Recognition of modified forms of ribonuclease A by the ubiquitin system.
The substrate specificity of the ubiquitin (Ub) conjugation system was explored with regard to recognition of unfolded conformation and/or oxidized methionine residues in six derivatives of bovine RNase A. Based on the following observations, ubiquitination of RNase A substrates by the enzymes in a rabbit reticulocyte extract appears to correlate with unfolded conformation rather than with methionine oxidation. 1) Methionine oxidation in already unfolded forms of RNase A does not enhance ubiquitination. 2) Fluorescence measurements and iodoacetate trapping of free sulfhydryls show that the disulfide bonds of MetSO-RNase A, in which the 4 methionine residues are oxidized to the sulfoxide, are reduced by 2 mM dithiothreitol (DTT) in standard Ub conjugation assays so that this derivative also is unfolded. 3) Although MetSO-RNase A is ubiquitinated in the absence of DTT, its intrinsic fluorescence, cation-exchange properties, and susceptibility to reduction indicate a non-native conformation. 4) Methionine sulfoxide-containing peptides that mimic regions of RNase A fail to inhibit conjugation of 125I-Ub to MetSO-RNase A. Ub adducts to two of the six derivatives (MetSO- and reduced/carboxamidomethylated MetSO-RNase A) increase when DTT is omitted from the reactions. Ubaldehyde, an inhibitor of isopeptidases that disassemble Ub-protein conjugates, increased product yields and reduced or abolished the DTT effect, suggesting that an isopeptidase specific for these two RNase A derivatives may be inactivated by oxidation. Ub conjugates of the other RNase A derivatives also increase with Ub-aldehyde but are unaffected by DTT.
Dunten
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RL
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Cohen
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000032:analysis; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D008715:Methionine; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D004220:Disulfides; D014452:Ubiquitins; D008715:Methionine; D012259:Ribonuclease, Pancreatic; D004229:Dithiothreitol