Ubiquitin carboxyl-terminal peptides. Substrates for ubiquitin activating enzyme.
The carboxyl terminus of ubiquitin is activated in the presence of ATP to enter the ubiquitin cycle in cells. Peptides corresponding to the COOH-terminal region of ubiquitin were synthesized to investigate their effects on the ATP/ubiquitin-dependent proteolytic pathway. Their activities in the PPi exchange assay with ubiquitin activating enzyme (E1) were proportional to their length. The hexapeptide Ac-Leu-Arg-Leu-Arg-Gly-Gly reacted with ATP to form an enzyme-adenylate-hexapeptide complex and at high concentrations was 20-25% as active as human ubiquitin in the PPi exchange assay with E1. However, the hexapeptide was not transferred to the sulfhydryl "thiol" site on E1. In addition, the COOH-terminal peptides did not support the degradation of 125I-bovine serum albumin in the reticulocyte lysate system. A nonhomologous peptide of equivalent length was inactive in all assays. Thus, synthetic COOH-terminal peptide(s) of ubiquitin can partially substitute for ubiquitin in its reactions with E1 but do not support subsequent steps of the energy-dependent proteolytic pathway. These results show that it may be possible to design small molecules that either serve as substrates or inhibitors for other specific steps in ubiquitin-dependent pathways.
Jonnalagadda
S
S
Department of Molecular Pharmacology, Smith Kline and French Laboratories, Philadelphia, Pennsylvania 19101.
Ecker
D J
DJ
Sternberg
E J
EJ
Butt
T R
TR
Crooke
S T
ST
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D006868:Hydrolysis; D008025:Ligases / Q000378:metabolism*; D008956:Models, Chemical; D009842:Oligopeptides / Q000378:metabolism*; D012156:Reticulocytes / Q000201:enzymology; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D009842:Oligopeptides; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes