Using anti-malondialdehyde-modified peptide adduct autoantibodies in serum of taiwanese women to diagnose primary Sjogren's syndrome.

BACKGROUND: Sjogren's syndrome (SS) is a systemic autoimmune disease featured with a dry mouth and dry eyes. Several autoantibodies, including anti-SSA, anti-SSB, antinuclear antibodies can be detected in patients with SS. Oxidation-specific epitopes (OSEs) can be formed from malondialdehyde (MDA)-modified protein adducts and trigger chronic inflammation. In this study, our purposes were used serum levels of anti-MDA-modified peptide adducts autoantibodies to evaluate predictive performance by machine learning algorithms in primary Sjögren's syndrome (pSS) and assess the association between pSS and healthy controls. METHODS: Three novel MDA-modified peptide adducts, including immunoglobulin (Ig) gamma heavy chain 1 (IGHG1)102-131, complement factor H (CFAH)1045-1062, and Ig heavy constant alpha 1 (IGHA1)307-327 were identified and validated. Serum levels of protein, MDA-modified protein adducts, MDA, and autoantibodies recognizing unmodified peptides and MDA-modified peptide adducts were measured. Statistically significance in correlations and odds ratios (ORs) were estimated. RESULTS: The random forest classifier utilized autoantibodies combination composed of IgM anti-IGHG1102-131, IgM anti-IGHG1102-131 MDA and IgM anti-IGHA1307-327 achieved predictive performance as an accuracy of 88.0%, a sensitivity of 93.7%, and a specificity of 84.4% which may be as potential diagnostic biomarkers to differentiate patients with pSS from rheumatoid arthritis (RA), and secondary SS in RA and HCs. CONCLUSIONS: Our findings imply that low levels of IgA anti-IGHG1102-131 MDA (OR = 2.646), IgA anti-IGHG1102-131 (OR = 2.408), IgA anti-CFAH1045-1062 (OR = 2.571), and IgA anti-IGHA1307-327 (OR = 2.905) may denote developing risks of pSS, respectively.

Comparative O-GlcNAc Proteomic Analysis Reveals a Role of O-GlcNAcylated SAM68 in Lung Cancer Aggressiveness.

O-GlcNAcylation is a reversible and dynamic post-translational protein modification catalyzed by O-GlcNAc transferase (OGT). Despite the reported association of O-GlcNAcylation with cancer metastasis, the O-GlcNAc proteome profile for cancer aggressiveness remains largely uncharacterized. Here, we report our comparative O-GlcNAc proteome profiling of two differentially invasive lung adenocarcinoma cell lines, which identified 158 down-regulated and 106 up-regulated candidates in highly invasive cells. Among these differential proteins, a nuclear RNA-binding protein, SAM68 (SRC associated in mitosis of 68 kDa), was further investigated. Results showed that SAM68 is O-GlcNAcylated and may interact with OGT in the nucleus. Eleven O-GlcNAcylation sites were identified, and data from mutant analysis suggested that multiple serine residues in the N-terminal region are important for O-GlcNAcylation and the function of SAM68 in modulating cancer cell migration and invasion. Analysis of clinical specimens found that high SAM68 expression was associated with late cancer stages, and patients with high-OGT/high-SAM68 expression in their tumors had poorer overall survival compared to those with low-OGT/low-SAM68 expression. Our study revealed an invasiveness-associated O-GlcNAc proteome profile and connected O-GlcNAcylated SAM68 to lung cancer aggressiveness.

Proteomic Analysis of Aqueous Humor Proteins in Association with Cataract Risks: Diabetes and Smoking.

Cataracts are one of the most common eye diseases that can cause blindness. Discovering susceptibility factors in the proteome that contribute to cataract development would be helpful in gaining new insights in the molecular mechanisms of the cataract process. We used label-free nanoflow ultra-high-performance liquid chromatography-tandem mass spectrometry to compare aqueous humor protein expressions in cataract patients with different cataract risk factors such as diabetes mellitus (DM) and smoking and in controls (with cataract) without risk exposure. Eight patients with diabetes and who smoked (with double risk factors), five patients with diabetes and five patients who smoked (both with a single risk factor), and nine aged-matched cataract controls patients (non-risk exposure) were enrolled. In total, 136 aqueous humor proteins were identified, of which only alpha-2-Heremans-Schmid (HS)-glycoprotein was considered to be significantly risk-associated because it was differentially expressed in these three groups and exhibited increased expression with increasing risk factors. Significant changes in the aqueous humor level of alpha-2-HS-glycoprotein between DM and control samples and between smoking and control samples were confirmed using ELISA. The alpha-2-HS-glycoprotein, called fetuin-a, could be a potential aqueous biomarker associated with DM and smoking, which were cataract risk factors.

Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis.

Protein O-GlcNAcylation is a dynamic post-translational modification involving the attachment of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of Ser/Thr residues on numerous nucleocytoplasmic proteins. Two enzymes are responsible for O-GlcNAc cycling on substrate proteins: O-GlcNAc transferase (OGT) catalyzes the addition while O-GlcNAcase (OGA) helps the removal of GlcNAc. O-GlcNAcylation modifies protein functions; therefore, dysregulation of O-GlcNAcylation affects cell physiology and contributes to pathogenesis. To maintain homeostasis of cellular O-GlcNAcylation, there exists feedback regulation of OGT and OGA expression responding to fluctuations of O-GlcNAc levels; yet, little is known about the molecular mechanisms involved. In this study, we investigated the O-GlcNAc-feedback regulation of OGT and OGA expression in lung cancer cells. Results suggest that, upon alterations in O-GlcNAcylation, the regulation of OGA expression occurs at the mRNA level and likely involves epigenetic mechanisms, while modulation of OGT expression is through translation control. Further analyses revealed that the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) contributes to the downregulation of OGT induced by hyper-O-GlcNAcylation; the S5A/S6A O-GlcNAcylation-site mutant of 4E-BP1 cannot support this regulation, suggesting an important role of O-GlcNAcylation. The results provide additional insight into the molecular mechanisms through which cells may fine-tune intracellular O-GlcNAc levels to maintain homeostasis.

Co-activation of Akt, Nrf2, and NF-κB signals under UPRER in torpid Myotis ricketti bats for survival.

Bats hibernate to survive stressful conditions. Examination of whole cell and mitochondrial proteomes of the liver of Myotis ricketti revealed that torpid bats had endoplasmic reticulum unfolded protein response (UPRER), global reduction in glycolysis, enhancement of lipolysis, and selective amino acid metabolism. Compared to active bats, torpid bats had higher amounts of phosphorylated serine/threonine kinase (p-Akt) and UPRER markers such as PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4). Torpid bats also had lower amounts of the complex of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (p65)/I-κBα. Cellular redistribution of 78 kDa glucose-regulated protein (GRP78) and reduced binding between PERK and GRP78 were also seen in torpid bats. Evidence of such was not observed in fasted, cold-treated, or normal mice. These data indicated that bats activate Akt, Nrf2, and NF-κB via the PERK-ATF4 regulatory axis against endoplasmic reticulum stresses during hibernation.

Targeting protein palmitoylation decreases palmitate­induced sphere formation of human liver cancer cells.

