Inhibition of protein synthesis by didemnin B: how EF-1alpha mediates inhibition of translocation.
The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.
Ahuja
D
D
Willard H. Dow Laboratory, Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Vera
M D
MD
SirDeshpande
B V
BV
Morimoto
H
H
Williams
P G
PG
Joullié
M M
MM
Toogood
P L
PL
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D047630:Depsipeptides; D004167:Diphtheria Toxin / Q000494:pharmacology; D005672:Fusidic Acid / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D020128:Inhibitory Concentration 50; D008954:Models, Biological; D020648:Peptide Elongation Factor 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D020652:Peptide Elongation Factor 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits; D012270:Ribosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D012996:Solutions; D013816:Thermodynamics
D000970:Antineoplastic Agents; D047630:Depsipeptides; D004167:Diphtheria Toxin; D020648:Peptide Elongation Factor 1; D020652:Peptide Elongation Factor 2; D010456:Peptides, Cyclic; D011500:Protein Synthesis Inhibitors; D012996:Solutions; D006153:Guanosine Diphosphate; C030051:didemnins; D005672:Fusidic Acid; D011189:Potassium Chloride; D006160:Guanosine Triphosphate