Proteasomal Degradation of Spermidine/Spermine N 1-Acetyltransferase Requires the Carboxyl-terminal Glutamic Acid Residues

Abstract

The rapid turnover of spermidine/spermine N 1 -acetyltransferase (SSAT), a key enzyme in the regulation of polyamine levels, was found to be mediated via ubiquitination and the proteasomal system. SSAT degradation was blocked by the binding of polyamines or of the polyamine analog, N 1 ,N 12 -bis(ethyl)spermine (BE-3-4-3), to the protein, providing a mechanism for the increase of SSAT activity in response to these agents. Site-directed mutagenesis indicated that a number of residues including arginine 19, cysteine 122, histidine 126, glutamic acid 152, arginine 155, and methionine 167 were needed for protection of SSAT by BE-3-4-3. These residues have previously been shown to reduce the affinity for the binding of polyamines to the SSAT protein, and these results indicate that the change in protein configuration brought about by this binding renders the protein resistant to proteasomal degradation. Mutations to alanines of residues arginine 7, cysteine 14, and lysine 141 also prevented the protection by BE-3-4-3, and these residues may be required for the formation of the protected conformation. The rapid degradation of SSAT required the carboxyl-terminal region of the protein, and the two terminal glutamic acid residues at positions 170 and 171 were found to be of critical importance. Truncation of the protein to remove these residues or the mutation of either of these acidic residues to glutamine completely abolished the rapid degradation of SSAT. The addition of two extra lysine residues at the carboxyl terminus or the conversion of the glutamic acids at positions 170 and 171 to lysines also prevented SSAT degradation by the proteasome. These results show the key role of the acidic residues at the carboxyl terminus of the protein in reacting with the proteasome. In contrast, mutation of lysine 166 to alanine, which extends the length of the acidic region in the carboxyl-terminal fragment of SSAT, actually increased the rate of degradation of SSAT without affecting its stabilization by BE-3-4-3. The binding of BE-3-4-3 or polyamines is therefore likely to change the configuration of the SSAT protein in a way that prevents the exposure of the carboxyl-terminal region of the ubiquitinated protein to the proteasome.Spermidine/spermine N 1 -acetyltransferase (SSAT), 1 which converts spermidine and spermine into their N 1 -acetyl derivatives, is an important enzyme in mammalian cells that prevents the overaccumulation of polyamines by facilitating their excretion and degradation (1-3). Alterations in SSAT activity are brought about by changes in amount of enzyme protein (4, 5), and the enzyme is highly inducible by a variety of hormones, physiological stimuli, drugs, and toxic agents. It is also strongly induced by polyamines, and it has been suggested that a rise in the free polyamine content is an intermediary in the induction by other agents (6). The most potent inducers of SSAT are polyamine analogs that have substitutions on the terminal nitrogens and, therefore, are not substrates for acetylation (1,(7)(8)(9)...

Structures of wild-type and mutant human spermidine/spermine N 1 -acetyltransferase, a potential therapeutic drug target

“…Our assays did not detect an effect of self-acetylation on the activity of purified SSAT, although it is possible that activity could be modulated through interaction with one or more additional factors present in vivo. An attractive idea would be that self-acetylation somehow directs SSAT to the known pathway for degradation of SSAT by the proteasome (18). However, initial tests in vitro by using a rabbit reticulocyte system (19) did not detect a difference in stability between wild-type and K26R SSAT.…”

“…SSAT is rapidly inducible by a polyamine-dependent pathway and typically has a short biological half-life, being efficiently ubiquitylated and subsequently degraded by the proteasome (16)(17)(18)(19). Under normal conditions, SSAT protein levels are very low, and at least part of the increase in SSAT levels caused by BE-3-3-3 is due to an increase in half-life, presumably because the bound inhibitor reduces ubiquitylation and thus degradation.…”

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

“…Exposure to BE-3-4-3 greatly increases the half-life of SSAT in i o [5,6,15] and in reticulocyte lysate degradation systems [8]. Such stabilization is a major factor in the drug-induced increase in SSAT activity.…”

“…Despite its rapid turnover, SSAT has no PESTrich sequence [9], although the C-terminal sequence MAT(A)EE has been shown to be required for rapid turnover, and may serve this function [8]. Purified SSAT has been reported to be phosphorylated by casein kinase II [10], but the stoichiometry and significance of this observation in terms of enzymic activity or stability is not known.…”

“…30 min in the absence of inducers [7,29]. The rapid turnover of SSAT can also be readily demonstrated in itro in reticulocyte lysate-derived systems [8]. In both systems, the degradation of SSAT is blocked by the proteasomal inhibitor MG132 (carbobenzoxy--leucyl--leucyl--leucinal), indicating that it is mediated via the proteasome, and in reticulocyte lysates the formation of ladders of high-molecular-mass bands, thought to be ubiquitin (Ub) conjugates, was demonstrated [7,29].…”