UV-triggered affinity capture identifies interactions between the Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) and antimalarial agents in live parasitized cells.

A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615.

The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG.

C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-ß-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including ß-galactosidases, ß-N-Acetylhexosaminidases and α-mannosidases.

Comparative analysis of the exoproteomes of Listeria monocytogenes strains grown at low temperatures.

This article presents the major differences in the exoproteomes of Listeria monocytogenes strains grown at 11°C and 20°C, and their comparison to 37°C, the optimal temperature of growth of this foodborne pathogenic bacteria. A set of four strains previously characterized and representing the genetic diversity of the species was used. Two were virulent, of which one was persistent, and two were low virulent strains. The proteins secreted by the strains grown in minimal medium were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by liquid chromatography tandem mass spectrometry. The heterogeneity among the four strains concerning the 15 major proteins detected was noticed. No clear association of exoproteome with virulence or genotype was found. Cluster analysis of the protein patterns of the strains suggests an increasing differentiation of strain response with low temperatures, highlighting the importance of the study of the exoproteomes. The main finding was the lack of some proteins in the exoproteome of the persistent strain, namely, flagellin (FlaA) and of OppA/oligopeptide ABC transporter, when compared to the other strains. In fact, these two proteins differ in abundance between strains grown at low temperature. Moreover, FlaA was the only glycoprotein identified in the exoproteomes. An attempt is made here to assess the relevance of the major exoproteins differentially detected. The investigation of the exoproteomes of other persistent and sporadic strains will allow identification of proteins involved in adaptation of particular L. monocytogenes strains to low temperatures in use throughout the food chain.

The genome and surface proteome of Capnocytophaga canimorsus reveal a key role of glycan foraging systems in host glycoproteins deglycosylation.

Capnocytophaga canimorsus are commensal Gram-negative bacteria from dog's mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the Flavobacteria-Bacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus-like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection.

Inhibition of protein synthesis by TOR inactivation revealed a conserved regulatory mechanism of the BiP chaperone in Chlamydomonas.

The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.

Quantitative proteomic (iTRAQ) analysis of 1st trimester maternal plasma samples in pregnancies at risk for preeclampsia.

A current major obstacle is that no reliable screening markers exist to detect pregnancies at risk for preeclampsia. Quantitative proteomic analysis employing isobaric labelling (iTRAQ) has been suggested to be suitable for the detection of potential plasma biomarkers, a feature we recently verified in analysis of pregnancies with Down syndrome foetuses. We have now examined whether this approach could yield biomarkers to screen pregnancies at risk for preeclampsia. In our study, we used maternal plasma samples obtained at 12 weeks of gestation, six from women who subsequently developed preeclampsia and six with uncomplicated deliveries. In our analysis, we observed elevations in 10 proteins out of 64 proteins in the preeclampsia study group when compared to the healthy control group. These proteins included clusterin, fibrinogen, fibronectin, and angiotensinogen, increased levels of which are known to be associated with preeclampsia. An elevation in the immune-modulatory molecule, galectin 3 binding protein, was also noted. Our pilot study, therefore, indicates that quantitative proteomic iTRAQ analysis could be a useful tool for the detection of new preeclampsia screening markers.

The Potential Tumor-Suppressor DHRS7 Inversely Correlates with EGFR Expression in Prostate Cancer Cells and Tumor Samples.

Prostate cancer (PCa), one of the most common malignancies in men, typically responds to initial treatment, but resistance to therapy often leads to metastases and death. The dehydrogenase/reductase 7 (DHRS7, SDR34C1) is an "orphan" enzyme without known physiological function. DHRS7 was previously found to be decreased in higher-stage PCa, and siRNA-mediated knockdown increased the aggressiveness of LNCaP cells. To further explore the role of DHRS7 in PCa, we analyzed the proteome of LNCaP cells following DHRS7 knockdown to assess potentially altered pathways. Although DHRS7 is able to inactivate 5α-dihydrotestosterone, DHRS7 knockdown did not affect androgen receptor (AR) target gene expression, and its effect on PCa cells seems to be androgen-independent. Importantly, proteome analyses revealed increased expression of epidermal growth factor receptor (EGFR), which was confirmed by RT-qPCR and Western blotting. Comparison of AR-positive LNCaP with AR-negative PC-3 and DU145 PCa cell lines revealed a negative correlation between DHRS7 and EGFR expression. Conversely, EGFR knockdown enhanced DHRS7 expression in these cells. Importantly, analysis of patient samples revealed a negative correlation between DHRS7 and EGFR expression, both at the mRNA and protein levels, and DHRS7 expression correlated positively with patient survival rates. These results suggest a protective role for DHRS7 in PCa.

