Proteomic Differences Between Hepatocellular Carcinoma and Nontumorous Liver Tissue Investigated by a Combined Gel-based and Label-free Quantitative Proteomics Study

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.Hepatocellular carcinoma (HCC)¹ currently is the fifth most common malignancy worldwide with an annual incidence up to 500 per 100,000 individuals depending on the geographic region investigated. Whereas 80% of new cases occur in developing countries, the incidence increases in industrialized nations including Western Europe, Japan, and the United States (1). To manage patients with HCC, tumor markers are very important tools for diagnosis, indicators of disease progression, outcome prediction, and evaluation of treatment efficacy. Several tumor markers have been reported for HCC, including α-fetoprotein (AFP) (2), Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) (3), and des-γ-carboxyl prothrombin (DCP) (4). However, none of these tumor markers show 100% sensitivity or specificity, which calls for new and better biomarkers.To identify novel biomarkers of HCC, many clinical studies using “omics”-based methods have been reported over the past decade (5–6). In particular, the proteomics-based approach has turned out to be a promising one, offering several quantification techniques to reveal differences in protein expression that are caused by a particular disease. In most studies, the well-established 2D-DIGE technique has been applied for protein quantification followed by identification via mass spectrometry (7–15). Even if the quantification is very accurate and sensitive in this gel-based approach, the relatively high amount of protein sample necessary for protein identification is the major disadvantage of this technique. Several mass-spectrometry-based quantitative studies using labeling-techniques like SILAC (stable isotope labeling by amino acids in cell culture) or iTRAQ (isobaric tags for relative and absolute quantification) have also been carried out for biomarker discovery of HCC (16–18). Here, the concomitant protein quantification and identification in a mass spectrometer allows high-throughput analyses. However, such experiments imply additional labeling reactions (in case of iTRAQ) or are limited to tissue culture systems (in case of SILAC). In the latter case, one can overcome the limitation by using the isotope-labeled proteins obtained from tissue culture as an internal standard added to a corresponding tissue sample. This approach is known as CDIT (culture-derived isotope tags) and was applied in a HCC study, very recently (19). Label-free proteomics approaches based on quantification by ion-intensities or spectral counting offer another possibility for biomarker discovery. These approaches are relatively cheap compared with the labeling approaches, because they do not require any labeling reagents and furthermore they allow for high-throughput and sensitive analyses in a mass spectrometer. A quantitative study of HCC using spectral counting has been reported (20), whereas to our knowledge an ion-intensity-based study has not been performed yet. Apart from these quantification strategies, protein alterations in HCC have been studied by MALDI imaging, as well. Here, the authors could show that based on its proteomic signature, hepatocellular carcinoma can be discriminated with high accuracy from liver metastasis samples or other cancer types (21) as well as liver cirrhosis (22). Based on these results, it could be assumed that MALDI imaging might be a promising alternative to standard histological methods in the future.Here, we report a quantitative proteomic study that combines two different techniques, namely the well-established 2D-DIGE approach and a label-free ion-intensity-based quantification via mass spectrometry and liquid chromatography. To our knowledge this is the first time such a combined study was performed with regard to hepatocellular carcinoma. By comparing the results of both studies, we aim to identify high-confident biomarker candidates of HCC, as gel- and LC-MS-based techniques are complementary. To verify the differential protein expressions detected in our proteomic studies we performed additional immunological verifications for selected proteins within two different sample sets (Fig. 1).Open in a separate windowFig. 1.Schematic representation of the applied workflow.     

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

Small heat-shock protein chaperones are important players in the protein quality control system of the cell, because they can immediately respond to partially unfolded proteins, thereby protecting the cell from harmful aggregates. The small heat-shock proteins can form large polydisperse oligomers that are exceptionally dynamic, which is implicated in their function of protecting substrate proteins from aggregation. Yet the mechanism of substrate recognition remains poorly understood, and little is known about what parts of the small heat-shock proteins interact with substrates and what parts of a partially unfolded substrate protein interact with the small heat-shock proteins. The transient nature of the interactions that prevent substrate aggregation rationalize probing this interaction by crosslinking mass spectrometry. Here, we used a workflow with lysine-specific crosslinking and offline nano-liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry to explore the interaction between the plant small heat-shock protein Hsp21 and a thermosensitive model substrate protein, malate dehydrogenase. The identified crosslinks point at an interaction between the disordered N-terminal region of Hsp21 and the C-terminal presumably unfolding part of the substrate protein.     

Role of NAD(P)H:quinone oxidoreductase polymorphism at codon 187 in susceptibility to lung,laryngeal and oral/pharyngeal cancers

NAD(P)H:quinone oxidoreductase (NQO1) has been proposed to play a protective role against the toxic effects of benzo[a]pyrene quinones. The C⁶⁰⁹T base change in the NQO1 gene, resulting in a Pro¹⁸⁷Ser amino acid change in the protein, has been associated with deficient enzyme activity. We examined whether this polymorphism modified the risks of smoking-related cancers in a case-control study involving patients with lung cancer (n = 150), laryngeal cancer (n = 129), oral/pharyngeal cancer (n = 121) and control individuals (n = 172), all Caucasian smokers. No statistically significant associations were observed between the NQO1 genotypes and smoking-related cancers, although the Ser/Ser genotype was associated with a tendency towards increased risk for lung cancer (odds ratio [OR] = 2.2, 95% confidence interval [CI] 0.7-6.7) and for oral/pharyngeal cancer (OR = 2.3, 95% CI 0.6-8.2). No significant interaction between the NQO1 genotype and either smoking exposure or GSTM1 genotype was found. Our results are consistent with the hypothesis that lack of NQO1 activity may be involved in some smoking-related cancers. However, they were based on small numbers of individuals with the putative atrisk genotype, and the associations did not reach statistical significance. Moreover, these results contrast with those observed in some other ethnic populations, where a protective effect of the NQO1 Ser allele was found. Further studies are therefore clearly needed for a better understanding of the potential role of NQO1 activity in tobacco-related cancers.     

PARma: identification of microRNA target sites in AGO-PAR-CLIP data

PARma is a complete data analysis software for AGO-PAR-CLIP experiments to identify target sites of microRNAs as well as the microRNA binding to these sites. It integrates specific characteristics of the experiments into a generative model. The model and a novel pattern discovery tool are iteratively applied to data to estimate seed activity probabilities, cluster confidence scores and to assign the most probable microRNA. Based on differential PAR-CLIP analysis and comparison to RIP-Chip data, we show that PARma is more accurate than existing approaches. PARma is available from http://www.bio.ifi.lmu.de/PARma     

Imbricaric Acid and Perlatolic Acid: Multi-Targeting Anti-Inflammatory Depsides from Cetrelia monachorum

In vitro screening of 17 Alpine lichen species for their inhibitory activity against 5-lipoxygenase, microsomal prostaglandin E₂ synthase-1 and nuclear factor kappa B revealed Cetrelia monachorum (Zahlbr.) W.L. Culb. & C.F. Culb. As conceivable source for novel anti-inflammatory compounds. Phytochemical investigation of the ethanolic crude extract resulted in the isolation and identification of 11 constituents, belonging to depsides and derivatives of orsellinic acid, olivetolic acid and olivetol. The two depsides imbricaric acid (4) and perlatolic acid (5) approved dual inhibitory activities on microsomal prostaglandin E₂ synthase-1 (IC₅₀ = 1.9 and 0.4 µM, resp.) and on 5-lipoxygenase tested in a cell-based assay (IC₅₀ = 5.3 and 1.8 µM, resp.) and on purified enzyme (IC₅₀ = 3.5 and 0.4 µM, resp.). Additionally, these two main constituents quantified in the extract with 15.22% (4) and 9.10% (5) showed significant inhibition of tumor necrosis factor alpha-induced nuclear factor kappa B activation in luciferase reporter cells with IC₅₀ values of 2.0 and 7.0 µM, respectively. In a murine in vivo model of inflammation, 5 impaired the inflammatory, thioglycollate-induced recruitment of leukocytes to the peritoneum. The potent inhibitory effects on the three identified targets attest 4 and 5 a pronounced multi-target anti-inflammatory profile which warrants further investigation on their pharmacokinetics and in vivo efficacy.     

Kinase Activity of ArcB from Escherichia coli Is Subject to Regulation by Both Ubiquinone and Demethylmenaquinone

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in ‘respiratory’ electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.     

Micro-Geographical Heterogeneity in Schistosoma mansoni and S. haematobium Infection and Morbidity in a Co-Endemic Community in Northern Senegal

Background

Schistosoma mansoni and S. haematobium are co-endemic in many areas in Africa. Yet, little is known about the micro-geographical distribution of these two infections or associated disease within such foci. Such knowledge could give important insights into the drivers of infection and disease and as such better tailor schistosomiasis control and elimination efforts.

Methodology

In a co-endemic farming community in northern Senegal (346 children (0–19 y) and 253 adults (20–85 y); n = 599 in total), we studied the spatial distribution of S. mansoni and S. haematobium single and mixed infections (by microscopy), S. mansoni-specific hepatic fibrosis, S. haematobium-specific urinary tract morbidity (by ultrasound) and water contact behavior (by questionnaire). The Kulldorff''s scan statistic was used to detect spatial clusters of infection and morbidity, adjusted for the spatial distribution of gender and age.

Principal Findings

Schistosoma mansoni and S. haematobium infection densities clustered in different sections of the community (p = 0.002 and p = 0.023, respectively), possibly related to heterogeneities in the use of different water contact sites. While the distribution of urinary tract morbidity was homogeneous, a strong geospatial cluster was found for severe hepatic fibrosis (p = 0.001). Particularly those people living adjacent to the most frequently used water contact site were more at risk for more advanced morbidity (RR = 6.3; p = 0.043).

Conclusions/Significance

Schistosoma infection and associated disease showed important micro-geographical heterogeneities with divergent patterns for S. mansoni and S. haematobium in this Senegalese community. Further in depth investigations are needed to confirm and explain our observations. The present study indicates that local geospatial patterns should be taken into account in both research and control of schistosomiasis. The observed extreme focality of schistosomiasis even at community level, suggests that current strategies may not suffice to move from morbidity control to elimination of schistosomiasis, and calls for less uniform measures at a finer scale.     

Real-Time Imaging of the Intracellular Glutathione Redox Potential in the Malaria Parasite Plasmodium falciparum

In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (E _GSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects E _GSH changes induced by oxidative and nitrosative stress. The cytosolic basal E _GSH of 3D7 and Dd2 were estimated to be −314.2±3.1 mV and −313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in E _GSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites'' E _GSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular E _GSH in P. falciparum. Applying this technique in further studies will enhance our understanding of redox regulation and mechanisms of drug action and resistance in Plasmodium and might also stimulate redox research in other pathogens.     

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.