dictyNews
Electronic Edition
Volume 38, number 29
November 16, 2012
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Abstracts
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Structure and function of a unique pore-forming protein from a
pathogenic acanthamoeba
Matthias Michalek1,2, Frank D. Sönnichsen3, Rainer Wechselberger4,
Andrew J. Dingley5, Chien-Wen Hung6, Annika Kopp7, Hans Wienk4,
Maren Simanski1, Rosa Herbst1, Inken Lorenzen2, Francine Marciano-Cabral8,
Christoph Gelhaus1, Thomas Gutsmann7, Andreas Tholey6,
Joachim Grötzinger2, Matthias Leippe1*
1 Zoological Institute, Zoophysiology, Christian-Albrechts Universität zu Kiel,
Olshausenstr. 40, 24098 Kiel, Germany.
2 Institute of Biochemistry, Christian-Albrechts Universität zu Kiel,
Olshausenstr. 40, 24098 Kiel, Germany.
3 Otto Diels Institute for Organic Chemistry, Christian-Albrechts Universität zu Kiel,
Olshausenstr. 40, 24098 Kiel, Germany.
4 NMR Spectroscopy Research Group, Bijvoet Center for Biomolecular Research,
Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
5 School of Chemical Sciences and School of Biological Sciences,
The University of Auckland, New Zealand.
6 Division of Systematic Proteome Research, Institute for Experimental Medicine,
Christian-Albrechts Universität zu Kiel, Niemannsweg 11, 24105 Kiel, Germany.
7 Division of Biophysics, Research Center Borstel, Center for Medicine and
Bioscience, 23845 Borstel, Germany.
8 Department of Microbiology and Immunology, Virginia Commonwealth University
School of Medicine, 1101 E. Marshall Street, Richmond, Virginia 23298-0678, USA.
* To whom correspondence should be addressed: [log in to unmask]
Nature Chemical Biology, in press
Human pathogens often produce soluble protein toxins that generate pores inside
membranes, resulting in death of target cells and tissue damage. In pathogenic
amoebae, this has been exemplified with amoebapores of the enteric protozoan
parasite Entamoeba histolytica. Here we characterize acanthaporin, to our
knowledge the first pore-forming toxin to be described from acanthamoebae,
which are free-living, bacteria-feeding, unicellular organisms that are opportunistic
pathogens of increasing importance and cause severe and often fatal diseases.
We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by
tracking its pore-forming activity, molecularly cloned the gene of its precursor and
recombinantly expressed the mature protein in bacteria. Acanthaporin was
cytotoxic for human neuronal cells and exerted antimicrobial activity against a
variety of bacterial strains by permeabilizing their membranes. The tertiary
structures of acanthaporin´s active monomeric form and inactive dimeric form,
both solved by NMR spectroscopy, revealed a currently unknown protein fold
and a pH-dependent trigger mechanism of activation.
Submitted by Matthias Leippe [[log in to unmask]]
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Daydreamer, a Ras effector and GSK-3 substrate, is important for directional
sensing and cell motility
Verena Kölsch, Zhouxin Shen, Susan Lee, Katarzyna Plak, Pouya Lotfi,
Jessica Chang, Pascale G. Charest, Jesus Lacal Romero, Taeck J. Jeon,
Arjan Kortholt, Steven P. Briggs and Richard A. Firtel
Mol Bio Cell, in press
How independent signaling pathways are integrated to holistically control
a biological process is not well understood. We have identified Daydreamer
(DydA), a new member of the MRL (Mig10/RIAM/Lamellipodin) family of
adaptor proteins that localizes to the leading edge. DydA is a putative Ras
effector that is required for cell polarization and directional movement during
chemotaxis. dydA- cells exhibit elevated F-actin and assembled MyoII,
increased and extended PI3K activity, and extended phosphorylation of
the activation loop of PKB and PKBR1, suggesting that DydA is involved
in the negative regulation of these pathways. DydA is phosphorylated by
GSK-3, which is required for some, but not all, of DydA’s functions, including
the proper regulation of PKB and PKBR1 and MyoII assembly. gskA- cells
exhibit very strong chemotaxis phenotypes, as previously described, but
exhibit an increased rate of random motility. gskA- cells have a reduced
MyoII response, a reduced level of PI(3,4,5)P3 production but a highly
extended recruitment of PI3K to the plasma membrane and highly extended
kinetics of PKB and PKBR1 activation. Our results demonstrate that GSK-3
function is essential for chemotaxis, regulating multiple substrates and that
one of these effectors, DydA, plays a key function in the dynamic regulation
of chemotaxis.
Submitted by Rick Firtel [[log in to unmask]]
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