Primary Impacts of the Fungal Toxin Sporidesmin on HepG2 Cells: Altered Cell Adhesion without Oxidative Stress or Cell Death.

The fungal metabolite sporidesmin is responsible for severe necrotizing inflammation of biliary tract and liver of livestock grazing on pasture containing spores of Pithomyces chartarum that synthesizes the toxin. The toxin is secreted into bile causing the erosion of the biliary epithelium accompanied by inflammation and damage to surrounding tissues. Toxicity has been suggested to be due to cycles of reduction and oxidation of sporidesmin leading to oxidative damage from the formation of reactive oxygen species. The current work is the first test of the oxidative stress hypothesis using cultured cells. Oxidative stress could not be detected in HepG2 cells incubated with sporidesmin using a dichlorodihydrofluorescein diacetate assay or by use of two-dimensional electrophoresis to search for oxidized peroxiredoxins. There was also no evidence for necrosis or apoptosis, although there was a loss of cell adhesion that was accompanied by the disruption of intracellular actin microfilaments that have known roles in cell adhesion. The results are consistent with a model in which altered contact between cells in situ leads to altered permeability and subsequent inflammation and necrosis, potentially from the leakage of toxic bile into surrounding tissues. There is now a need for the further characterization of the damage processes in vivo, including the investigation of altered permeability and mechanisms of cell death in the biliary tract and other affected organs.

Localisation of clozapine during experimental autoimmune encephalomyelitis and its impact on dopamine and its receptors.

Multiple sclerosis is a disease characterised by axonal demyelination in the central nervous system (CNS). The atypical antipsychotic drug clozapine attenuates experimental autoimmune encephalomyelitis (EAE), a mouse model used to study multiple sclerosis, but the precise mechanism is unknown and could include both peripheral and CNS-mediated effects. To better understand where clozapine exerts its protective effects, we investigated the tissue distribution and localisation of clozapine using matrix-assisted laser desorption ionization imaging mass spectrometry and liquid chromatography-mass spectrometry. We found that clozapine was detectable in the brain and enriched in specific brain regions (cortex, thalamus and olfactory bulb), but the distribution was not altered by EAE. Furthermore, although not altered in other organs, clozapine levels were significantly elevated in serum during EAE. Because clozapine antagonises dopamine receptors, we analysed dopamine levels in serum and brain as well as dopamine receptor expression on brain-resident and infiltrating immune cells. While neither clozapine nor EAE significantly affected dopamine levels, we observed a significant downregulation of dopamine receptors 1 and 5 and up-regulation of dopamine receptor 2 on microglia and CD4+-infiltrating T cells during EAE. Together these findings provide insight into how neuroinflammation, as modelled by EAE, alters the distribution and downstream effects of clozapine.

Combination of laser microdissection, 2D-DIGE and MALDI-TOF MS to identify protein biomarkers to predict colorectal cancer spread.

Biomarkers are urgently required to support current histological staging to provide additional accuracy in stratifying colorectal cancer (CRC) patients according to risk of spread to properly assign adjuvant chemotherapy after surgery. Chemotherapy is given to patients with stage III to reduce the risk of recurrence but is controversial in stage II patients. Up to 25% of stage II patients will relapse within 5 years after tumor removal and when this occurs cure is seldom possible. The aim of this study was to identify protein biomarkers to stratify risk of spread of CRC patients. Laser micro-dissection was used to isolate cancer cells from primary colorectal tumors of stage II patients which did or did not metastasize within 5 years after surgical resection. Protein expression differences between two groups of tumors were profiled by 2D-DIGE with saturation CyDye labeling and identified using MALDI-TOF mass spectrometry. Evaluation of protein candidates was conducted using tissue micro array (TMA) immunohistochemistry on 125 colorectal tumor tissue samples of different stages. A total of 55 differentially expressed proteins were identified. Ten protein biomarkers were chosen based on p value and ratio between non metastasized and metastazised groups and evaluated on 125 tissues using TMA immunohistochemistry. Expression of HLAB, protein 14-3-3ß, LTBP3, ADAMTS2, JAG2 and NME2 on tumour cells was significantly associated with clinical parameters related to tumour progression, invasion and metastasis. Kaplan-Meier survival curve showed strong expression of six proteins was associated with good CRC specific survival. Expression of HLAB, ADAMTS2, LTBP3, JAG2 and NME2 on tumour cells, was associated with tumour progression and invasion, metastasis and CRC specific survival may serve as potential biomarkers to stratify CRC patients into low and high risk of tumour metastasis. Combined methods of laser microdissection, 2D DIGE with saturation labelling and MALDI-TOF MS proved to be resourceful techniques capable of identifying protein biomarkers to predict risk of spread of CRC to liver.

