Polyamine analogues inhibit the ubiquitination of spermidine/spermine N1-acetyltransferase and prevent its targeting to the proteasome for degradation

Abstract

Spermidine/spermine N1-acetyltransferase (SSAT), a key enzyme in mammalian polyamine catabolism, undergoes rapid turnover (half-life approx. 30min) 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.

Role of N-terminal residues in the ubiquitin-independent degradation of human thymidylate synthase

“…1.0 h, and was stabilized by the proteasome inhibitors MG132 and lactacystin ( Figure 1B). An in vitro reticulocyte lysate-based assay [20] verified that the 'lysine-less' TS polypeptide is stabilized by MG132 (results not shown).…”

Polyamine catabolism and disease

“…As indicated above, SSAT expression is the result of considerable post-transcriptional regulation, with the greatest increases in protein expression being a result of enhanced translation and protein stabilization [41, 114, 115]. By contrast, SMO expression appears to be regulated predominantly at the level of transcription and somewhat by message stabilization [116], but there is currently no evidence of post-translation regulation in response to any of the antitumor polyamines analogues examined.…”

Subcellular distribution of spermidine/spermine N1‐acetyltransferase

“…Polyamine analogue treatment greatly increases the half-life of SSAT (Fogel-Petrovic et al, 1996). Analogues appear to regulate the halflife by inhibiting ubiquitination of the SSAT enzyme and thereby preventing it targeting proteosomal degradation (Coleman and Pegg, 2001). Polyamine analogue treatment efficiently reduces the polyamine pools not only by stimulating degradation of the natural polyamines, but also by inhibiting their biosynthesis (Porter and Bergeron, 1988;Zagaja et al, 1998;Frydman et al, 2003a,b;Huang et al, 2003).…”