Although non­alcoholic fatty liver disease (NAFLD) is considered a benign disorder, hepatic steatosis has been proposed to be involved in the tumorigenesis of liver cancer. However, the underlying mechanism for carcinogenesis in fatty liver diseases remains unclear. Cancer stem cells (CSCs) have been hypothesized to serve a key role in tumorigenesis. Tumor formation begins with a subset of heterogeneous cells that share properties with stem cells, such as self­renewal and undifferentiated properties. Our previous study reported that the saturated fatty acid palmitate (PA) significantly enhanced the CSC properties of the HepG2 human liver cancer cell line; however, its underlying mechanisms are unknown. In the present study, a proteomic approach was used to investigate the palmitoylation of proteins in HepG2 CSCs. CSC behavior was induced in HepG2 cells via 200 µM PA. Proteomic analysis was performed to identify post­transcriptional modifications of proteins in HepG2 CSCs in response to PA treatment. The present study identified proteins modified by palmitoylation in HepG2 CSC spheres formed following PA treatment. It was therefore hypothesized that palmitoylation may be crucial for CSC sphere formation. Furthermore, the present study demonstrated that two palmitoylation inhibitors, tunicamycin (5, 10 and 25 µg/ml) and 2­bromohexadecanoic acid (25, 50 and 150 µM), significantly decreased CSC sphere formation without affecting cell viability. An association was identified between sphere formation capacity and tumor­initiating capacity of CSCs. The results of the present study demonstrated that protein palmitoylation may influence the PA­induced CSC tumor­initiating capacity, and that the inhibition of palmitoylation may be a suitable chemopreventive strategy for treating patients with NAFLD.

Mevalonate Pathway Enzyme HMGCS1 Contributes to Gastric Cancer Progression.

The 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) is a potential regulatory node in the mevalonate pathway that is frequently dysregulated in tumors. This study found that HMGCS1 expression is upregulated in stomach adenocarcinoma samples of patients and tumorspheres of gastric cancer cells. HMGCS1 elevates the expression levels of the pluripotency genes Oct4 and SOX-2 and contributes to tumorsphere formation ability in gastric cancer cells. HMGCS1 also promotes in vitro cell growth and progression and the in vivo tumor growth and lung metastasis of gastric cancer cells. After blocking the mevalonate pathway by statin and dipyridamole, HMGCS1 exerts nonmetabolic functions in enhancing gastric cancer progression. Furthermore, the level and nuclear translocation of HMGCS1 in gastric cancer cells are induced by serum deprivation. HMGCS1 binds to and activates Oct4 and SOX-2 promoters. HMGCS1 also enhances the integrated stress response (ISR) and interacts with the endoplasmic reticulum (ER) stress transducer protein kinase RNA-like endoplasmic reticulum kinase (PERK). Our results reveal that HMGCS1 contributes to gastric cancer progression in both metabolic and nonmetabolic manners.

Utilizing proteomic approach to identify nuclear translocation related serine kinase phosphorylation site of GNMT as downstream effector for benzo[a]pyrene.

Glycine N-methyltransferase (GNMT) protein is highly expressed in certain tissues, such as liver, pancreas, and prostate. GNMT serves multiple roles which include a methyl group transfer enzyme and a liver tumor suppressor. Benzo(a)pyrene (BaP), a family member of polycyclic aromatic hydrocarbon (PAH), is a known environmental carcinogen found in coal tar, tobacco smoke, barbecued food and incomplete combustion of auto fuel. BaP recruits cytochrome P450 to transform itself into benzo(a)pyrene-7,8-diol-9,10-epoxide (B(a)PDE), which covalently interacts with DNA causing tumorigenesis. BaP can be detoxified through GNMT and induces GNMT translocation into the cellular nucleus. GNMT translocation is accompanied by phosphorylation, but the role of phosphorylation in GNMT remains to be explored. Using liquid chromatography coupled with tandem mass spectrometry, this study identified serine 9 of GNMT as the phosphorylation site upon BaP treatment. When serine 9 was mutated and lost the capability to be phosphorylated, the occurrence of BaP-induced GNMT nuclear translocation was dramatically decreased. Also, this mutant from of GNMT lost the ability of phosphorylation and increased cytochrome P450 1A1 (Cyp1a) expression upon BaP treatment. In addition, protein kinase C (PKC) and c-Jun NH2-terminal kinase (JNK) may be required for such phosphorylation. Further characterization of phosphorylated GNMT for its link to BaP may bring new insights into chemical detoxification.

Low Levels of IgM and IgA Recognizing Acetylated C1-Inhibitor Peptides Are Associated with Systemic Lupus Erythematosus in Taiwanese Women.