Role of DGAT enzymes in triacylglycerol metabolism.

The esterification of a fatty acyl moiety to diacylglycerol to form triacylglycerol (TAG) is catalysed by two diacylglycerol O-acyltransferases (DGATs) encoded by genes belonging to two distinct gene families. The enzymes are referred to as DGAT1 and DGAT2 in order of their identification. Both proteins are transmembrane proteins localized in the endoplasmic reticulum. Their membrane topologies are however significantly different. This difference is hypothesized to give the two isozymes different abilities to interact with other proteins and organelles and access to different pools of fatty acids, thereby creating a distinction between the enzymes in terms of their role and contribution to lipid metabolism. DGAT1 is proposed to have dual topology contributing to TAG synthesis on both sides of the ER membrane and esterifying only the pre-formed fatty acids. There is evidence to suggest that DGAT2 translocates to the lipid droplet (LD), associates with other proteins, and synthesizes cytosolic and luminal apolipoprotein B associated LD-TAG from both endogenous and exogenous fatty acids. The aim of this review is to differentiate between the two DGAT enzymes by comparing the genes that encode them, their proposed topologies, the proteins they interact with, and their roles in lipid metabolism.

Proteins Annexin A2 and PSA in Prostate Cancer Biopsies Do Not Predict Biochemical Failure.

BACKGROUND/AIM: We previously reported the use of mass spectrometry and western blotting to identify proteins from tumour regions of formalin-fixed paraffin-embedded biopsies from 16 men who presented with apparently localized prostate cancer, and found that annexin A2 (ANXA2) appeared to be a better predictor of subsequent biochemical failure than prostate-specific antigen (PSA). MATERIALS AND METHODS: In this follow-up study, ANXA2 and PSA were measured using western blotting of proteins extracted from biopsies from 37 men from a subsequent prostate cancer trial. RESULTS: No significant differences in ANXA2 and PSA levels were observed between men with and without biochemical failure. The statistical effect sizes were small, d=0.116 for ANXA2, and 0.266 for PSA. CONCLUSION: ANXA2 and PSA proteins measured from biopsy tumour regions are unlikely to be good biomarkers for prediction of the clinical outcome of prostate cancer presenting with apparently localized disease.

Identification of a gene involved in the regulation of hyphal growth of Epichloë festucae during symbiosis.

Secreted proteins, those involved in cell wall biogenesis, are likely to play a role in communication in the symbiotic interaction between the fungal endophyte Epichloë festucae with perennial ryegrass (Lolium perenne), particularly given the close association between fungal hyphae and the plant cell wall. Our hypothesis was that secreted proteins are likely to be responsible for establishing and maintaining a normal symbiotic relationship. We analyzed an endophyte EST database for genes with predicted signal peptide sequences. Here, we report the identification and characterization of rhgA; a gene involved in the regulation of hyphal growth in planta In planta analysis of ΔrhgA mutants showed that disruption of rhgA resulted in extensive unregulated hyphal growth. This phenotype was fully complemented by insertion of the rhgA gene and suggests that rhgA is important for maintaining normal hyphal growth during symbiosis.

Proteins from formalin-fixed paraffin-embedded prostate cancer sections that predict the risk of metastatic disease.

