Metabolite of SIR2 reaction modulates TRPM2 Ion channel

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins are protein deacetylases that regulate gene expression, apoptosis, metabolism, and aging. These NAD+-dependent enzymes catalyze a reaction in which the acetyl group from substrate is transferred to the ADP-ribose portion of NAD+ to form deacetylated product, nicotinamide, and the metabolite OAADPr, whose functions remain elusive. Using cell-based assays and RNA interference, we show that puromycin-induced cell death is greatly diminished by nicotinamide (a potent sirtuin inhibitor), and by decreased expression of sirtuins SIRT2 and SIRT3. Furthermore, we demonstrate using channel current recordings and binding assays that OAADPr directly binds to the cytoplasmic domain of TRPM2 and activates the TRPM2 channel. ADP-ribose binds TRPM2 with similarly affinity, whereas NAD+ displays almost negligible binding. These studies provide the first evidence for the potential role of sirtuin-generated OAADPr in TRPM2 channel gatin

Design and Synthesis of a Quintessential Self-Transmissible IncX1 Plasmid, pX1.0

DNA exchange in bacteria via conjugative plasmids is believed to be among the most important contributing factors to the rapid evolution- and diversification rates observed in bacterial species. The IncX1 plasmids are particularly interesting in relation to enteric bacteria, and typically carry genetic loads like antibiotic resistance genes and virulence factors. So far, however, a “pure” version of these molecular parasites, without genetic loads, has yet to be isolated from the environment. Here we report the construction of pX1.0, a fully synthesized IncX1 plasmid capable of horizontal transfer between different enteric bacteria. The designed pX1.0 sequence was derived from the consensus gene content of five IncX1 plasmids and three other, more divergent, members of the same phylogenetic group. The pX1.0 plasmid was shown to replicate stably in E. coli with a plasmid DNA per total DNA ratio corresponding to approximately 3–9 plasmids per chromosome depending on the growth phase of the host. Through conjugation, pX1.0 was able to self-transfer horizontally into an isogenic strain of E. coli as well as into two additional species belonging to the family Enterobacteriaceae. Our results demonstrate the immediate applicability of recent advances made within the field of synthetic biology for designing and constructing DNA systems, previously existing only in silica

Genetic map of pX1.0.

<p>The GC-content is depicted in the central part of the map. Open reading frames are shown as colour coded arrows, indicating their function and transcribed direction. The ORFs are divided into the following modules: gem (gene expression modulation), tra (conjugal transfer), mob1 (mobilization 1), rep/stb (replication initiation/toxin-antitoxin plasmid addiction), mob2 (mobilization 2), par (plasmid partitioning) and res (resistance marker). Unique restriction sites are shown at the module junctions. MCS represents the restriction sites <i>Pme</i>I, <i>Abs</i>I, <i>Mre</i>I, <i>Kpn</i>I, <i>Swa</i>I, <i>Not</i>I and <i>Sgr</i>DI.</p

Genetic maps of p2ESCUM, pOU1114, pSE34, pOLA52, and pOU1115.

<p>The GC-content is depicted in the central part of the maps. Open reading frames (ORFs) proven to be conserved within the IncX1 plasmids constitute the putative IncX1 backbone and are presented as green boxes, whereas red boxes represent ORFs comprising the genetic load.</p