The objective of this study was to identify novel acetylation (Ac) modifications of the C1-inhibitor (C1-INH) and explain the association of the levels of autoantibodies against acetylated C1-INH peptides with the risk of developing systemic lupus erythematosus (SLE). Ac modifications of the C1-INH were identified and validated through in-gel digestion, nano-liquid chromatography-tandem mass spectrometry, immunoprecipitation, and Western blotting by using serum protein samples obtained from patients with SLE and age-matched healthy controls (HCs). In addition, the levels of serum C1-INH, Ac-protein adducts, and autoantibodies against unmodified and acetylated C1-INH peptides were measured. C1-INH levels in patients with SLE were significantly lower than those in HCs by 1.53-fold (p = 0.0008); however, Ac-protein adduct concentrations in patients with SLE were significantly higher than those in HCs by 1.35-fold (p = 0.0009). Moreover, immunoglobulin M (IgM) anti-C1-INH367-385 Ac and IgA anti-C1-INH367-385 Ac levels in patients with SLE were significantly lower than those in HCs. The low levels of IgM anti-C1-INH367-385 (odds ratio [OR] = 4.725, p < 0.001), IgM anti-C1-INH367-385 Ac (OR = 4.089, p = 0.001), and IgA anti-C1-INH367-385 Ac (OR = 5.566, p < 0.001) indicated increased risks for the development of SLE compared with HCs.

Isotypes of autoantibodies against differentially expressed novel malondialdehyde-modified peptide adducts in serum of Taiwanese women with rheumatoid arthritis.

This study identified and validated four differentially expressed novel malondialdehyde (MDA)-modified peptide adducts and evaluated autoantibodies against native and MDA-modified peptides among Taiwanese women patients with rheumatoid arthritis (RA), osteoarthritis (OA) and healthy controls (HCs). Ig kappa chain C region76-99, alpha-1-antitrypsin284-298, alpha-2-macroglobulin824-841, and apolipoprotein B-1004022-4040 exhibiting 2-fold differences in relative modification ratios were identified by concanavalin A (Con A) affinity chromatography, 1D SDS-PAGE, in-gel digestion, nano-LC/MS/MS and nano-LC/MS using pooled serum-derived Con A-captured proteins from 9 RA and 9 age-matched HCs. Furthermore, the levels of proteins, serum MDA, and MDA-modified protein adducts were further validated against individual serum from 20 RA and 20 HCs, and autoantibodies against native and their MDA-modified peptides used 45 RA, 30 OA and 45 HCs. Levels of serum MDA and MDA-modified protein adducts were significantly higher in RA than HCs but protein levels were not significantly different. Serum Igs G and M against MDA-modified peptides showed better diagnostic performance in differentiating among patients with RA, OA and HCs, with an area under the receiver operating characteristic curve of 0.96-0.98, sensitivity of 88.9%-97.8%, and specificity of 88.9%-100%. Autoantibodies against MDA-modified epitopes become useful clinical biomarkers for RA. BIOLOGICAL SIGNIFICANCE: By using a label-free relative quantitative proteomic analysis of concanavalin A (Con A)-bound serum samples, the current study discovered and validated malondialdehyde (MDA)-modified peptide adducts as novel biomarkers for differentiating between rheumatoid arthritis (RA) patients and healthy controls (HCs). In addition, the serum levels of MDA, proteins, and MDA-modified protein adducts as well as the MDA modification of proteins were determined. Isotypes of autoantibodies against MDA-modified peptide adducts can be used as serological biomarkers for further discriminating among RA patients, osteoarthritis patients and HCs. This strategy can become the basis for identifying potential diagnostic and pathological biomarkers for RA.

Characterization of a new murine cell line of sarcomatoid hepatocellular carcinoma and its application for biomarker/therapy development.