BACKGROUND: Prostate cancer is the most frequently diagnosed cancer in men and the third leading cause of cancer related deaths among men living in developed countries. Biomarkers that predict disease outcome at the time of initial diagnosis would substantially aid disease management. RESULTS: Proteins extracted from formalin-fixed paraffin-embedded tissue were identified using nanoflow liquid chromatography-MALDI MS/MS or after separation by one- or two-dimensional electrophoresis. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000963. A list of potential biomarker candidates, based on proposed associations with prostate cancer, was derived from the 320 identified proteins. Candidate biomarkers were then examined by multiplexed Western blotting of archival specimens from men with premetastatic disease and subsequent disease outcome data. Annexin A2 provided the best prediction of risk of metastatic disease (log-rank Chi squared p = 0. 025). A tumor/control tissue >2-fold relative abundance increase predicted early biochemical failure, while <2-fold change predicted late or no biochemical failure. CONCLUSIONS: This study confirms the potential for use of archival FFPE specimens in the search for prognostic biomarkers for prostate cancer and suggests that annexin A2 abundance in diagnostic biopsies is predictive for metastatic potential. Protein profiling each cancer may lead to an overall reduction in mortality from metastatic prostate cancer as well as reduced treatment associated morbidity.

Phenotypic population screen identifies a new mutation in bovine DGAT1 responsible for unsaturated milk fat.

Selective breeding has strongly reduced the genetic diversity in livestock species, and contemporary breeding practices exclude potentially beneficial rare genetic variation from the future gene pool. Here we test whether important traits arising by new mutations can be identified and rescued in highly selected populations. We screened milks from 2.5 million cows to identify an exceptional individual which produced milk with reduced saturated fat content, and improved unsaturated and omega-3 fatty acid concentrations. The milk traits were transmitted dominantly to her offspring, and genetic mapping and genome sequencing revealed a new mutation in a previously unknown splice enhancer of the DGAT1 gene. Homozygous carriers show features of human diarrheal disorders, and may be useful for the development of therapeutic strategies. Our study demonstrates that high-throughput phenotypic screening can uncover rich genetic diversity even in inbred populations, and introduces a novel strategy to develop novel milks with improved nutritional properties.

The Cytosolic Oligosaccharide-Degrading Proteome of Butyrivibrio Proteoclasticus.

The growth and productivity of ruminants depends on a complex microbial community found in their fore-stomach (rumen), which is able to breakdown plant polysaccharides and ferment the released sugars. Butyrivibrio proteoclasticus B316T is a Gram-positive polysaccharide-degrading, butyrate-producing bacterium that is present at high numbers in the rumen of animals consuming pasture or grass silage based diets. B316T is one of a small number of rumen fibrolytic microbes capable of efficiently degrading and utilizing xylan, as well as being capable of utilizing arabinose, xylose, pectin and starch. We have therefore carried out a proteomic analysis of B316T to identify intracellular enzymes that are implicated in the metabolism of internalized xylan. Three hundred and ninety four proteins were identified including enzymes that have potential to metabolize assimilated products of extracellular xylan digestion. Identified enzymes included arabinosidases, esterases, an endoxylanase, and ß-xylosidase. The presence of intracellular debranching enzymes indicated that some hemicellulosic side-chains may not be removed until oligosaccharides liberated by extracellular digestion have been assimilated by the cells. The results support a model of extracellular digestion of hemicellulose to oligosaccharides that are then transported to the cytoplasm for further digestion by intracellular enzymes.

Cathepsin D Expression in Colorectal Cancer: From Proteomic Discovery through Validation Using Western Blotting, Immunohistochemistry, and Tissue Microarrays.

Despite recent advances in surgical techniques and therapeutic treatments, survival from colorectal cancer (CRC) remains disappointing with some 40-50% of newly diagnosed patients ultimately dying of metastatic disease. Current staging by light microscopy alone is not sufficiently predictive of prognosis and would benefit from additional support from biomarkers in order to stratify patients appropriately for adjuvant therapy. We have identified that cathepsin D expression was significantly greater in cells from invasive front (IF) area and liver metastasis (LM) than those from main tumour body (MTB). Cathepsin D expression was subsequently examined by immunohistochemistry in tissue microarrays from 119 patients with CRC. Strong expression in tumour cells at the IF did not correlate significantly with any clinico-pathological parameters examined or patient survival. However, cathepsin D expression in cells from the MTB was highly elevated in late stage CRC and showed significant correlation with subsequent distant metastasis and shorter cancer-specific survival. We also found that macrophages surrounding tumour cells stained strongly for cathepsin D but there was no significant correlation found between cathepsin D in macrophages at IF and MTB of CRC patient with the clinic-pathological parameters examined.

Proteomic phenotyping of Novosphingobium nitrogenifigens reveals a robust capacity for simultaneous nitrogen fixation, polyhydroxyalkanoate production, and resistance to reactive oxygen species.

Novosphingobium nitrogenifigens Y88(T) (Y88) is a free-living, diazotrophic Alphaproteobacterium, capable of producing 80% of its biomass as the biopolymer polyhydroxybutyrate (PHB). We explored the potential utility of this species as a polyhydroxybutyrate production strain, correlating the effects of glucose, nitrogen availability, dissolved oxygen concentration, and extracellular pH with polyhydroxybutyrate production and changes in the Y88 proteomic profile. Using two-dimensional differential in-gel electrophoresis and tandem mass spectrometry, we identified 217 unique proteins from six growth conditions. We observed reproducible, characteristic proteomic signatures for each of the physiological states we examined. We identified proteins that changed in abundance in correlation with either nitrogen fixation, dissolved oxygen concentration, or acidification of the growth medium. The proteins that correlated with nitrogen fixation were identified either as known nitrogen fixation proteins or as novel proteins that we predict play roles in aspects of nitrogen fixation based on their proteomic profiles. In contrast, the proteins involved in central carbon and polyhydroxybutyrate metabolism were constitutively abundant, consistent with the constitutive polyhydroxybutyrate production that we observed in this species. Three proteins with roles in detoxification of reactive oxygen species were identified in this obligate aerobe. The most abundant protein in all experiments was a polyhydroxyalkanoate granule-associated protein, phasin. The full-length isoform of this protein has a long, intrinsically disordered Ala/Pro/Lys-rich N-terminal segment, a feature that appears to be unique to sphingomonad phasins. The data suggest that Y88 has potential as a PHB production strain due to its aerobic tolerance and metabolic orientation toward polyhydroxybutyrate accumulation, even in low-nitrogen growth medium.

Metabolic proteomics of the liver and mammary gland during lactation.

The liver and the mammary gland have complementary metabolic roles during lactation. Glucose synthesized by the liver is released into the circulation and is taken up by the mammary gland where major metabolic products of glucose include milk sugar (lactose) and the glycerol backbone of milk fat (triglycerides). Hepatic synthesis of glucose is often accompanied by ß-oxidation in that organ to provide energy for glucose synthesis, while mammary gland synthesizes rather than oxidizes fat during lactation. We have therefore compared enzyme abundances between the liver and mammary gland of lactating Friesian cows where metabolic output is well established. Quantitative differences in protein amount were assessed using two-dimensional differential in-gel electrophoresis. As predicted, the abundances of enzymes catalysing gluconeogenesis and ß-oxidation were greatest in the liver, and enzyme abundances in mammary tissue were consistent with fat synthesis rather than ß-oxidation.

Proteomic changes at 8 weeks after infection are associated with chronic liver pathology in experimental schistosomiasis.

