Correction to: Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease.

[This corrects the article DOI: 10.1186/s12953-018-0131-y.].

Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease.

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients. Methods: Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE-/-) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients. Results: Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r = 0.520, p < 0.0001). Conclusion: Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted.

The role of S100P in the invasion of pancreatic cancer cells is mediated through cytoskeletal changes and regulation of cathepsin D.

Up-regulation of S100P, a member of the S100 calcium-binding protein family, is an early molecular event in the development of pancreatic cancer and it is expressed at high levels in both precursor lesions and invasive cancer. To gain more insight into the molecular mechanisms underlying the functional roles of this protein, we stably overexpressed S100P in the Panc1 pancreatic cancer cell line and identified the consequent changes in global protein expression by two-dimensional difference in-gel electrophoresis. The observed changes in target proteins were confirmed by Western blot analysis and immunofluorescence, whereas their functional effect was investigated using motility and invasion assays. In this study, we have shown that overexpression of S100P led to changes in the expression levels of several cytoskeletal proteins, including cytokeratins 8, 18, and 19. We have also shown disorganization of the actin cytoskeleton network and changes in the phosphorylation status of the actin regulatory protein cofilin. Additionally, we have shown that overexpression of S100P leads to increased expression of another early pancreatic cancer marker, S100A6, as well as the aspartic protease cathepsin D, both of which are involved in cellular invasion. Functional studies showed that the increased invasive potential of S100P-overexpressing cells was at least partially due to the increase in cathepsin D expression. In summary, our data suggest that these changes could contribute to the metastatic spread of pancreatic cancer and may explain the devastating prognosis of this disease.

AGR2, a unique tumor-associated antigen, is a promising candidate for antibody targeting.

Anterior gradient 2 (AGR2), a protein disulfide isomerase, shows two subcellular localizations: intracellular (iAGR2) and extracellular (eAGR2). In healthy cells that express AGR2, the predominant form is iAGR2, which resides in the endoplasmic reticulum. In contrast, cancer cells secrete and express eAGR2 on the cell surface. We wanted to test if AGR2 is a cancer-specific tumor-associated antigen. We utilized two AGR2 antibodies, P3A5 and P1G4, for in vivo tumor localization and tumor growth inhibition. The monoclonal antibodies recognized both human AGR2 and mouse Agr2. Biodistribution experiments using a syngeneic mouse model showed high uptake of P3A5 AGR2 antibody in xenografted eAgr2+ pancreatic tumors, with limited uptake in normal tissues. In implanted human patient-derived eAGR2+ pancreatic cancer xenografts, tumor growth inhibition was evaluated with antibodies and Gemcitabine (Gem). Inhibition was more potent by P1G4 + Gem combination than Gem alone or P3A5 + Gem. We converted these two antibodies to human:mouse chimeric forms: the constructed P3A5 and P1G4 chimeric mVLhCκ and mVHhCγ (γ1, γ2, γ4) genes were inserted in a single mammalian expression plasmid vector, and transfected into human 293F cells. Expressed human:mouse chimeric IgG1, IgG2 and IgG4 antibodies retained AGR2 binding. Increase in IgG yield by transfected cells could be obtained with serial transfection of vectors with different drug resistance. These chimeric antibodies, when incubated with human blood, effectively lysed eAGR2+ PC3 prostate cancer cells. We have, thus, produced humanized anti-AGR2 antibodies that, after further testing, might be suitable for treatment against a variety of eAGR2+ solid tumors.

Mass spectrometry identification of circulating alpha-1-B glycoprotein, increased in aged female C57BL/6 mice.

In this study, we surveyed the profiles of mouse circulating proteins by 2-dimensional SDS-PAGE in different strains, sexes and ages. Among visible protein spots on 2-D gels with silver-staining, we identified a unique set of 7 seemingly-related proteins whose levels were consistently elevated in older C57BL/6 female mice. This set of 7 proteins was absent in C57BL/6 males or in BALB/c mice of either sex of any age. When C57BL/6 female mice were crossed with BALB/c males, the age-related increase of these proteins became sporadic and not linear in the F1 offspring. All 7 spots of this protein group were picked and subjected to identification by mass spectrometric analysis after tryptic digestion. The results showed that all 7 spots were different isoforms of alpha(1)B-glycoprotein with different degrees of post-translational modifications, such as phosphorylation. These results suggest that alpha(1)B-glycoprotein changes in mice in a sex and age dependent manner.

Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis.

BACKGROUND: Kveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4-6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins. METHODS: Kv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay. RESULTS: We observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin or alpha-actinin-4. CONCLUSIONS: Stimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis.

Cell surface nucleolin on developing muscle is a potential ligand for the axonal receptor protein tyrosine phosphatase-sigma.

Reversible tyrosine phosphorylation, catalyzed by receptor tyrosine kinases and receptor tyrosine phosphatases, plays an essential part in cell signaling during axonal development. Receptor protein tyrosine phosphatase-sigma has been implicated in the growth, guidance and repair of retinal axons. This phosphatase has also been implicated in motor axon growth and innervation. Insect orthologs of receptor protein tyrosine phosphatase-sigma are also implicated in the recognition of muscle target cells. A potential extracellular ligand for vertebrate receptor protein tyrosine phosphatase-sigma has been previously localized in developing skeletal muscle. The identity of this muscle ligand is currently unknown, but it appears to be unrelated to the heparan sulfate ligands of receptor protein tyrosine phosphatase-sigma. In this study, we have used affinity chromatography and tandem MS to identify nucleolin as a binding partner for receptor protein tyrosine phosphatase-sigma in skeletal muscle tissue. Nucleolin, both from tissue lysates and in purified form, binds to receptor protein tyrosine phosphatase-sigma ectodomains. Its expression pattern also overlaps with that of the receptor protein tyrosine phosphatase-sigma-binding partner previously localized in muscle, and nucleolin can also be found in retinal basement membranes. We demonstrate that a significant amount of muscle-associated nucleolin is present on the cell surface of developing myotubes, and that two nucleolin-binding components, lactoferrin and the HB-19 peptide, can block the interaction of receptor protein tyrosine phosphatase-sigma ectodomains with muscle and retinal basement membranes in tissue sections. These data suggest that muscle cell surface-associated nucleolin represents at least part of the muscle binding site for axonal receptor protein tyrosine phosphatase-sigma and that nucleolin may also be a necessary component of basement membrane binding sites of receptor protein tyrosine phosphatase-sigma.

Intra-Tumor Genetic Heterogeneity in Wilms Tumor: Clonal Evolution and Clinical Implications.

The evolution of pediatric solid tumors is poorly understood. There is conflicting evidence of intra-tumor genetic homogeneity vs. heterogeneity (ITGH) in a small number of studies in pediatric solid tumors. A number of copy number aberrations (CNA) are proposed as prognostic biomarkers to stratify patients, for example 1q+ in Wilms tumor (WT); current clinical trials use only one sample per tumor to profile this genetic biomarker. We multisampled 20 WT cases and assessed genome-wide allele-specific CNA and loss of heterozygosity, and inferred tumor evolution, using Illumina CytoSNP12v2.1 arrays, a custom analysis pipeline, and the MEDICC algorithm. We found remarkable diversity of ITGH and evolutionary trajectories in WT. 1q+ is heterogeneous in the majority of tumors with this change, with variable evolutionary timing. We estimate that at least three samples per tumor are needed to detect >95% of cases with 1q+. In contrast, somatic 11p15 LOH is uniformly an early event in WT development. We find evidence of two separate tumor origins in unilateral disease with divergent histology, and in bilateral WT. We also show subclonal changes related to differential response to chemotherapy. Rational trial design to include biomarkers in risk stratification requires tumor multisampling and reliable delineation of ITGH and tumor evolution.

Sarcoidosis and tuberculosis cytokine profiles: indistinguishable in bronchoalveolar lavage but different in blood.

