Both sphingomyelin synthases SMS1 and SMS2 are required for sphingomyelin homeostasis and growth in human HeLa cells

Sphingomyelin (SM) is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The mammalian genome encodes two known SM synthase (SMS) isoforms, SMS1 and SMS2. However, the relative contributions of these enzymes to SM production in mammalian cells remained to be established. Here we show that SMS1 and SMS2 are co-expressed in a variety of cell types and function as the key Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. RNA interference-mediated depletion of either SMS1 or SMS2 caused a substantial decrease in SM production levels, an accumulation of ceramides, and a block in cell growth. Although SMS-depleted cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the biological role of SM synthases goes beyond formation of SM.     

Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry

Untargeted metabolomics aims to gather information on as many metabolites as possible in biological systems by taking into account all information present in the data sets. Here we describe a detailed protocol for large-scale untargeted metabolomics of plant tissues, based on reversed phase liquid chromatography coupled to high-resolution mass spectrometry (LC-QTOF MS) of aqueous methanol extracts. Dedicated software, MetAlign, is used for automated baseline correction and alignment of all extracted mass peaks across all samples, producing detailed information on the relative abundance of thousands of mass signals representing hundreds of metabolites. Subsequent statistics and bioinformatics tools can be used to provide a detailed view on the differences and similarities between (groups of) samples or to link metabolomics data to other systems biology information, genetic markers and/or specific quality parameters. The complete procedure from metabolite extraction to assembly of a data matrix with aligned mass signal intensities takes about 6 days for 50 samples.     

Fachnachricht Molekulargenetische Methoden

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Fachnachricht Molekulargenetische Methoden     

Temperature effect on the sulfur isotope fractionation during sulfate reduction by two strains of the hyperthermophilic Archaeoglobus fulgidus

Sulfur isotope fractionation during dissimilatory sulfate reduction by two strains of the thermophilic archaeon Archaeoglobus fulgidus (strains VC‐16 and Z) was explored over the entire temperature range of growth. The optimal cell‐specific sulfate reduction rate (14 fmol cell^?1 h^?1) was found at 82–84°C but growth was measured as low as 54°C. The fractionation ranged between 0.52‰ and 27‰, with largest fractionations were found at intermediate temperatures and the smallest fractionations at the lowest and highest temperatures. There was an inverse relationship between the cell‐specific sulfate reduction rate and fractionation, and the cell‐specific rate was a good indicator of the expected fractionations regardless of whether temperature or substrate concentrations controlled the rate. Comparison of the fractionation trend found in this study with similar measurements for seven other sulfate‐reducers showed that sulfate‐reducing organisms respond to temperature in three different ways and this correlated with their maximum fractionation value, but not with the cell‐specific sulfate reduction rate. A sulfur isotope model was used to reproduce the observed variation of fractionation with temperature. This approach predicted the rate of internal sulfur transformations as having the major influence on the observed fractionations in the intermediate temperature range, whereas the exchange of sulfate across the cell membrane controls fractionation at low and high temperatures.     

Contractions but not AICAR increase FABPpm content in rat muscle sarcolemma

In the present study, it was investigated whether acute muscle contractions in rat skeletal muscle increased the protein content of FABPpm in the plasma membrane. Furthermore, the effect of AICAR stimulation on FAT/CD36 and FABPpm protein content in sarcolemma of rat skeletal muscle was evaluated. Methods Male wistar rats (150 g) were anesthetized and either subjected to in situ electrically induced contractions (hindlimb muscles: 20 min, 10–20 V, 200 ms trains, 100 Hz) or stimulated with the pharmacological activator of AMPK, AICAR. To investigate changes in the content of FABPpm and FAT/CD36 in the plasma membrane by these stimuli, the giant sarcolemma vesicle (GSV) technique was applied. The hindlimb muscles were removed and used for the production of GSV and lysates. All samples were analyzed using the western blotting technique. Results Electrical stimulation of rat hindlimb muscle resulted in an increase in FABPpm protein content in the GSV of 61% (P < 0.05) and in FAT/CD36 protein content in the GSV of 33% (P < 0.05). AICAR stimulation increased FAT/CD36 protein content in GSV by 22% (P < 0.05), whereas FABPpm protein content in GSV was unaffected by AICAR treatment. There was no change in total FAT/CD36 and FABPpm protein expression, measured in lysates with western blotting, by either stimulus. AMPK thr172 and ERK1/2 thr202/204 phosphorylation were significantly increased with muscle contractions (P < 0.05), whereas only AMPK thr172 phosphorylation was increased with AICAR stimulation (P < 0.05). Conclusion These data show that contractions increase both FAT/CD36 and FABPpm protein content in skeletal muscle plasma membrane, whereas only FAT/CD36 protein content is increased when muscle are stimulated with AICAR. This suggests that AMPK is involved in regulation of FAT/CD36, but not FABPpm in skeletal muscle. However, since both ERK1/2 thr202/204 and AMPK thr172 phosphorylation are increased during muscle contractions, the present study cannot rule out that both could play a significant role in regulation of FAT/CD36 and FABPpm during muscle contractions.     