Chronic Schistosoma mansoni infection can present as a moderate or severe disease, termed intestinal or hepatosplenic schistosomiasis, respectively. Similarly, either moderate splenomegaly or hypersplenomegaly syndrome develops in CBA/J mice by 20weeks of infection and is similar to intestinal or hepatosplenic schistosomiasis respectively. Using this mouse model and two-dimensional differential in gel electrophoresis, the liver proteomic signatures of uninfected mice and mice infected for 6, 8, 12, or 20weeks were compared, and significant protein spots identified using mass spectrometry. We found the greatest number of changes at 12weeks suggesting that this period represents the peak time of change. Pathway analysis identified specific proteins and pathways that correlated to the pathological changes indicative of severe disease, and these pathways were involved as early as 8weeks after infection. These findings provide insight into the development of severe liver pathology in schistosomiasis and may aid in developing biomarkers for hepatosplenic schistosomiasis.

Extracellular polysaccharide-degrading proteome of Butyrivibrio proteoclasticus.

Plant polysaccharide-degrading rumen microbes are fundamental to the health and productivity of ruminant animals. Butyrivibrio proteoclasticus B316(T) is a gram-positive, butyrate-producing anaerobic bacterium with a key role in hemicellulose degradation in the rumen. Gel-based proteomics was used to examine the growth-phase-dependent abundance patterns of secreted proteins recovered from cells grown in vitro with xylan or xylose provided as the sole supplementary carbon source. Five polysaccharidases and two carbohydrate-binding proteins (CBPs) were among 30 identified secreted proteins. The endo-1,4-ß-xylanase Xyn10B was 17.5-fold more abundant in the culture medium of xylan-grown cells, which suggests it plays an important role in hemicellulose degradation. The secretion of three nonxylanolytic enzymes and two CBPs implies they augment hemicellulose degradation by hydrolysis or disruption of associated structural polysaccharides. Sixteen ATP-binding cassette (ABC) transporter substrate-binding proteins were identified, several of which had altered relative abundance levels between growth conditions, which suggests they are important for oligosaccharide uptake. This study demonstrates that B. proteoclasticus modulates the secretion of hemicellulose-degrading enzymes and ATP-dependent sugar uptake systems in response to growth substrate and supports the notion that this organism makes an important contribution to polysaccharide degradation in the rumen.

Carbohydrate transporting membrane proteins of the rumen bacterium, Butyrivibrio proteoclasticus.

The research was aimed at finding which membrane proteins of the rumen bacterium Butyrivibrio proteoclasticus are involved in the uptake of carbohydrates resulting from extracellular enzymatic degradation of hemicellulose and fructan. The proteomic analysis of cells grown with fructose or xylan as the sole substrate identified 13 membrane proteins predicted to function as carbohydrate transporters. One protein detected was the membrane component of a fructose-specific phosphoenolpyruvate:sugar phosphotransferase system believed to be involved in the fructose uptake following extracellular fructan breakdown. The other 12 proteins were all ABC transport system substrate-binding proteins, nine of which belong to functional category COG1653 that includes proteins predicted to transport oligosaccharides. Four of the SBPs were significantly upregulated in xylan grown cells, and three of these were found in polysaccharide utilisation loci where they are clustered with other genes involved in hemicellulose breakdown and metabolism. It is possible that the carbon source available regulates a wider network of genes. The information on the mechanisms used by rumen bacteria to take up carbohydrates from their environment may improve our understanding of the ruminant digestion and facilitate strategies for improved pasture and stored feed utilisation.

Characterization of the Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative Standard using SDS-PAGE shotgun proteomics.

Although there are now multiple methods for the analysis of membrane proteomes, there is relatively little systematic characterization of proteomic workflows for membrane proteins. The Asia Oceania Human Proteome Organisation (AOHUPO) has therefore embarked on a Membrane Proteomics Initiative (MPI) using a large range of workflows. Here, we describe the characterization of the MPI mouse liver microsomal membrane Standard using SDS-PAGE prior to in-gel tryptic digestion and LC-ESI-MS/MS. The Na(2) CO(3) wash followed by SDS-PAGE prior to in-gel tryptic digestion and LC-MS/MS strategy was effective for the detection of membrane proteins with 47.1% of the identified proteins being transmembrane proteins. Gene Ontology term enrichment analysis showed that biological processes involving transport, lipid metabolism, cell communication, cell adhesion, and cellular component organization were significantly enriched. Comparison of the present data with the previously published reports on mouse liver proteomes confirmed that the MPI Standard provides an excellent resource for the analysis of membrane proteins in the AOHUPO MPI.