Proteins induced by telomere dysfunction and DNA damage represent biomarkers of human aging and disease.

Telomere dysfunction limits the proliferative capacity of human cells by activation of DNA damage responses, inducing senescence or apoptosis. In humans, telomere shortening occurs in the vast majority of tissues during aging, and telomere shortening is accelerated in chronic diseases that increase the rate of cell turnover. Yet, the functional role of telomere dysfunction and DNA damage in human aging and diseases remains under debate. Here, we identified marker proteins (i.e., CRAMP, stathmin, EF-1alpha, and chitinase) that are secreted from telomere-dysfunctional bone-marrow cells of late generation telomerase knockout mice (G4mTerc(-/-)). The expression levels of these proteins increase in blood and in various tissues of aging G4mTerc(-/-) mice but not in aging mice with long telomere reserves. Orthologs of these proteins are up-regulated in late-passage presenescent human fibroblasts and in early passage human cells in response to gamma-irradiation. The study shows that the expression level of these marker proteins increases in the blood plasma of aging humans and shows a further increase in geriatric patients with aging-associated diseases. Moreover, there was a significant increase in the expression of the biomarkers in the blood plasma of patients with chronic diseases that are associated with increased rates of cell turnover and telomere shortening, such as cirrhosis and myelodysplastic syndromes (MDS). Analysis of blinded test samples validated the effectiveness of the biomarkers to discriminate between young and old, and between disease groups (MDS, cirrhosis) and healthy controls. These results support the concept that telomere dysfunction and DNA damage are interconnected pathways that are activated during human aging and disease.

Human T cell receptor gammadelta cells recognize endogenous mevalonate metabolites in tumor cells.

T lymphocytes expressing the T cell receptor (TCR)-gammadelta recognize unknown antigens on tumor cells. Here we identify metabolites of the mevalonate pathway as the tumor ligands that activate TCR-gammadelta cells. In tumor cells, blockade of hydroxy-methylglutaryl-CoA reductase (HMGR), the rate limiting enzyme of the mevalonate pathway, prevents both accumulation of mevalonate metabolites and recognition by TCR-gammadelta cells. When metabolite accumulation is induced by overexpressing HMGR or by treatment with nitrogen-containing bisphosphonate drugs, tumor cells derived from many tissues acquire the capacity to stimulate the same TCR-gammadelta population. Accumulation of mevalonate metabolites in tumor cells is a powerful danger signal that activates the immune response and may represent a novel target of tumor immunotherapy.

Quantitative proteomics analysis of maternal plasma in Down syndrome pregnancies using isobaric tagging reagent (iTRAQ).

Currently no specific biomarkers exist for the screening of pregnancies at risk for Down syndrome (DS). Since a quantitative proteomic approach with isobaric labelling (iTRAQ) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing DS fetuses in comparison to normal healthy babies. In our study, we used plasma from six women with DS pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. In our quantitative proteomics analysis we were able to detect 178 proteins using iTRAQ labelling in conjunction with 4800 MALDI TOF/TOF. Amongst these we observed changes in betaHCG, a known screening marker for DS, indicating that our assay was functional. We found a number of elevated proteins Ig lambda chain C region, serum amyloid P-component, amyloid beta A4, and under expressed proteins like gamma-actin and titin in DS pregnancies. These proteins are also found in the sera of patients with Alzheimer disease, which share similar pathologies of DS. Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers.

