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dictyNews Volume 42 Number 28
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.
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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 [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]
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[End dictyNews, volume 42, number 28]
