dictyNews

Electronic Edition

Volume 42, number 28

December 2, 2016



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Abstracts

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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 [[log in to unmask]]

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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 [[log in to unmask]]

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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  [[log in to unmask]]

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[End dictyNews, volume 42, number 28]