Characterization of new cell permeable C3-like proteins that inactivate Rho and stimulate neurite outgrowth on inhibitory substrates.
The activation state of Rho is an important determinant of axon growth and regeneration in neurons. Axons can extend neurites on growth inhibitory substrates when Rho is inactivated by C3-ADP-ribosyltransferase (C3). We found by Rho-GTP pull-down assay that inhibitory substrates activate Rho. To inactivate Rho, scrape-loading of C3 was necessary because it does not freely enter cells. To overcome the poor permeability of C3, we made and characterized five new recombinant C3-like chimeric proteins designed to cross the cell membrane by receptor-independent mechanisms. These proteins were constructed by the addition of short transport peptides to the carboxyl-terminal of C3 and tested using a bioassay measuring neurite outgrowth of PC-12 cells plated on growth inhibitory substrates. All five constructs stimulated neurite outgrowth but with different dose-response profiles. Biochemical properties of the chimeric proteins were examined using C3-05, the most effective construct tested. Gel shift assays showed that C3-05 retained the ability to ADP-ribosylate Rho. Western blots and immunocytochemistry were used to verify the presence of C3 inside treated cells. C3-05 was also effective at promoting neurite outgrowth in primary neuronal cultures, as well as causing the disassembly of actin stress fibers and focal adhesions complexes in fibroblasts. These studies demonstrate that the new C3-like proteins are effective in delivering biologically active C3 into different cell types, thereby, inactivating Rho.
Winton
Matthew J
MJ
Département de Pathologie et Biologie Cellulaire, Université de Montréal and BioAxone Thérapeutique Inc., 2900 Edouard Montpetit, P-906 Pavillon Principal, Montréal, Québec H3T 1J4, Canada.
Dubreuil
Catherine I
CI
Lasko
Dana
D
Leclerc
Nicole
N
McKerracher
Lisa
L
D016475:3T3 Cells; D036002:ADP Ribose Transferases / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D001905:Botulinum Toxins; D016466:CHO Cells; D002455:Cell Division; D002478:Cells, Cultured; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007150:Immunohistochemistry; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D016716:PC12 Cells; D010455:Peptides / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D013997:Time Factors; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D018076:DNA, Complementary; D010455:Peptides; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D000244:Adenosine Diphosphate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins