dictyNews Electronic Edition Volume 42, number 28 December 2, 2016 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Interspecies comparison of peptide substrate reporter metabolism using compartment-based modeling Allison J. Tierney, Nhat Pham, Kunwei Yang, Brooks K. Emerick, and Michelle L. Kovarik Analytical and Bioanalytical Chemistry, in press Peptide substrate reporters are fluorescently labeled peptides that can be acted upon by one or more enzymes of interest. Peptide substrates are readily synthesized and more easily separated than full-length protein substrates; however, they are often more rapidly degraded by peptidases. As a result, peptide reporters must be made resistant to proteolysis in order to study enzymes in intact cells and lysates. This is typically achieved by optimizing the reporter sequence in a single cell type or model organism, but studies of reporter stability in a variety of organisms are needed to establish the robustness and broader utility of these molecular tools. We measured peptidase activity toward a peptide substrate reporter for protein kinase B (Akt) in E. coli, D. discoideum, and S. cerevisiae using capillary electrophoresis with laser-induced fluorescence (CE-LIF). Using compartment-based modeling, we determined individual rate constants for all potential peptidase reactions and explored how these rate constants differed between species. We found the reporter to be stable in D. discoideum (t1/2 = 82-103 min) and S. cerevisiae (t1/2 = 279-314 min), but less stable in E. coli (t1/2 = 21-44 min). These data suggest that the reporter is sufficiently stable to be used for kinase assays in eukaryotic cell types while also demonstrating the potential utility of compartment-based models in peptide substrate reporter design. submitted by: Michelle Kovarik [michelle.kovarik@trincoll.edu] ——————————————————————————————————————— Xpf suppresses mutagenic consequences of bacterial phagocytosis in Dictyostelium Lucas B. Pontel, Judith Langenick, Ivan V. Rosado, Xiao-Yin Zhang1,4, David Traynor, Robert R. Kay and Ketan J. Patel Journal of Cell Science, in press As time passes, mutations accumulate in the genomes of all living organisms. These changes promote genetic diversity, but also precipitate ageing and the initiation of cancer. Food is a common source of mutagens, but little is known about how nutritional factors cause lasting genetic changes in the consuming organism. Here, we describe an unusual genetic interaction between DNA repair in the unicellular amoeba – Dictyostelium discoideum – and its natural bacterial food source. Dictyostelium deficient in the DNA repair nuclease Xpf displays a severe and specific growth defect when feeding on bacteria. Despite being proficient in the phagocytosis and digestion of bacteria, over time, xpf- Dictyostelium feeding on bacteria ceases to grow and in many instances die. The Xpf nuclease activity is required for sustained growth using a bacterial food source. Furthermore, the ingestion of this food source leads to a striking accumulation of mutations in the genome of xpf- Dictyostelium. This work therefore establishes Dictyostelium as a model genetic system to dissect nutritional genotoxicity, providing insight into how phagocytosis can induce mutagenesis and compromise survival fitness. submitted by: Lucas Pontel [lpontel@mrc-lmb.cam.ac.uk] ——————————————————————————————————————— SodC modulates Ras and PKB signaling in Dictyostelium. Boris Castillo, Seon-Hee Kim, Mujataba Sharief, Tong Sun, Lou W. Kim European Journal of Cell Biology, in press We have previously reported that the basal RasG activity is aberrantly high in cells lacking Superoxide dismutase C (SodC). Here we report that other Ras proteins such as RasC and RasD activities are not affected in sodC− cells and mutagenesis studies showed that the presence of the Cys118 in the Ras proteins is essential for the superoxide-mediated activation of Ras proteins in Dictyostelium. In addition to the loss of SodC, lack of extracellular magnesium ions increased the level of intracellular superoxide and active RasG proteins. Aberrantly active Ras proteins in sodC− cells persistently localized at the plasma membrane, but those in wild type cells under magnesium deficient medium exhibited intracellular vesicular localization. Interestingly, the aberrantly activated Ras proteins in wild type cells were largely insulated from their normal downstream events such as Phosphatidylinositol-3,4,5-P3 (PIP3) accumulation, Protein Kinase B (PKB) activation, and PKBs substrates phosphorylation. Intriguingly, however, aberrantly activated Ras proteins in sodC− cells were still engaged in signaling to their downstream targets, and thus excessive PKBs substrates phosphorylation persisted. In summary, we suggest that SodC and RasG proteins are essential part of a novel inhibitory mechanism that discourages oxidatively stressed cells from chemotaxis and thus inhibits the delivery of potentially damaged genome to the next generation. submitted by: Lou Kim [kiml@fiu.edu] ============================================================== [End dictyNews, volume 42, number 28]