Post-transcriptional regulation of the content of spermidine/spermine N1-acetyltransferase by N1N12-bis(ethyl)spermine

Abstract

Spermidine/spermine N1-acetyltransferase (SSAT) is the rate-limiting enzyme for the degradation and excretion of polyamines in mammalian cells, and its activity is known to be increased enormously on exposure to polyamines and polyamine analogues. The mechanism by which such an analogue, BESM [N1N12-bis(ethyl)spermine], increases the content of SSAT was investigated by transfecting COS-7 cells with plasmids containing SSAT cDNA in the pEUK expression vector. Despite a large increase in mRNA production, there was only a very small increase in SSAT activity in the transfected cells. When BESM was added at 36 h after transfection, there was a large and very rapid increase in SSAT protein amounting to 380-fold in 12 h without any increase in the mRNA. SSAT protein turned over very rapidly, with a half-life of about 20 min. In the presence of BESM, this turnover was greatly reduced, and the half-life increased to more than 13 h. However, this increase was not sufficient to account for all of the increase in SSAT protein, suggesting that there is also regulation of the translation of the mRNA by BESM. Further evidence for such translation regulation was obtained by studying the polysomal distribution of the SSAT mRNA. In the absence of BESM, most of the mRNA was present in fractions which sedimented more slowly than the monoribosome peak. In BESM-treated cells, a significant proportion of the SSAT mRNA was moved into the small-polysome region of the gradient. The expression of SSAT and the effects of BESM on the polysomal distribution of SSAT mRNA were not affected by the 5'- or 3'-untranslated regions of the mRNA, since constructs which lacked all of these regions gave similar results to constructs containing the entire mRNA sequence. These results show that the increased transcription of the SSAT gene that occurs in the presence of polyamine analogues such as BESM is not sufficient for SSAT expression and that post-transcriptional regulation is critical for the control of SSAT content.

Differential post‐transcriptional control of ornithine decarboxylase and spermidine‐spermine N1‐acetyltransferase by polyamines

“…Enhanced translation may also play a role in analog post-transcriptional effects. Parry et al [18] used transfected COS-7 cells which overexpress SSAT mRNA to show that increases in SSAT activity by N 1,N12-di(ethyl)spermine was partially due to enhancement of SSAT mRNA translation [18]. Northern blot data presented here suggest that analog effects on mRNA levels must also be considered.…”

“…This problem was at least partially obviated by the finding that certain polyamine analogs can bring about several hundred-fold increases in enzyme protein and/or activity in some cell types [29]. The use of analogs for studying mechanisms of induction, however, is complicated by the multiplicity of their effects on SSAT gene expression [12,13,[15][16][17][18] and by uncertainty whether enzyme responses are truly representative of those produced by the natural polyamines. As we have shown here, SSAT mRNA can be rapidly induced by inhibitors of protein synthesis to levels comparable to those produced by the most potent analog, DENSPM [12,13].…”

“…More specifically, the near 1 : 1 correlation between increased activity and available transcripts strongly suggests that regulation involves enhanced mRNA translation as opposed to enzyme stabilization since the latter would be expected to substantially increase this ratio. As an example, analogs such as DENSPM are known to greatly prolong enzyme protein half-life [15,16,18] and, as discussed below, produce activity to mRNA ratios in the range of 8:1. Since there is no evidence that natural polyamines can similarly stabilize SSAT protein and since they induce enzyme activity in proportion to available mRNA, translational regulation is strongly implicated.…”

“…Analogs of spermine (SPM) such as N1,Nn-bis -(ethyl)norspermine (DENSPM) increase enzyme protein up to several hundred-fold and produce a 10-20-fold accumulation of SSAT mRNA due to increased gene transcription and mRNA stabilization [12,13]. Because the mRNA response is much less than the increase in SSAT activity, post-transcriptional mechanisms such as stabilization of SSAT protein and enhanced translation have also been implicated [15][16][17][18]. By contrast, the natural polyamine SPM brings about a ,-,7-fold increase in SSAT protein (or activity) which is accompanied by a low level increase in SSAT mRNA [13,14].…”

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

“…It is very difficult to measure the turnover of SSAT accurately in cells with low polyamine levels because the content of SSAT protein is extremely small under such noninducing conditions. However, estimates of a half-life of less than 1 h have been made (15)(16)(17), and this half-life is increased by more than …”