title: Distinct biological activities of C3 and ADP-ribosyltransferase-deficient C3-E174Q.
abstract: Low-molecular-weight GTP-binding proteins of the Rho family control the organization of the actin cytoskeleton in eukaryotic cells. Dramatic reorganization of the actin cytoskeleton is caused by the C3 exoenzyme derived from Clostridium botulinum (C3), based on ADP-ribosylation of RhoA/B/C. In addition, wild-type as well as ADP-ribosyltransferase-deficient C3-E174Q induce axonal outgrowth of primary murine hippocampal neurons and prevent growth cone collapse, indicating a non-enzymatic mode of action. In this study, we compared the effects of C3-E174Q and wild-type C3 in the murine hippocampal cell line HT22. Treatment of HT22 cells with C3 resulted in Rho ADP-ribosylation and cell rounding. The ADP-ribosyltransferase-deficient mutant C3-E174Q did not induce either Rho ADP-ribosylation or morphological changes. C3 as well as C3-E174Q treatment resulted in growth arrest, reduced expression of cyclin D levels, and increased expression of RhoB, a negative regulator of cell-cycle progression. Serum starvation induced apoptosis in HT22 cells, as determined on the basis of increased expression of caspase-9 and Bax. C3 but not C3-E174Q protected serum-starved HT22 cells from apoptosis. This is the first study separating ADP-ribosyltransferase-dependent from ADP-ribosyltransferase-independent effects of C3. While morphological changes and anti-apoptotic activity strictly depend on ADP-ribosyltransferase activity, the anti-proliferative effects are independent of ADP-ribosyltransferase activity.
authors.affiliation: Institute of Toxicology, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, Hanover, Germany. rohrbeck.astrid@mh-hannover.de
authors.forename: Astrid
authors.initials: A
authors.lastname: Rohrbeck
authors.forename: Tanja
authors.initials: T
authors.lastname: Kolbe
authors.forename: Sandra
authors.initials: S
authors.lastname: Hagemann
authors.forename: Harald
authors.initials: H
authors.lastname: Genth
authors.forename: Ingo
authors.initials: I
authors.lastname: Just
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003014:Clostridium botulinum / Q000201:enzymology / Q000235:genetics; D056741:Cyclin D / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D007700:Kinetics; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D020743:rhoB GTP-Binding Protein / Q000378:metabolism
chemical_list: D056741:Cyclin D; D012333:RNA, Messenger; D011994:Recombinant Proteins; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D020169:Caspases; D001905:Botulinum Toxins; D020743:rhoB GTP-Binding Protein