Transcriptional Alterations in X-Linked Dystonia-Parkinsonism Caused by the SVA Retrotransposon.

X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of the TAF1 gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the TAF1 intron retention transcript TAF1-32i can be found in XDP cells as compared to healthy controls. Here, we investigate the sequence of the deep intronic region included in this transcript and show that it is also present in cells from healthy individuals, albeit in lower amounts than in XDP cells, and that it undergoes degradation by nonsense-mediated mRNA decay. Furthermore, we investigate epigenetic marks (e.g., DNA methylation and histone modifications) present in this intronic region and the spanning sequence. Finally, we show that the SVA evinces regulatory potential, as demonstrated by its ability to repress the TAF1 promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons.

Linking CREB function with altered metabolism in murine fibroblast-based model cell lines.

The cAMP-responsive element binding protein CREB is frequently overexpressed and activated in tumors of distinct histology, leading to enhanced proliferation, migration, invasion and angiogenesis as well as reduced apoptosis. The de-regulated expression of CREB might be linked with transcriptional as well as post-transcriptional regulation mechanisms. We show here that altered CREB expression levels and function are associated with changes in the cellular metabolism. Using comparative proteome-based analysis an altered expression pattern of proteins involved in the cellular metabolism in particular in glycolysis was found upon CREB down-regulation in HER-2/neu-transfected cell lines. This was associated with diminished expression levels of the glucose transporter 1, reduced glucose uptake and reduced glycolytic activity in HER-2/neu-transfected cells with down-regulated CREB when compared to HER-2/neu+ cells. Furthermore, hypoxia-induced CREB activity resulted in changes of the metabolism in HER-2/neu transfected cells. Low pH values in the supernatant of HER-2/neu transformants were restored by CREB down-regulation, but further decreased by hypoxia. The altered intracellular pH values were associated with a distinct expression of lactate dehydrogenase, and its substrate lactate. Moreover, enhanced phosphorylation of CREB on residue Ser133 was accompanied by a down-regulation of pERK and an up-regulation of pAKT. CREB promotes the detoxification of ROS by catalase, therefore protecting the mitochondrial activity under oxidative stress. These data suggest that there might exists a link between CREB function and the altered metabolism in HER-2/neu-transformed cells. Thus, targeting these altered metabolic pathways might represent an attractive therapeutic approach at least for the treatment of patients with HER-2/neu overexpressing tumors.

VHL-dependent alterations in the secretome of renal cell carcinoma: Association with immune cell response?

Secreted proteins could modulate the interaction between tumor, stroma and immune cells within the tumor microenvironment thereby mounting an immunosuppressive tumor microenvironment. In order to determine the secretome-mediated, von Hippel Lindau (VHL)-regulated cross-talk between tumor cells and T lymphocytes peripheral blood mononuclear cells (PBMC) from healthy donors were either cultured in conditioned media obtained from normoxic and hypoxic human VHL-deficient renal cell carcinoma (RCC) cell line (786-0VHL-) and its wild type (wt) VHL-transfected counterpart (786-0VHL+) or directly co-cultured with both cell lines. An increased T cell proliferation was detected in the presence of 786-0VHL+-conditioned medium. By applying a quantitative proteomic-based approach using differential gel electrophoresis followed by mass spectrometry fourteen proteins were identified to be differentially expressed within the secretome of 786-0VHL- cells when compared to that of 786-0VHL+ cells. All proteins identified were involved in multiple tumor-associated biological functions including immune responses. Functional studies on manganese superoxide dismutase 2 (MnSOD2) demonstrated that it was a regulator of T cell activation-induced oxidative signaling and cell death. Direct effects of soluble MnSOD2 on the growth properties and interleukin 2 (IL-2) secretion of T cells could be demonstrated underlining the critical role of extracellular MnSOD2 levels for T cell proliferation and activation.

Distinct von Hippel-Lindau gene and hypoxia-regulated alterations in gene and protein expression patterns of renal cell carcinoma and their effects on metabolism.

During the last decade the knowledge about the molecular mechanisms of the cellular adaption to hypoxia and the function of the "von Hippel Lindau" (VHL) protein in renal cell carcinoma (RCC) has increased, but there exists little information about the overlap and differences in gene/protein expression of both processes. Therefore the aim of this study was to dissect VHL- and hypoxia-regulated alterations in the metabolism of human RCC using ome-based strategies. The effect of the VHL- and hypoxia-regulated altered gene/protein expression pattern on the cellular metabolism was analyzed by determination of glucose uptake, lactate secretion, extracellular pH, lactate dehydrogenase activity, amino acid content and ATP levels. By employing VHL-/VHL(+) RCC cells cultured under normoxic and hypoxic conditions, VHL-dependent, HIF-dependent as well as VHL-/HIF-independent alterations in the gene and protein expression patterns were identified and further validated in other RCC cell lines. The genes/proteins differentially expressed under these distinct conditions were mainly involved in the cellular metabolism, which was accompanied by an altered metabolism as well as changes in the abundance of amino acids in VHL-deficient cells. In conclusion, the study reveals similarities, but also differences in the genes and proteins controlled by VHL functionality and hypoxia thereby demonstrating differences in the metabolic switch of RCC under these conditions.

Methanogenic food web in the gut contents of methane-emitting earthworm Eudrilus eugeniae from Brazil.

The anoxic saccharide-rich conditions of the earthworm gut provide an ideal transient habitat for ingested microbes capable of anaerobiosis. It was recently discovered that the earthworm Eudrilus eugeniae from Brazil can emit methane (CH4) and that ingested methanogens might be associated with this emission. The objective of this study was to resolve trophic interactions of bacteria and methanogens in the methanogenic food web in the gut contents of E. eugeniae. RNA-based stable isotope probing of bacterial 16S rRNA as well as mcrA and mrtA (the alpha subunit of methyl-CoM reductase and its isoenzyme, respectively) of methanogens was performed with [(13)C]-glucose as a model saccharide in the gut contents. Concomitant fermentations were augmented by the rapid consumption of glucose, yielding numerous products, including molecular hydrogen (H2), carbon dioxide (CO2), formate, acetate, ethanol, lactate, succinate and propionate. Aeromonadaceae-affiliated facultative aerobes, and obligate anaerobes affiliated to Lachnospiraceae, Veillonellaceae and Ruminococcaceae were associated with the diverse fermentations. Methanogenesis was ongoing during incubations, and (13)C-labeling of CH4 verified that supplemental [(13)C]-glucose derived carbon was dissimilated to CH4. Hydrogenotrophic methanogens affiliated with Methanobacteriaceae and Methanoregulaceae were linked to methanogenesis, and acetogens related to Peptostreptoccocaceae were likewise found to be participants in the methanogenic food web. H2 rather than acetate stimulated methanogenesis in the methanogenic gut content enrichments, and acetogens appeared to dissimilate supplemental H2 to acetate in methanogenic enrichments. These findings provide insight on the processes and associated taxa potentially linked to methanogenesis and the turnover of organic carbon in the alimentary canal of methane-emitting E. eugeniae.

Identification of 14-3-3ß gene as a novel miR-152 target using a proteome-based approach.

Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G(+), miR-152(low) cells, and their miR-152-overexpressing (miR(high)) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein ß/α/YWHAB (14-3-3ß) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3ß expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3ß expression in miR-152(high) cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients' outcome, a link between 14-3-3ß and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3ß and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity.

Vitamin D receptor regulates intestinal proteins involved in cell proliferation, migration and stress response.

BACKGROUND: Genome-wide association studies found low plasma levels of 25-hydroxyvitamin D and vitamin D receptor (VDR) polymorphisms associated with a higher prevalence of pathological changes in the intestine such as chronic inflammatory bowel diseases. METHODS: In this study, a proteomic approach was applied to understand the overall physiological importance of vitamin D in the small intestine, beyond its function in calcium and phosphate absorption. RESULTS: In total, 569 protein spots could be detected by two-dimensional-difference in-gel electrophoresis (2D-DIGE), and 82 proteins were considered as differentially regulated in the intestinal mucosa of VDR-deficient mice compared to that of wildtype (WT) mice. Fourteen clearly detectable proteins were identified by MS/MS and further analyzed by western blot and/or real-time RT-PCR. The differentially expressed proteins are functionally involved in cell proliferation, cell adhesion and cell migration, stress response and lipid transport. Mice lacking VDR revealed higher levels of intestinal proteins associated with proliferation and migration such as the 37/67 kDa laminin receptor, collagen type VI (alpha 1 chain), keratin-19, tropomyosin-3, adseverin and higher levels of proteins involved in protein trafficking and stress response than WT mice. In contrast, proteins that are involved in transport of bile and fatty acids were down-regulated in small intestine of mice lacking VDR compared to WT mice. However, plasma and liver concentrations of cholesterol and triglycerides were not different between the two groups of mice. CONCLUSION: Collectively, these data imply VDR as an important factor for controlling cell proliferation, migration and stress response in the small intestine.

Towards defining biomarkers indicating resistances to targeted therapies.

An impressive, but often short objective response was obtained in many tumor patients treated with different targeted therapies, but most of the patients develop resistances against these drugs. So far, a number of distinct mechanisms leading to intrinsic as well as acquired resistances have been identified in tumors of distinct origin. These can arise from genetic alterations, like mutations, truncations, and amplifications or due to deregulated expression of various proteins and signal transduction pathways, but also from cellular heterogeneity within tumors after an initial response. Therefore, biomarkers are urgently needed for cancer prognosis and personalized cancer medicine. The application of "ome"-based technologies including cancer (epi)genomics, next generation sequencing, cDNA microarrays and proteomics might led to the predictive or prognostic stratification of patients to categorize resistance mechanisms and to postulate combinations of treatment strategies. This review discusses the implementation of proteome-based analysis to identify markers of pathway (in)activation in tumors and the resistance mechanisms, which represent major clinical problems as a tool to optimize individually tailored therapies based on targeted drugs. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Reduced immunosuppressive properties of axitinib in comparison with other tyrosine kinase inhibitors.

The multikinase inhibitors sunitinib, sorafenib, and axitinib have an impact not only on tumor growth and angiogenesis, but also on the activity and function of immune effector cells. In this study, a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. Tyrosine kinase inhibitor treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib, axitinib did not affect the mitochondrial membrane potential (Δψm) but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl-1), leading to an irreversible arrest in the G2/M cell cycle phase and delayed apoptosis. Furthermore, the sorafenib-mediated suppression of immune effector cells, in particular the reduction of the CD8(+) T cell subset along with the down-regulation of key immune cell markers such as chemokine CC motif receptor 7 (CCR7), CD26, CD69, CD25, and CXCR3, was not observed in axitinib-treated immune effector cells. Therefore, axitinib rather than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy.

Emission of methane by Eudrilus eugeniae and other earthworms from Brazil.

Earthworms emit denitrification-derived nitrous oxide and fermentation-derived molecular hydrogen. The present study demonstrated that the earthworm Eudrilus eugeniae, obtained in Brazil, emitted methane. Other worms displayed a lesser or no capacity to emit methane. Gene and transcript analyses of mcrA (encoding the alpha subunit of methyl-CoM reductase) in gut contents of E. eugeniae suggested that Methanosarcinaceae, Methanobacteriaceae, and Methanomicrobiaceae might be associated with this emission.

Unexpected oligomeric structure of the FocA formate channel of Escherichia coli : a paradigm for the formate-nitrite transporter family of integral membrane proteins.

FocA is a predicted formate channel with a deduced mass of 31 kDa that catalyzes the bidirectional movement of formate across the cytoplasmic membrane of Escherichia coli and is the archetype of the formate-nitrite transporter (FNT) family. Overproduced FocA variants with either an N- or a C-terminal Strep-tag increased formate import into anaerobic E. coli cells as determined by the enhanced activity of a single-copy formate-dependent fdhF::lacZ fusion. Using anti-FocA antibodies, we could show that both FocA variants were integrated into the cytoplasmic membrane. Circular dichroism spectroscopy of purified FocA(Strep-N) revealed a high alpha-helical content of 56% consistent with the predicted six transmembrane helices present in the protein. Analysis of the oligomeric state by blue-native polyacrylamide gel electrophoresis revealed FocA to have an unexpected pentameric quaternary structure. This study reports the first isolation of an FNT family member.