Sarcomatoid hepatocellular carcinoma (SHC) is a rare type of HCC with significantly poorer survival than ordinary HCC. Little is known about the mechanism associated with SHC and its biomarkers and therapy. Here, we established a mouse liver cancer cell line and designated as Ymac-1. A sarcomatous appearance was observed in the allograft tumor arose from Ymac-1. Liver-secreted plasma proteins were found in Ymac-1 cultured supernatant by proteomics analysis. The positive staining of CK7, CK8, Vimentin and the suppressed expression of AFP indicated that Ymac-1 is a SHC cell line. Compared to its original tumor, an elevated level of EMT markers, N-cadherin and Vimentin, was found in Ymac-1. Ymac-1 displayed a higher migration rate and side population percentage than a mouse ordinary HCC cell line-Hepa1-6. Microarray analysis was performed to identify potential biomarkers/therapeutic targets for SHC. G6pd, a vital enzyme in pentose phosphate pathway, is highly expressed in Ymac-1. Depletion of G6pd in Ymac-1 reduced CD133 expression and sphere formation. Positive correlations between G6PD and CD133 were observed in human specimen. Higher expression of both G6PD and CD133 in tumor were associated with poor survival. In summary Ymac-1 can be a useful SHC cell model for novel biomarker and therapy development.

Proteomics Analysis to Identify and Characterize the Biomarkers and Physical Activities of Non-Frail and Frail Older Adults.

Globally, the proportion of older adults is increasing. Older people face chronic conditions such as sarcopenia and functional decline, which are often associated with disability and frailty. Proteomics assay of potential serum biomarkers of frailty in older adults. Older adults were divided into non-frail and frail groups (n = 6 each; 3 males in each group) in accordance with the Chinese-Canadian Study of Health and Aging Clinical Frailty Scale. Adults were measured for grip power and the 6-min walk test for physical activity, and venous blood was sampled after adults fasted for 8 h. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used for proteomics assay. The groups were compared for levels of biomarkers by t test and Pearson correlation analysis. Non-frail and frail subjects had mean age 77.5±0.4 and 77.7±1.6 years, mean height 160.5±1.3 and 156.6±2.9 cm and mean weight 62.5±1.2 and 62.8±2.9 kg, respectively. Physical activity level was lower for frail than non-frail subjects (grip power: 13.8±0.4 vs 26.1±1.2 kg; 6-min walk test: 215.2±17.2 vs 438.3±17.2 m). Among 226 proteins detected, for 31, serum levels were significantly higher for frail than non-frail subjects; serum levels of Ig kappa chain V-III region WOL, COX7A2, and albumin were lower. The serum levels of ANGT, KG and AT were 2.05-, 1.76- and 2.22-fold lower (all p < 0.05; Figure 1A, 2A and 3A) for non-frail than frail subjects and were highly correlated with grip power (Figure 1B, 2B and 3B). Our study found that ANGT, KG and AT levels are known to increase with aging, so degenerated vascular function might be associated with frailty. In total, 226 proteins were revealed proteomics assay; levels of angiotensinogen (ANGT), kininogen-1 (KG) and antithrombin III (AT) were higher in frail than non-frail subjects (11.26±2.21 vs 5.09±0.74; 18.42±1.36 vs 11.64±1.36; 22.23±1.64 vs 9.52±0.95, respectively, p < 0.05). These 3 factors were highly correlated with grip power (p < 0.05), with higher correlations between grip power and serum levels of ANGT (r = -0.89), KG (r = -0.90), and AT (r = -0.84). In conclusion, this is the first study to demonstrate a serum proteomic profile characteristic of frailty in older adults. Serum ANGT, KG and AT levels could be potential biomarkers for monitoring the development and progression of frailty in older adults.

Characterization of the serum and liver proteomes in gut-microbiota-lacking mice.