Survival of pathogenic mycobacteria in macrophages is mediated through autophosphorylation of protein kinase G.

Pathogenic mycobacteria survive within macrophages through the inhibition of phagosome-lysosome fusion. A crucial factor for avoiding lysosomal degradation is the mycobacterial serine/threonine protein kinase G (PknG). PknG is released into the macrophage cytosol upon mycobacterial infection, suggesting that PknG might exert its activity by interfering with host signaling cascades, but the mode of action of PknG remains unknown. Here, we show that PknG undergoes autophosphorylation on threonine residues located at the N terminus. In contrast to all other mycobacterial kinases investigated thus far, autophosphorylation of PknG was not involved in the regulation of its kinase activity. However, autophosphorylation was crucial for the capacity of PknG to promote mycobacterial survival within macrophages. These results will contribute to a better understanding of the virulence mechanisms of pathogenic mycobacteria and may help to design improved inhibitors of PknG to be developed as antimycobacterial compounds.

Quantitative proteomics reveals reduction of endocytic machinery components in gliomas.

BACKGROUND: Gliomas are the most frequent and aggressive malignancies of the central nervous system. Decades of molecular analyses have demonstrated that gliomas accumulate genetic alterations that culminate in enhanced activity of receptor tyrosine kinases and downstream mediators. While the genetic alterations, like gene amplification or loss, have been well characterized, little information exists about changes in the proteome of gliomas of different grades. METHODS: We performed unbiased quantitative proteomics of human glioma biopsies by mass spectrometry followed by bioinformatic analysis. FINDINGS: Various pathways were found to be up- or downregulated. In particular, endocytosis as pathway was affected by a vast and concomitant reduction of multiple machinery components involved in initiation, formation, and scission of endocytic carriers. Both clathrin-dependent and -independent endocytosis were changed, since not only clathrin, AP-2 adaptins, and endophilins were downregulated, but also dynamin that is shared by both pathways. The reduction of endocytic machinery components caused increased receptor cell surface levels, a prominent phenotype of defective endocytosis. Analysis of additional biopsies revealed that depletion of endocytic machinery components was a common trait of various glioma grades and subclasses. INTERPRETATION: We propose that impaired endocytosis creates a selective advantage in glioma tumor progression due to prolonged receptor tyrosine kinase signaling from the cell surface. FUND: This work was supported by Grants 316030-164105 (to P. Jenö), 31003A-162643 (to M. Spiess) and PP00P3-176974 (to G. Hutter) from the Swiss National Science Foundation. Further funding was received by the Department of Surgery from the University Hospital Basel.

Proteomic Landscape of Aldosterone-Producing Adenoma.

Primary aldosteronism is a disease of excessive production of adrenal steroid hormones and the most common cause of endocrine hypertension. Primary aldosteronism results mainly from bilateral adrenal hyperplasia or unilateral aldosterone-producing adenoma (APA). Primary aldosteronism cause at the molecular level is incompletely understood and a targeted treatment preventing excessive adrenal steroid production is not available. Here, we perform deep quantitative proteomic and phosphoproteomic profiling of 6 pairs of APA and adjacent nontumoral adrenal cortex. We show that increased steroidogenesis in APA is accompanied by upregulation of steroidogenic enzymes (HSD3B2, CYP21A2, CYP11B2) and of proteins involved in cholesterol uptake (LSR). We demonstrate that HSD3B2 is phosphorylated at Ser95 or 96 and identify a novel phosphorylation site, Ser489, in CYP21A2, suggesting that steroidogenic enzymes are regulated by phosphorylation. Our analysis also reveals altered ECM (extracellular matrix) composition in APA that affects ECM-cell surface interactions and actin cytoskeleton rearrangements. We show that RHOC, a GTPase controlling actin organization in response to extracellular stimuli, is upregulated in APA and promotes expression of the aldosterone synthase gene CYP11B2. Our data also indicate deregulation of protein N-glycosylation and GABAergic signaling in APAs. Finally, we find that mTORC1 (mammalian target of rapamycin complex 1) signaling is the major pathway deregulated in APA. Our study provides a rich resource for future research on the molecular mechanisms of primary aldosteronism.

Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1.

Diffuse gliomas progress by invading neighboring brain tissue to promote postoperative relapse. Transcription factor SOX2 is highly expressed in invasive gliomas and maps to chromosome region 3q26 together with the genes for PI3K/AKT signaling activator PIK3CA and effector molecules of mitochondria fusion and cell invasion, MFN1 and OPA1. Gene copy number analysis at 3q26 from 129 glioma patient biopsies revealed mutually exclusive SOX2 amplifications (26%) and OPA1 losses (19%). Both forced SOX2 expression and OPA1 inactivation increased LN319 glioma cell invasion in vitro and promoted cell dispersion in vivo in xenotransplanted D. rerio embryos. While PI3 kinase activity sustained SOX2 expression, pharmacological PI3K/AKT pathway inhibition decreased invasion and resulted in SOX2 nucleus-to-cytoplasm translocation in an mTORC1-independent manner. Chromatin immunoprecipitation and luciferase reporter gene assays together demonstrated that SOX2 trans-activates PIK3CA and OPA1. Thus, SOX2 activates PI3K/AKT signaling in a positive feedback loop, while OPA1 deletion is interpreted to counteract OPA1 trans-activation. Remarkably, neuroimaging of human gliomas with high SOX2 or low OPA1 genomic imbalances revealed significantly larger necrotic tumor zone volumes, corresponding to higher invasive capacities of tumors, while autologous necrotic cells are capable of inducing higher invasion in SOX2 overexpressing or OPA1 knocked-down relative to parental LN319. We thus propose necrosis volume as a surrogate marker for the assessment of glioma invasive potential. Whereas glioma invasion is activated by a PI3K/AKT-SOX2 loop, it is reduced by a cryptic invasion suppressor SOX2-OPA1 pathway. Thus, PI3K/AKT-SOX2 and mitochondria fission represent connected signaling networks regulating glioma invasion.

Insulin resistance causes inflammation in adipose tissue.

Obesity is a major risk factor for insulin resistance and type 2 diabetes. In adipose tissue, obesity-mediated insulin resistance correlates with the accumulation of proinflammatory macrophages and inflammation. However, the causal relationship of these events is unclear. Here, we report that obesity-induced insulin resistance in mice precedes macrophage accumulation and inflammation in adipose tissue. Using a mouse model that combines genetically induced, adipose-specific insulin resistance (mTORC2-knockout) and diet-induced obesity, we found that insulin resistance causes local accumulation of proinflammatory macrophages. Mechanistically, insulin resistance in adipocytes results in production of the chemokine monocyte chemoattractant protein 1 (MCP1), which recruits monocytes and activates proinflammatory macrophages. Finally, insulin resistance (high homeostatic model assessment of insulin resistance [HOMA-IR]) correlated with reduced insulin/mTORC2 signaling and elevated MCP1 production in visceral adipose tissue from obese human subjects. Our findings suggest that insulin resistance in adipose tissue leads to inflammation rather than vice versa.

A proteomic study to identify soya allergens--the human response to transgenic versus non-transgenic soya samples.

BACKGROUND: In spite of being among the main foods responsible for allergic reactions worldwide, soybean (Glycine max)-derived products continue to be increasingly widespread in a variety of food products due to their well-documented health benefits. Soybean also continues to be one of the elected target crops for genetic modification. The aim of this study was to characterize the soya proteome and, specifically, IgE-reactive proteins as well as to compare the IgE response in soya-allergic individuals to genetically modified Roundup Ready soya versus its non-transgenic control. METHODS: We performed two-dimensional gel electrophoresis of protein extracts from a 5% genetically modified Roundup Ready flour sample and its non-transgenic control followed by Western blotting with plasma from 5 soya-sensitive individuals. We used peptide tandem mass spectrometry to identify soya proteins (55 protein matches), specifically IgE-binding ones, and to evaluate differences between transgenic and non-transgenic samples. RESULTS: We identified 2 new potential soybean allergens--one is maturation associated and seems to be part of the late embryogenesis abundant proteins group and the other is a cysteine proteinase inhibitor. None of the individuals tested reacted differentially to the transgenic versus non-transgenic samples under study. CONCLUSION: Soybean endogenous allergen expression does not seem to be altered after genetic modification. Proteomics should be considered a powerful tool for functional characterization of plants and for food safety assessment.

