[22] Preparation of native and recombinant Clostridium botulinum C3 ADP-ribosyltransferase and identification of Rho proteins by ADP-ribosylation

Morii, Narito; Narumiya, Shuh

doi:10.1016/0076-6879(95)56024-6

Cited by 51 publications

(48 citation statements)

References 32 publications

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“…2B-E show the morphology of the Swiss 3T3 cells treated with the wild type and mutant C3 exoenzymes. As reported previously [12], cells treated with the wild type enzyme demonstrated a marked cell rounding, with beaded dendritic processes (Fig. 2C).…”

Section: Resultssupporting

confidence: 54%

“…C3 exoenzyme has been used to elucidate the role of rho p21 in various cellular functions [5][6][7][8][9][10][11][12][24][25][26][27][28][29]. There have been, however, no reports using site-directed enzyme mutants to demonstrate the relationship between its ADP-ribosylation activity and the biological response.…”

Section: Resultsmentioning

confidence: 99%

“…The ADP-ribosylation buffer contained 100 mM Tris-HC1 (pH 8.0), 10 mM thymidine, 10 mM nicotinamide, 10 mM dithiothreitol, 5 mM MgCI 2, and [32P]NAD (1000 dpm/pmol) (Dupont New England Nuclear). Purified wild type or mutant C3 exoenzyme was incubated with 1 ,ug of recombinant GST-rho A p21 (fusion protein) or with 100/tg protein of crude cell homogenate in this buffer and quantification of the ADP-ribosylation was carried out as previously described [12].…”

Section: Adp-ribosylation and Cell Culturementioning

confidence: 99%

“…Clostridium botulinum C3 exoenzyme [3] specifically ADPribosylates the small molecular weight GTP binding protein rho p21 at the Asn 41 residue located in a putative effector binding domain [4]. Treatment of cells with C3 exoenzyme inhibits stimulus-induced actin filament organization and cell adhesion [5][6][7][8][9][10][11][12], indicating that rho p21 mediates these cellular responses as a molecular switch and that C3 exoenzyme inhibits these functions of rho p21. Identification of the amino acid residues of the C3 exoenzyme interacting with NAD is important in elucidating the structure of its catalytic site and in understanding a mechanism of the ADP-ribosylation.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Glu¹⁷³ as the critical amino acid residue for the ADP‐ribosyltransferase activity of Clostridium botulinum C3 exoenzyme

et al. 1995

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Clostridium botulinum C3 exoenzyme specifically ADP-ribosylates rho-p21 in eukaryotic cells. Trp Is and Glu 173 of this enzyme were substituted with other amino acids via sitedirected mutagenesis. All substitutions at Glu 173 caused a significant reduction in affinity for NAD and diminished ADPribosyltransferase activity. On the other hand, the activity of enzymes with the substitution at Trp Is remained intact. Swiss 3T3 cells treated with the enzyme with the Trp TM substitution showed the typical morphologic changes of the C3 exoenzyme phenotype. In contrast, no changes were found in cells incubated with the Glu~73-substituted enzyme. These results indicate that the Glu 173 residue of the C3 exoenzyme plays a key role in interacting with NAD and in expression of ADP-ribosyltransferase activity, which is essential for the phenotypic change by C3 exoenzyme treatment.

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Section: Resultssupporting

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Adp-ribosylation and Cell Culturementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of Glu¹⁷³ as the critical amino acid residue for the ADP‐ribosyltransferase activity of Clostridium botulinum C3 exoenzyme

et al. 1995

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“…3D). This bacterial enzyme inhibits the activity of Rho without affecting that of other members of the Rho subfamily of small GTPases such as Cdc42 or Rac (27). Stimulation of exoenzyme C3-pretreated acini with CCK also induced the formation of vacuoles, which were not coated with F-actin (Fig.…”

Section: Resultsmentioning

confidence: 99%

Stabilization of Exocytosis by Dynamic F-actin Coating of Zymogen Granules in Pancreatic Acini

Nemoto

Kojima

Oshima

et al. 2004

Journal of Biological Chemistry

128

164

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Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca 2؉ -dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a cagedCa 2؉ compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.

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Direkte Modulation von Aktivität und Funktion kleiner GTPasen

et al. 2015

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Kleine GTPasen bilden eine Familie von GDP‐/GTP‐bindenden Proteinen, die als biomolekulare Schalter innerhalb von Zellen wirken. Fehlfunktionen oder Fehlregulierungen kleiner GTPasen stehen in Verbindung mit zahlreichen menschlichen Erkrankungen, was diese Proteine zu einem vielversprechenden Ziel der pharmazeutischen Forschung macht. Allerdings werden kleine GTPasen oftmals als pharmakologisch nicht adressierbar klassifiziert, da jahrzehntelange Bemühungen keinerlei klinisch relevante Verbindungen liefern konnten, die die Aktivität kleiner GTPasen direkt modulieren. Die Entdeckung neuer, transienter Bindetaschen an den Proteinoberflächen und die Anwendung neuartiger Techniken haben dem Feld jedoch kürzlich neuen Schwung verliehen. Neben Ras‐Proteinen rücken zudem vermehrt andere kleine GTPasen in den Fokus der Forschung. Dieser Artikel bietet einen Überblick über die relevanten Aspekte der Superfamilie kleiner GTPasen und fasst die jüngsten Fortschritte und Perspektiven für die direkte Modulierung dieser herausfordernden Proteinklasse zusammen.

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[22] Preparation of native and recombinant Clostridium botulinum C3 ADP-ribosyltransferase and identification of Rho proteins by ADP-ribosylation

Cited by 51 publications

References 32 publications

Identification of Glu¹⁷³ as the critical amino acid residue for the ADP‐ribosyltransferase activity of Clostridium botulinum C3 exoenzyme

Identification of Glu¹⁷³ as the critical amino acid residue for the ADP‐ribosyltransferase activity of Clostridium botulinum C3 exoenzyme

Stabilization of Exocytosis by Dynamic F-actin Coating of Zymogen Granules in Pancreatic Acini

Direkte Modulation von Aktivität und Funktion kleiner GTPasen

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[22] Preparation of native and recombinant Clostridium botulinum C3 ADP-ribosyltransferase and identification of Rho proteins by ADP-ribosylation

Cited by 51 publications

References 32 publications

Identification of Glu173 as the critical amino acid residue for the ADP‐ribosyltransferase activity of Clostridium botulinum C3 exoenzyme

Identification of Glu173 as the critical amino acid residue for the ADP‐ribosyltransferase activity of Clostridium botulinum C3 exoenzyme

Stabilization of Exocytosis by Dynamic F-actin Coating of Zymogen Granules in Pancreatic Acini

Direkte Modulation von Aktivität und Funktion kleiner GTPasen

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Identification of Glu¹⁷³ as the critical amino acid residue for the ADP‐ribosyltransferase activity of Clostridium botulinum C3 exoenzyme

Identification of Glu¹⁷³ as the critical amino acid residue for the ADP‐ribosyltransferase activity of Clostridium botulinum C3 exoenzyme