Current nutrition research is focusing on health promotion, disease prevention, and performance improvement for individuals and communities around the world. The humans with required nutritional ingredients depend on both how well the individual is provided with balanced foods and what state of gut microbiota the host has. Studying the mutually beneficial relationships between gut microbiome and host is an increasing attention in biomedical science. The purpose of this study is to understand the role of gut microbiota and to study interactions between gut microbiota and host. In this study, we used a shotgun proteomic approach to reveal the serum and liver proteomes in gut-microbiota-lacking mice. For serum, 15 and 8 proteins were uniquely detected in specific-pathogen-free (SPF) and germ-free (GF) mice, respectively, as well as the 3 and 20 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Among the proteins of the serum, major urinary protein 1 (MUP-1) of GF mice was significantly decreased compared to SPF mice. In addition, MUP-1 expression is primarily regulated by testosterone. Lacking in gut flora has been implicated in many adverse effects, and now we have found its pathogenic root maybe gut bacteria can regulate the sex-hormone testosterone levels. In the liver, 8 and 22 proteins were uniquely detected in GF mice and SPF mice, respectively, as well as the 14 and 30 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Furthermore, ingenuity pathway analysis (IPA) indicated that gut microbiota influence the host in cancer, organismal injury and abnormalities, respiratory disease; cell cycle, cellular movement and tissue development; cardiovascular disease, reproductive system disease; and lipid metabolism, molecular transport and small molecule biochemistry. Our findings provide more detailed information of the role of gut microbiota and will be useful to help study gut bacteria and disease prevention.

FNDC3B promotes cell migration and tumor metastasis in hepatocellular carcinoma.

Recurrence and metastasis are common in hepatocellular carcinoma (HCC) and correlate with poor prognosis. We investigated the role of fibronectin type III domain containing 3B (FNDC3B) in HCC metastasis. Overexpression of FNDC3B in HCC cell lines enhanced cell migration and invasion. On the other hand, knockdown of FNDC3B using short-hairpin RNA reduced tumor nodule formation in both intra- and extra-hepatic metastasis. High levels of FNDC3B were observed in metastatic HCCs and correlated with poor patient survival and shorter recurrence time. Mutagenesis and LC-MS/MS analyses showed that FNDC3B promotes cell migration by cooperating with annexin A2 (ANXA2). Furthermore, FNDC3B and ANXA2 expression correlated negatively with patient survival. Our results indicate that FNDC3B behaves like an oncogene by promoting cell migration. This suggests FNDC3B could serve as a biomarker and therapeutic target for HCC metastasis.

The rapid immunosuppression in phytohemagglutinin-activated human T cells is inhibited by the proliferative Ca(2+) influx induced by progesterone and analogs.

Progesterone, an endogenous immunomodulator, suppresses human T-cell activation during pregnancy. A sustained Ca(2 +) influx is an important signal for T-cell proliferation after crosslinking of T-cell receptor/CD3 complexes by anti-CD3 antibodies or phytohemagglutinin (PHA). Progesterone targets cell membrane sites inducing rapid responses including elevated intracellular free calcium concentration ([Ca(2+)]i) and suppressed T-cell PHA-activated proliferation. Interestingly, both PHA and progesterone induce [Ca(2+)]i elevation, but it remains unclear whether the PHA-induced Ca(2+) influx is affected by progesterone leading to T-cell immunosuppression. Primary T-cells were isolated from human peripheral blood and the quench effect on intracellular fura-2 fluorescence of Mn(2+) was used to explore the responses to Ca(2+) influx with cell proliferation being determined by MTT assay. PHA-stimulated Ca(2+) influx was dose-dependently suppressed by progesterone and its agonist R5020, which correlated with PHA-activated T-cell proliferation inhibition. A similar dose-dependent suppression effect on cellular Ca(2+) influx and proliferation occurred with the TRPC channel inhibitor BTP2 and selective TRPC3 channel inhibitor Pyr3. In addition, two progesterone analogs, Org OD 02-0 and 20α-hydroxyprogesterone (20α-OHP), also produced dose-dependent suppression of Ca(2+) influx, but had no effect on proliferation. Finally, inhibition of PHA-activated T-cell proliferation by progesterone is further suppressed by 20α-OHP, but not by Org OD 02-0. Overall, progesterone and R5020 are able to rapidly decrease PHA-stimulated sustained Ca(2+) influx, probably via blockade of TRPC3 channels, which suppresses T-cell proliferation. Taken together, the roles of progesterone and its analogs regarding the rapid response Ca(2+) influx need to be further explored in relation to cytokine secretion and proliferation in activated T-cells.