Identification of cytokeratin 18 as a biomarker of mouse and human hepatosplenic schistosomiasis.

Previously, we demonstrated unique protein expression patterns in 20-week-Schistosoma mansoni-infected CBA/J mice with moderate splenomegaly syndrome (MSS) or hypersplemomegaly syndrome (HSS). To better understand the development of severe pathology, we compared the two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic signatures of livers from uninfected mice and mice infected for 6, 8, 12, or 20 weeks and found significant changes in collagen isoforms, interleukin-2 (IL-2), cytokeratin 18, hydroxyproline, S. mansoni phosphoenolpyruvate carboxykinase, major urinary protein isoforms, and peroxiredoxin 6. Cytokeratin 18, hydroxyproline, and connective tissue growth factor (CTGF) were chosen for analysis in mouse and human sera using targeted biochemical assays. Consistent with the liver analysis, cytokeratin 18, CTGF, and hydroxyproline were significantly elevated in sera from mice with HSS compared to those from uninfected mice or mice with MSS. Moreover, cytokeratin 18 and CTGF were found to be markers for subjects with hepatosplenic and intestinal schistosomiasis, respectively, while serum hydroxyproline was a strong indicator of fibrosis for severe HS. These findings indicate that schistosome-associated changes to the liver can be detected in the serum and reveal the potential for cytokeratin 18 to be used as a diagnostic marker for early detection of hepatosplenic schistosomiasis.

Effects of the microtubule stabilizing agent peloruside A on the proteome of HL-60 cells.

Peloruside A, isolated from the marine sponge Mycale hentscheli, has a similar mechanism of action to paclitaxel (Taxol®), a drug used clinically to treat tumors of the breast, ovary and lung. Paclitaxel and peloruside stabilize the polymerized form of tubulin and arrest cells in G2/M of the cell cycle. We have therefore used two-dimensional electrophoresis of proteins to examine the effect of peloruside A on the human HL-60 promyeloid leukemic cell line. Our goals included investigation whether affected proteins could be mapped onto pathways that predicted the cellular effects of this compound. In response to 100 nM peloruside A treatment for 24 h, seventeen identified proteins showed significant change in abundance with fourteen increases and three decreases. Use of Ingenuity Pathways Analysis confirmed that peloruside A affected pathways consistent with the known effects on microtubules and apoptosis. Our results also indicate a potential role of c-Myc in the cellular actions of peloruside consistent with an induction of aneuploidy seen at low concentrations of peloruside.

Proteomic analysis of membrane preparations from developing Pinus radiata compression wood.

For coniferous gymnosperms, few data exist as to the contribution of the membrane-associated proteome to cell wall and wood formation. In this study, we begin to address this knowledge deficiency by examining the proteomic profile of Golgi-enriched membrane preparations derived from developing Pinus radiata compression wood. These membrane preparations were generated by a combination of discontinuous sucrose gradient centrifugation and Triton X-114-based phase separation. Fractionation by phase separation removed contaminating proteins associated with the cytoskeleton and enabled the discrimination between soluble and membrane-bound/integral proteins. The proteomic analysis of the resulting aqueous and detergent phases using high-performance liquid chromatography-tandem mass spectrometry resulted in the identification of 175 proteins. The majority of the identified proteins were membrane bound/integral and originated from cellular components such as the nucleus, plastids, endoplasmic reticulum, plasma membrane and Golgi vesicles. On the basis of bioinformatic analysis, many of the identified proteins were predicted to be involved either in the regulation of wood formation or in cell wall biosynthesis, which indicated that the proteomic analysis of non-cytosolic proteins in developing xylem is a useful strategy to investigate the molecular aspects of wood formation in pine.

The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate-washed membrane standard.

The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate-washing step enriches for integral and lipid-anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress.