Polyamine analogues inhibit the ubiquitination of spermidine/spermine N-1-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(ethy l)spermine (BE-3-4-3), which greatly stabilize the enzyme. Rapid degradatio n of SSAT in reticulocyte lysates was preceded by formation of a ladder of ubiquitinated forms, and required the production of high-molecular-mass com plexes with ubiquitin (HMM-SSAT-Ubs). Mutation of all 11 lysines in SSAT se parately to arginine demonstrated that no single lysine residue is critical for its degradation in vitro, but mutant K87R had a significantly longer h alf-life, suggesting that lysine-87 may be the preferred site for ubiquitin ation. Mutations at the C-terininus of SSAT, such as E171Q, resulted in mar ked stabilization of the protein, due to the lack of formation of the HMM-S SAT-Ubs. Addition of BE-3-4-3 prevented the accumulation of ubiquitin conju gates and the proteasomal degradation of wild-type SSAT. These results indi cate that conformational changes brought about by the binding of polyamine analogues prevent the efficient polyubiquitination of SSAT, leading to a ma jor increase in the amount of SSAT protein, and that alteration of the C-te rminal end of the protein has a similar effect in preventing the productive interaction with an E2 or E3 component of the ubiquitin pathway.