Proteomics Analysis to Identify and Characterize the Molecular Signatures of Hepatic Steatosis in Ovariectomized Rats as a Model of Postmenopausal Status.

Postmenopausal women are particularly at increased risk of developing non-alcoholic fatty liver disease (NAFLD). Here we aimed to determine the impact of postmenopausal-induced NAFLD (PM-NAFLD) in an ovariectomized rat model. Sixteen six-week-old Sprague-Dawley female rats were randomly divided into two groups (eight per group), for sham-operation (Sham) or bilateral ovariectomy (Ovx). Four months after surgery, indices of liver damage and liver histomorphometry were measured. Both serum aspartate aminotransferase (AST) and alanine aminotranferease (ALT) levels were significantly higher in the Ovx than Sham group. We performed quantitative LC-MS/MS-based proteomic profiling of livers from rats with PM-NAFLD to provide baseline knowledge of the PM-NAFLD proteome and to investigate proteins involved in PM-NAFLD by ingenuity pathways analysis (IPA) to provide corroborative evidence for differential regulation of molecular and cellular functions affecting metabolic processes. Of the 586 identified proteins, the levels of 59 (10.0%) and 48 (8.2%) were significantly higher and lower, respectively, in the Ovx group compared to the Sham group. In conclusion, the changes in regulation of proteins implicated in PM-NAFLD may affect other vital biological processes in the body apart from causing postmenopause-mediated liver dysfunction. Our quantitative proteomics analysis may also suggest potential biomarkers and further clinical applications for PM-NAFLD.

Analysis of differentially expressed novel post-translational modifications of plasma apolipoprotein E in Taiwanese females with breast cancer.

APOE ε2 or ε4 alleles being used as indicators of breast cancer risk are controversial in Taiwanese females. We provide a concept for relative comparisons of post-translational modifications (PTMs) of plasma apolipoprotein E (ApoE) between normal controls and breast cancer patients to investigate the association of ApoE with breast cancer risk. APOE polymorphisms (ApoE isoforms) were not assessed in this study. The relative modification ratio (%) of 15 targeted and 21 modified peptides were evaluated by 1D SDS-PAGE, in-gel digestion, and label-free nano-LC/MS to compare normal controls with breast cancer patients. Plasma levels of the ApoE protein did not significantly differ between normal controls and breast cancer patients. Eleven sites with novel PTMs were identified from 7 pairs of differentially expressed targeted and modified peptides according to the relative modification ratio including methylation at the E3 (↑1.45-fold), E7 (↑1.45-fold), E11 (↑1.19-fold), E77 (↑2.02-fold), E87 (↑2.02-fold), and Q98 (↑1.62-fold) residues; dimethylation at the Q187 (↑1.44-fold) residue; dihydroxylation at the R92 (↑1.25-fold), K95 (↑1.25-fold), and R103 (↑1.25-fold) residues; and glycosylation at the S129 (↑1.14-fold) residue. The clustered methylation and dihydroxylation of plasma ApoE proteins may play a role in breast cancer.

Secretion of one adipokine Nampt/Visfatin suppresses the inflammatory stress-induced NF-κB activity and affects Nampt-dependent cell viability in Huh-7 cells.

Nampt/visfatin acts in both intracellular and extracellular compartments to regulate multiple biological roles, including NAD metabolism, cancer, inflammation, and senescence. However, its function in chronic inflammation and carcinogenesis in hepatocellular carcinoma (HCC) has not been well-defined. Here we use Huh-7 hepatoma cells as a model to determine how Nampt/visfatin affects cellular survival under oxidative stress. We found that the transition of Nampt/visfatin from intracellular into extracellular form was induced by H2O2 treatment in 293T cells and confirmed that this phenomenon was not due to cell death but through the secretion of Nampt/visfatin. In addition, Nampt/visfatin suppressed cell viability in oxidative treatment in Huh-7 cells and acted on the inhibition of hepatoma cell growth. Oxidative stress also reduced the Nampt-mediated activation of NF-κB gene expression. In this study, we identify a novel feature of Nampt/visfatin which functions as an adipokine that can be secreted upon cellular stress. Our results provide an example to understand how adipokine interacts with chemotherapeutic treatment by oxidative stress in HCC.

Dendritic cells respond to nasopharygeal carcinoma cells through annexin A2-recognizing DC-SIGN.

Dendritic cells (DCs) play an essential role in immunity and are used in cancer immunotherapy. However, these cells can be tuned by tumors with immunosuppressive responses. DC-specific intercellular adhesion molecule 3-Grabbing Nonintegrin (DC-SIGN), a C-type lectin expressed on DCs, recognizes certain carbohydrate structures which can be found on cancer cells. Nasopharyngeal carcinoma (NPC) is an epithelial cell-derived malignant tumor, in which immune response remains unclear. This research is to reveal the molecular link on NPC cells that induces the immunosuppressive responses in DCs. In this article, we report identification of annexin A2 (ANXA2) on NPC cells as a ligand for DC-SIGN on DCs. N-linked mannose-rich glycan on ANXA2 may mediate the interaction. ANXA2 was abundantly expressed in NPC, and knockdown of ANXA2 suppressed NPC xenograft in mice, suggesting a crucial role of ANXA2 in NPC growth. Interaction with NPC cells caused DC-SIGN activation in DCs. Consequently DC maturation and the proinflammatory interleukin (IL)-12 production were inhibited, and the immunosuppressive IL-10 production was promoted. Blockage of either DC-SIGN or ANXA2 eliminated the production of IL-10 from DCs. This report suggests that suppression of ANXA2 at its expression or glycosylation on NPC may improve DC-mediated immunotherapy for the tumor.

Comparative proteomics, network analysis and post-translational modification identification reveal differential profiles of plasma Con A-bound glycoprotein biomarkers in gastric cancer.

In the study, we used Con A affinity chromatography, 1-D gel electrophoresis, and nano-LC-MS/MS to screen biomarker candidates in plasma samples obtained from 30 patients with gastric cancer and 30 healthy volunteers. First, we pooled plasma samples matched by age and sex. We identified 17 differentially expressed Con A-bound glycoproteins, including 10 upregulated proteins and 7 downregulated proteins; these differences were significant (Student's t-test, p-value<0.05). Furthermore, 2 of the upregulated proteins displayed expression levels that were increased by 2-fold or more in gastric cancer samples when compared with normal control samples. These proteins included leucine-rich alpha-2-glycoprotein (LRG1) and inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), and the expression levels were validated by Western blot analysis. Pathway and network analysis of the differentially expressed proteins by Ingenuity Pathway Analysis revealed vital canonical pathways involving acute phase response signaling, the complement system, LXR/RXR activation, hematopoiesis from pluripotent stem cells, and primary immunodeficiency signaling. Our results suggest that Con A-bound LRG1 and ITIH3 may not be practically applicable as a robust biomarker for the early detection of gastric cancer. Additionally, three novel PTMs in ITIH3 were identified and include hexose-N-acetyl-hexosamine at asparagine-(41), trimethylation at aspartic acid-(290), and flavin adenine dinucleotide at histidine-(335). BIOLOGICAL SIGNIFICANCE: Our study was to describe a combinatorial approach of Con A affinity chromatography, 1-D SDS-PAGE, and nano-LC/MS/MS that provides a label-free, comparative glycoproteomic quantification strategy for the investigation of glycoprotein profiles in plasma from gastric cancer patients versus healthy volunteers and to identify glycoprotein biomarkers for the early clinical detection of gastric cancer. Three novel PTMs, HexHexNAc, trimethylation and FAD, in Con A-bound ITIH3 were identified and built in molecular modeling. The aspartic acid-(290) trimethylation site was located in a metal ion-dependent adhesion site (MIDAS motif; (290)-DXSXS…T…D-(313)) that may influence important function for binding protein ligands.