Invited
Speaker
Malaria functional genomics: A Plasmodium receptor for C kinase
(PfRack) modulates calcium signaling in mammalian cells
Celia Garcia
Brazil
- Malaria is the world’s most lethal parasitic disease, causing
up to 2.7 million deaths annually. Recent data from our and other
laboratories support the concept that Ca2+ pathway is exploited
by the Plasmodium parasite to control crucial physiological
functions, as invasion (Gazarini et al, 2003). RACK (Receptor for
Activated C Kinase) is a mammalian scaffold protein that, among other
functions, is responsible for stabilizing PKC in its active form.
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In this study we have used a synthetic, codon-optimized form
of the PfRACK gene to express this protein in the HEK 293 and PC12
mammalian cell lines and primary hepatocytes, in order to test whether
this malarial protein can couple with mammalian signaling machinery.
Endogenous RACK1 expression was also knocked down using RNAi technology
to isolate the effect of the expression of the heterologous gene.
By performing time-lapse confocal microscopy, we observed that ATP
(100µM)-induced calcium signals were reduced by 83,3 +3,8%
in HEK 293 cells expressing PfRACK and by 89,6 +7,8% in cells expressing
PfRACK and treated with siRNA for mammalian RACK1, although no statistical
difference was found in the SiRNA-only treated group. Similar results
were observed in PC12 cells. To test more directly if PfRACK acts
on InsP3 receptors, we performed experiments using a permeable caged
InsP3. Upon 2-photon encaging, the InsP3 failed to increase Ca2+
in cells expressing PfRACK. Confocal immunolocalization of
PfRACK in transfected HEK 293 cells and hepatocytes showed partial
colocalization of PfRACK with its mammalian ortholog.
- These data demonstrate that PfRACK is able to interfere with
the host’s signaling machinery, specifically with InsP3 receptors.
Moreover, this study demonstrates for the first time that synthesis
of codon-optimized genes can be a useful technique to investigate
the function of malaria parasite proteins in established models of
the mammalian system.
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