oxide that can affect activities of many proteins. We recently discovered that excessive S-nitrosylation from GSNOR deficiency in mice under inflammation inactivates the key DNA repair protein O(6) -alkylguanine-DNA alkyltransferase and promotes both spontaneous and carcinogen-induced hepatocellular carcinoma. To explore further the mechanism of tumorigenesis due to GSNOR deficiency, we compared the protein expression profiles in the livers of wild-type and GSNOR-deficient (GSNOR(-/-) ) mice that were challenged with lipopolysaccharide to induce inflammation and expression of inducible nitric oxide synthase (iNOS). Two-dimensional difference gel electrophoresis analysis identified 38 protein spots of significantly increased intensity and 31 protein spots of significantly decreased intensity in the GSNOR(-/-) mice compared to those in the wild-type mice. We subsequently identified 19 upregulated and 19 downregulated proteins in GSNOR(-/-) mice using mass spectrometry. Immunoblot analysis confirmed in GSNOR(-/-) mice a large increase in the expression of the pro-inflammatory mediator S100A9, a protein previously implicated in human liver carcinogenesis. We also found a decrease in the expression of multiple members of the protein disulfide-isomerase (PDI) family and an alteration in the expression pattern of the endoplasmic reticulum (ER) chaperones in GSNOR(-/-) mice. Furthermore, altered expression of these proteins from GSNOR deficiency was prevented in mice lacking both GSNOR and iNOS. In addition, we detected S-nitrosylation of two members of the PDI protein family. These results suggest that S-nitrosylation resulting from GSNOR deficiency may promote carcinogenesis under inflammatory conditions in part through the disruption of inflammatory and ER stress responses.

Proteomic analysis of S-nitrosylation induced by 1-methyl-4-phenylpyridinium (MPP+).

BACKGROUND: Nitric oxide (NO) mediates its function through the direct modification of various cellular targets. S-nitrosylation is a post-translational modification of cysteine residues by NO that regulates protein function. Recently, an imbalance of S-nitrosylation has also been linked to neurodegeneration through the impairment of pro-survival proteins by S-nitrosylation. RESULTS: In the present study, we used two-dimensional gel electrophoresis in conjunction with the modified biotin switch assay for protein S-nitrosothiols using resin-assisted capture (SNO-RAC) to identify proteins that are S-nitrosylated more intensively in neuroblastoma cells treated with a mitochondrial complex I inhibitor, 1-methyl-4-phenylpyridinium (MPP+). We identified 14 proteins for which S-nitrosylation was upregulated and seven proteins for which it was downregulated in MPP+-treated neuroblastoma cells. Immunoblot analysis following SNO-RAC confirmed a large increase in the S-nitrosylation of esterase D (ESD), serine-threonine kinase receptor-associated protein (STRAP) and T-complex protein 1 subunit γ (TCP-1 γ) in MPP+-treated neuroblastoma cells, whereas S-nitrosylation of thioredoxin domain-containing protein 5 precursor (ERp46) was decreased. CONCLUSIONS: These results suggest that S-nitrosylation resulting from mitochondrial dysfunction can compromise neuronal survival through altering multiple signal transduction pathways and might be a potential therapeutic target for neurodegenerative diseases.