Development and characterization of a pseudo multiple reaction monitoring method for the quantification of human uromodulin in urine.

BACKGROUND: Uromodulin is the most abundant protein in healthy human urine. Recently it has been suggested as a specific biomarker of renal tubular damage. We have developed a novel pseudo multiple reaction monitoring (pseudo MRM) for the protein's quantification in human urine. RESULTS: Selection of two peptides allowed quantification of uromodulin in human urine. The pseudo MRM quantified uromodulin in healthy individuals between 21 and 1344 nM and in autosomal dominant tubulointerstitial kidney disease-UMOD patients between 2 and 25 nM. CONCLUSION: The pseudo MRM allows greater confidence in assay specificity than traditional MRM methods and quantified uromodulin at concentrations higher than achievable by ELISA. Differences in urinary uromodulin concentration related to the rs4293393 promoter variant in the UMOD gene was confirmed. This method will be used to further investigate uromodulin as a biomarker of renal injury.

Identification and structure of a putative Ca2+-binding domain at the C terminus of AQP1.

Aquaporin-1 (AQP1) is the first functionally identified aquaporin of a growing family of membrane water channels found in all forms of life. Recently, a possible secondary function as a cyclic guanosine monophosphate (cGMP) gated ion channel was attributed to AQP1. We have reconstituted purified protein from bovine and human red blood cell membranes into highly ordered 2D crystals. The topography of both AQP1s was determined by electron microscopy from freeze-dried, unidirectionally metal-shadowed 2D crystals as well as from surface topographs of native crystals recorded in buffer solution with the atomic force microscope (AFM). In spite of the high level of sequence homology between bovine and human AQP1, the surfaces showed distinct differences. Alignment of both sequences and comparison of the acquired surface topographies with the atomic model of human AQP1 revealed the topographic changes on the surface of bovine AQP1 to be induced by a few amino acid substitutions. A striking degree of sequence homology was found between the carboxyl-terminal domains of AQP1s from different organisms and EF-hands from Ca2+-binding proteins belonging to the calmodulin superfamily, suggesting the existence of a Ca2+-binding site at the C terminus of AQP1 instead of the putative cGMP-binding site reported previously. To unveil its position on the acquired surface topographies, 2D crystals of AQP1 were digested with carboxypeptidase Y, which cleaves off the intracellular C terminus. Difference maps of AFM topographs between the native and the peptidase-treated AQP1s showed the carboxylic tail to be close to the 4-fold symmetry axis of the tetramer. SDS-PAGE and matrix-assisted laser desorption/ionisation mass spectrometry of native and decarboxylated bovine and human AQP1 revealed that the EF-hand motif found at the C terminus of AQP1 was partially resistant to peptidase digestion. The importance of the C-terminal domain is implicated by structural instability of decarboxylated AQP1. A possible role of the C terminus and calcium in translocation of AQP1 in cholangiocytes from intracellular vesicles to the plasma membrane and in triggering its fusion is discussed. Functional studies are now required to identify the physiological role of the Ca2+-binding site.

A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1.

Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O2) and releases hydrogen peroxide (H2O2), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys(208)-Cys(241) disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H2O2 production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O2 is reduced to H2O2. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys(208)/Cys(241)-dependent mixed-disulfide complex with Ero1α. The H2O2-detoxifying glutathione peroxidase 8 also binds to the Cys(208)/Cys(241) loop region. Supported by O2 diffusion simulations, these data describe the first enzymatically controlled O2 access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H2O2 production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.