Analysis of protein processing by N-terminal proteomics reveals novel species-specific substrate determinants of granzyme B orthologs

Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells.     

Accurate Prediction of DnaK-Peptide Binding via Homology Modelling and Experimental Data

Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. The prokaryotic heat-shock protein DnaK is the E. coli representative of the ubiquitous Hsp70 family, which specializes in the binding of exposed hydrophobic regions in unfolded polypeptides. Accurate prediction of DnaK binding sites in E. coli proteins is an essential prerequisite to understand the precise function of this chaperone and the properties of its substrate proteins. In order to map DnaK binding sites in protein sequences, we have developed an algorithm that combines sequence information from peptide binding experiments and structural parameters from homology modelling. We show that this combination significantly outperforms either single approach. The final predictor had a Matthews correlation coefficient (MCC) of 0.819 when assessed over the 144 tested peptide sequences to detect true positives and true negatives. To test the robustness of the learning set, we have conducted a simulated cross-validation, where we omit sequences from the learning sets and calculate the rate of repredicting them. This resulted in a surprisingly good MCC of 0.703. The algorithm was also able to perform equally well on a blind test set of binders and non-binders, of which there was no prior knowledge in the learning sets. The algorithm is freely available at http://limbo.vib.be.     

Antagonism of 5-HT1A receptors uncovers an excitatory effect of SSRIs on 5-HT neuronal activity, an action probably mediated by 5-HT7 receptors

Both microdialysis and electrophysiology were used to investigate whether another serotonin (5‐HT) receptor subtype next to the 5‐HT_1A autoreceptor is involved in the acute effects of a selective serotonin reuptake inhibitor on 5‐HT neuronal activity. On the basis of a previous study, we decided to investigate the involvement of the 5‐HT₇ receptors. Experiments were performed with the specific 5‐HT₇ antagonist SB 258741 and the putative 5‐HT₇ agonist AS19. In this study WAY 100.635 was used to block 5‐HT_1A receptors. Systemic administration of SB 258741 significantly reduced the effect of combined selective serotonin reuptake inhibitor and WAY 100.635 administration on extracellular 5‐HT in the ventral hippocampus as well as 5‐HT neuronal firing in the dorsal raphe nucleus. In the microdialysis study, co‐administration of AS19 and WAY 100.635 showed a biphasic effect on extracellular 5‐HT in ventral hippocampus, hinting at opposed 5‐HT₇ receptor mediated effects. In the electrophysiological experiments, systemic administration of AS19 alone displayed a bell‐shaped dose–effect curve: moderately increasing 5‐HT neuronal firing at lower doses while decreasing it at higher doses. SB 258741 was capable of blocking the effect of AS19 at a low dose. This is consistent with the pharmacological profile of AS19, displaying high affinity for 5‐HT₇ receptors and moderate affinity for 5‐HT_1A receptors. The data are in support of an excitatory effect of selective serotonin reuptake inhibitors on 5‐HT neuronal activity mediated by 5‐HT₇ receptors. It can be speculated, that the restoration of 5‐HT neuronal firing upon chronic antidepressant treatment, which is generally attributed to desensitization of 5‐HT_1A receptors alone, in fact results from a shift in balance between 5‐HT_1A and 5‐HT₇ receptor function.     

Promoter DNA Methylation of Oncostatin M receptor-β as a Novel Diagnostic and Therapeutic Marker in Colon Cancer

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging “cancer methylome”. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-β (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.