Comparative genomic context analyses of the genes encoding the TPS/P pathway via TPSP-fusions as well as single domain TPS and TPP proteins within the Archaea.

<p>Gene distributions and genomic organizations were analyzed using blast and genome region comparison tools at NCBI (<a href="http://blast.ncbi.nlm.nih.gov/" target="_blank">http://blast.ncbi.nlm.nih.gov/</a>) and IMG 2.0 (<a href="http://img.jgi.doe.gov/cgi-bin/w/main.cgi" target="_blank">http://img.jgi.doe.gov/cgi-bin/w/main.cgi</a>). As template the TPSP of <i>T. tenax</i> was chosen. Each arrow represents one gene with the arrow direction indicating the 5′-3′ orientation. Genes are drawn to scale. The genome region illustration is based on the 5′-3′ orientation of the <i>tps(p)</i> gene in the respective organism. Gene colors: <i>tps</i> (red); <i>tpp</i> (yellow), <i>gt</i> (light blue); <i>msc</i> (green), unknown (white), <i>glucose dehydrogenase</i> (pink), <i>glycoside hydrolase</i> (dark blue), <i>phosphomannomutase</i> (brown), <i>mannose-1-phosphate guanyltransferase</i> (grey). The broken red arrow represents an N-terminal <i>tps</i> gene fragment in <i>M. thermophila</i>.</p

Proposed model of stress response in <i>T.</i><i>tenax</i> involving TPSP (yellow, red), GT (blue) and MSC (green).

<p>In response to stress (e.g. high osmolarity), trehalose is synthesized via complex formation of TPSP and GT, resulting in an increased intracellular trehalose concentration (top right). Upon stress relief (e.g. hypo-osmotic shock) the MSC opens in response to changes in membrane tension resulting in trehalose efflux (down left).</p

The First Prokaryotic Trehalose Synthase Complex Identified in the Hyperthermophilic Crenarchaeon <i>Thermoproteus tenax</i>

<div><p>The role of the disaccharide trehalose, its biosynthesis pathways and their regulation in Archaea are still ambiguous. In <i>Thermoproteus tenax</i> a fused trehalose-6-phosphate synthase/phosphatase (TPSP), consisting of an N-terminal trehalose-6-phosphate synthase (TPS) and a C-terminal trehalose-6-phosphate phosphatase (TPP) domain, was identified. The <i>tpsp</i> gene is organized in an operon with a putative glycosyltransferase (GT) and a putative mechanosensitive channel (MSC). The <i>T. tenax</i> TPSP exhibits high phosphatase activity, but requires activation by the co-expressed GT for bifunctional synthase-phosphatase activity. The GT mediated activation of TPS activity relies on the fusion of both, TPS and TPP domain, in the TPSP enzyme. Activation is mediated by complex-formation <i>in vivo</i> as indicated by yeast two-hybrid and crude extract analysis. In combination with first evidence for MSC activity the results suggest a sophisticated stress response involving TPSP, GT and MSC in <i>T. tenax</i> and probably in other Thermoproteales species. The monophyletic prokaryotic TPSP proteins likely originated via a single fusion event in the Bacteroidetes with subsequent horizontal gene transfers to other Bacteria and Archaea. Furthermore, evidence for the origin of eukaryotic TPSP fusions via HGT from prokaryotes and therefore a monophyletic origin of eukaryotic and prokaryotic fused TPSPs is presented. This is the first report of a prokaryotic, archaeal trehalose synthase complex exhibiting a much more simple composition than the eukaryotic complex described in yeast. Thus, complex formation and a complex-associated regulatory potential might represent a more general feature of trehalose synthesizing proteins.</p></div

Trehalose and phosphate forming activity of the recombinant TPSP.

<p>(<b>A</b>) Trehalose formation from UDPG (uridine diphosphate-glucose) and G6P (glucose-6-phosphate) by TPSP and artificial TPP domain in combination with and without GT determined by thin layer chromatography on silica plates. (<b>B</b>) Specific activity of <i>T. tenax</i> TPSP and TPP at 80°C in the presence and absence of GT measured as P_i (inorganic phosphate) release from UDPG and G6P, i.e combined TPS and TPP activity (left panel) as well as from T6P (trehalose-6-phosphate), i.e. TPP activity, (right panel), respectively, via the malachite green assay. (<b>C</b>) Specific TPS activity of the <i>T. tenax</i> TPSP, TPS and TPP at 80°C in the presence and absence of GT measured as UDP release from UDPG and G6P in a discontinuous assay system. UDP was determined spectrophotometrically by monitoring the decrease in absorption due to oxidation of NADH via PK and LDH at 340 nm. (+) indicates the presence, (−) the absence of the respective protein.</p

DataSheet1_Analyzing the postulated inhibitory effect of Manumycin A on farnesyltransferase.docx

Manumycin A is postulated to be a specific inhibitor against the farnesyltransferase (FTase) since this effect has been shown in 1993 for yeast FTase. Since then, plenty of studies investigated Manumycin A in human cells as well as in model organisms like Caenorhabditis elegans. Some studies pointed to additional targets and pathways involved in Manumycin A effects like apoptosis. Therefore, these studies created doubt whether the main mechanism of action of Manumycin A is FTase inhibition. For some of these alternative targets half maximal inhibitory concentrations (IC50) of Manumycin A are available, but not for human and C. elegans FTase. So, we aimed to 1) characterize missing C. elegans FTase kinetics, 2) elucidate the IC50 and Ki values of Manumycin A on purified human and C. elegans FTase 3) investigate Manumycin A dependent expression of FTase and apoptosis genes in C. elegans. C. elegans FTase has its temperature optimum at 40°C with KM of 1.3 µM (farnesylpyrophosphate) and 1.7 µM (protein derivate). Whilst other targets are inhibitable by Manumycin A at the nanomolar level, we found that Manumycin A inhibits cell-free FTase in micromolar concentrations (Ki human 4.15 μM; KiC. elegans 3.16 μM). Furthermore, our gene expression results correlate with other studies indicating that thioredoxin reductase 1 is the main target of Manumycin A. According to our results, the ability of Manumycin A to inhibit the FTase at the micromolar level is rather neglectable for its cellular effects, so we postulate that the classification as a specific FTase inhibitor is no longer valid.</p

Complex formation of TPSP with GT <i>in vivo</i>.

<p>Yeast two-hybrid analysis of GT and TPSP interactions. Yeast strains (AH109) were co-transformed with pGADT7::<i>tpsp</i> and pGBKT7::<i>gt</i> and grown in SD medium (+2% (w/v) glucose, -leucine, -tryptophan) to an OD₆₀₀ of 1.5 and diluted to an OD₆₀₀ of 0.1. 5 µl of this suspension were dropped on SD-agar plates. <b>Lower panel</b>: -leucine, -tryptophan, -histidine, -adenine, + X-α-Gal. <b>Upper panel</b>: -leucine, -tryptophan. The plates were incubated for three days at 30°C. As positive control pGADT7::<i>rpok</i> and pGBKT7::<i>tfb3</i> were used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061354#pone.0061354-Paytubi1" target="_blank">[26]</a>. The negative controls are the empty vectors pGBKT7 and pGADT7 and the false positive controls are the empty vectors pGBKT7 and pGADT7::<i>gt</i>.</p