Polyamine analogues inhibit the ubiquitination of spermidine/spermine N1-acetyltransferase and prevent its targeting to the proteasome for degradation.
Spermidine/spermine N(1)-acetyltransferase (SSAT), a key enzyme in mammalian polyamine catabolism, undergoes rapid turnover (half-life approx. 30 min) and is highly inducible in response to polyamine analogues such as bis(ethyl)spermine (BE-3-4-3), which greatly stabilize the enzyme. Rapid degradation of SSAT in reticulocyte lysates was preceded by formation of a ladder of ubiquitinated forms, and required the production of high-molecular-mass complexes with ubiquitin (HMM-SSAT-Ubs). Mutation of all 11 lysines in SSAT separately to arginine demonstrated that no single lysine residue is critical for its degradation in vitro, but mutant K87R had a significantly longer half-life, suggesting that lysine-87 may be the preferred site for ubiquitination. Mutations at the C-terminus of SSAT, such as E171Q, resulted in marked stabilization of the protein, due to the lack of formation of the HMM-SSAT-Ubs. Addition of BE-3-4-3 prevented the accumulation of ubiquitin conjugates and the proteasomal degradation of wild-type SSAT. These results indicate that conformational changes brought about by the binding of polyamine analogues prevent the efficient polyubiquitination of SSAT, leading to a major increase in the amount of SSAT protein, and that alteration of the C-terminal end of the protein has a similar effect in preventing the productive interaction with an E2 or E3 component of the ubiquitin pathway.
Coleman
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Department of Cellular and Molecular Physiology, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, P.O. Box 850, Hershey, PA 17033, USA. csc6@psu.edu
Pegg
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D000123:Acetyltransferases / Q000737:chemistry*; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D008239:Lysine / Q000737:chemistry; D009097:Multienzyme Complexes / Q000737:chemistry*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011073:Polyamines; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000737:chemistry / Q000378:metabolism; D013095:Spermidine / Q000737:chemistry*; D013096:Spermine / Q000737:chemistry*; D013997:Time Factors; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D001120:Arginine; D003546:Cysteine Endopeptidases; D018076:DNA, Complementary; D008239:Lysine; D009097:Multienzyme Complexes; D011073:Polyamines; D046988:Proteasome Endopeptidase Complex; D013095:Spermidine; D013096:Spermine; D014452:Ubiquitins; D000099228:Diamine N-Acetyltransferase