BACKGROUND: The clinical, radiological and pathological similarities between sarcoidosis and tuberculosis can make disease differentiation challenging. A complicating factor is that some cases of sarcoidosis may be initiated by mycobacteria. We hypothesised that immunological profiling might provide insight into a possible relationship between the diseases or allow us to distinguish between them. METHODS: We analysed bronchoalveolar lavage (BAL) fluid in sarcoidosis (n = 18), tuberculosis (n = 12) and healthy volunteers (n = 16). We further investigated serum samples in the same groups; sarcoidosis (n = 40), tuberculosis (n = 15) and healthy volunteers (n = 40). A cross-sectional analysis of multiple cytokine profiles was performed and data used to discriminate between samples. RESULTS: We found that BAL profiles were indistinguishable between both diseases and significantly different from healthy volunteers. In sera, tuberculosis patients had significantly lower levels of the Th2 cytokine interleukin-4 (IL-4) than those with sarcoidosis (p = 0.004). Additional serum differences allowed us to create a linear regression model for disease differentiation (within-sample accuracy 91%, cross-validation accuracy 73%). CONCLUSIONS: These data warrant replication in independent cohorts to further develop and validate a serum cytokine signature that may be able to distinguish sarcoidosis from tuberculosis. Systemic Th2 cytokine differences between sarcoidosis and tuberculosis may also underly different disease outcomes to similar respiratory stimuli.

Proteomics and systems biology: current and future applications in the nutritional sciences.

In the last decade, advances in genomics, proteomics, and metabolomics have yielded large-scale datasets that have driven an interest in global analyses, with the objective of understanding biological systems as a whole. Systems biology integrates computational modeling and experimental biology to predict and characterize the dynamic properties of biological systems, which are viewed as complex signaling networks. Whereas the systems analysis of disease-perturbed networks holds promise for identification of drug targets for therapy, equally the identified critical network nodes may be targeted through nutritional intervention in either a preventative or therapeutic fashion. As such, in the context of the nutritional sciences, it is envisioned that systems analysis of normal and nutrient-perturbed signaling networks in combination with knowledge of underlying genetic polymorphisms will lead to a future in which the health of individuals will be improved through predictive and preventative nutrition. Although high-throughput transcriptomic microarray data were initially most readily available and amenable to systems analysis, recent technological and methodological advances in MS have contributed to a linear increase in proteomic investigations. It is now commonplace for combined proteomic technologies to generate complex, multi-faceted datasets, and these will be the keystone of future systems biology research. This review will define systems biology, outline current proteomic methodologies, highlight successful applications of proteomics in nutrition research, and discuss the challenges for future applications of systems biology approaches in the nutritional sciences.

AGR2 is a novel surface antigen that promotes the dissemination of pancreatic cancer cells through regulation of cathepsins B and D.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers largely due to disseminated disease at the time of presentation. Here, we investigated the role and mechanism of action of the metastasis-associated protein anterior gradient 2 (AGR2) in the pathogenesis of pancreatic cancer. AGR2 was induced in all sporadic and familial pancreatic intraepithelial precursor lesions (PanIN), PDACs, circulating tumor cells, and metastases studied. Confocal microscopy and flow cytometric analyses indicated that AGR2 localized to the endoplasmic reticulum (ER) and the external surface of tumor cells. Furthermore, induction of AGR2 in tumor cells regulated the expression of several ER chaperones (PDI, CALU, RCN1), proteins of the ubiquitin-proteasome degradation pathway (HIP2, PSMB2, PSMA3, PSMC3, and PSMB4), and lysosomal proteases [cathepsin B (CTSB) and cathepsin D (CTSD)], in addition to promoting the secretion of the precursor form pro-CTSD. Importantly, the invasiveness of pancreatic cancer cells was proportional to the level of AGR2 expression. Functional downstream targets of the proinvasive activity of AGR2 included CTSB and CTSD in vitro, and AGR2, CTSB, and CTSD were essential for the dissemination of pancreatic cancer cells in vivo. Taken together, the results suggest that AGR2 promotes dissemination of pancreatic cancer and that its cell surface targeting may permit new strategies for early detection as well as therapeutic management.

Urinary proteome profiling using 2D-DIGE and LC-MS/MS.

Proteomic methodologies have been at the forefront of cancer research for several years. The use of proteomic strategies to study all expressed genes aims to discover biomarkers indicative of the physiological state of cancer cells at specific time points, enabling early diagnosis, following cancer development/progression, screening and monitoring the efficacy of new therapeutic agents. Onco-proteomics has the potential to impact on oncology practice by delivering individualised highly selective clinical care. 2D-DIGE (2D difference in gel electrophoresis) enables simultaneous examination and comparison of multiple samples using cyanine dyes to label amino acid residues that are then separated based on charge and mass. These advantages combined with universal availability have until recently made 2D-DIGE a first method of choice in cancer proteome analysis of diverse specimens, including tissues, cell lines, blood and other body fluids.

Application of proteomics in cancer gene profiling: two-dimensional difference in gel electrophoresis (2D-DIGE).

In the post-genomic era, proteomic strategies are at the forefront of cancer research. By studying the complement of all expressed genes, proteomics aims to provide knowledge of biomarkers indicative of the physiological state of cancer cells at a specific time, enabling screening, early diagnosis, monitoring the course of cancer development/progression, and gauging the efficacy and safety of novel therapeutic agents. Onco-proteomics thus has the ability to revolutionize oncology practice by delivering highly selective and individualised clinical care. One of the proteomic techniques, two-dimensional (2D) difference in gel electrophoresis (DIGE) enables simultaneous examination and comparison of multiple samples using cyanine dyes to label amino acid residues that are then separated based on charge and mass. This technique reduces variability, improves reproducibility, and allows easier quantitation when compared with traditional 2D polyacrylamide gel electrophoresis (PAGE). These advantages combined with universal availability makes 2D-DIGE a first method of choice in cancer proteome analysis of diverse specimens, including tissues, cell lines, blood, and other body fluids.

Analysis of the urine proteome in patients with pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) accounts for over 213 000 deaths worldwide each year, largely due to late diagnosis. One of the risk factors for the development of PDAC is chronic pancreatitis (CP); the intense desmoplastic reaction makes differentiation between the two conditions extremely difficult. In order to identify biomarkers for noninvasive diagnosis, we performed 2-D DIGE analysis of urine samples from healthy individuals and patients with PDAC and CP. Despite considerable intersample heterogeneity, a total of 127 statistically valid (p<0.05), differentially expressed protein spots were detected, 101 of which were identified using MALDI-TOF MS. A number of these, including annexin A2, gelsolin and CD59 have already been associated with PDAC, however, their validation using immunoblotting proved challenging. This is probably due to extensive PTMs and processing thus indicating the need for raising specific antibodies for urinary proteins. Despite this, our study clearly demonstrates that urine is a valid source of noninvasive biomarkers in patients with pancreatic diseases.

A parallel proteomic and metabolomic analysis of the hydrogen peroxide- and Sty1p-dependent stress response in Schizosaccharomyces pombe.

Using an integrated approach incorporating proteomics, metabolomics and published mRNA data, we have investigated the effects of hydrogen peroxide on wild type and a Sty1p-deletion mutant of the fission yeast Schizosaccharomyces pombe. Differential protein expression analysis based on the modification of proteins with matched fluorescent labelling reagents (2-D-DIGE) is the foundation of the quantitative proteomics approach. This study identifies 260 differentially expressed protein isoforms from 2-D-DIGE gels using MALDI MS and reveals the complexity of the cellular response to oxidative stress and the dependency on the Sty1p stress-activated protein kinase. We show the relationship between these protein changes and mRNA expression levels identified in a parallel whole genome study, and discuss the regulatory mechanisms involved in protecting cells against hydrogen peroxide and the involvement of Sty1p-dependent stress-activated protein kinase signalling. Metabolomic profiling of 29 intermediates using 1H NMR was also conducted alongside the protein analysis using the same sample sets, allowing examination of how the protein changes might affect the metabolic pathways and biological processes involved in the oxidative stress response. This combined analysis identifies a number of interlinked metabolic pathways that exhibit stress- and Sty1-dependent patterns of regulation.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform.

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Global changes in gene expression observed at the transition from growth to stationary phase in Listeria monocytogenes ScottA batch culture.

Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually. The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L. monocytogenes adapts to environmental change are yet to be fully elucidated. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. We have utilized a proteomic approach to investigate the response of L. monocytogenes batch cultures to the transition from exponential to stationary growth phase. Proteomic analysis showed that batch cultures of L. monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone. Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase. We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase. The significance of these findings in terms of functionality and the mechanistic picture are discussed.