Properties of the Spermidine/SpermineN1-Acetyltransferase Mutant L156F That Decreases Cellular Sensitivity to the Polyamine AnalogueN1,N11-Bis(ethyl)norspermine

Abstract

Properties of a mutant form of spermidine/spermine N 1 -acetyltransferase, L156F (L156F-SSAT), that is present in Chinese hamster ovary cells selected for resistance to the polyamine analogue N 1, N 11 -bis(ethyl)norspermine (BE 3-3-3) were investigated. Increased K m values, decreased V max values, and decreased k cat values with both polyamine substrates, spermidine and spermine, indicated that L156F-SSAT is an inferior and less efficient acetyltransferase than wild-type SSAT. Transfection of L156F-SSAT into C55.7Res cells indicated that cellular SSAT activity per nanogram of SSAT protein correlated well with the in vitro data and was also ϳ20-fold less for the mutant protein than for wild-type SSAT. Increased expression of L156F-SSAT was unable to restore cellular sensitivity to BE 3-3-3 despite providing measurable basal SSAT activity. Only a 4-fold induction of L156F-SSAT activity resulted from the exposure of cells to the polyamine analogue, whereas wild-type SSAT was induced ϳ300-fold. Degradation studies indicated that BE 3-3-3 cannot prevent ubiquitination of L156F-SSAT and is therefore unable to protect the mutant protein from degradation. These studies indicate that the decreased cellular sensitivity to BE 3-3-3 is caused by the lack of SSAT activity induction in the presence of the analogue due to its inability to prevent the rapid degradation of the L156F-SSAT protein.

Spermidine/spermine-N1-acetyltransferase: a key metabolic regulator

“…The mutant CHO cells were found to contain a point mutation in the SSAT gene that alters the coding sequence, changing Leu 156 to Phe. This mutation not only greatly impairs the activity but also prevents BE-3-3-3 from protecting the mutant protein from polyubiquitination and degradation (97). Subsequent determination of the crystal structure of SSAT explains these results, since the L156F mutation would change the binding surface for the aliphatic carbons of the substrate/inducer at positions 8 -10 (15).…”

“…Such binding prevents the formation of polyubiquitinated SSAT (Fig. 4E) (15,35,97). This stabilization causes a large amplification of the effect provided by increased mRNA production and translation in response to the signal of polyamines or analogs.…”

Small Interfering RNA Suppression of Polyamine Analog-Induced Spermidine/SpermineN1-Acetyltransferase

“…In the second, McCloskey and Pegg (2000) found that a point mutation in SSAT gene was responsible for resistance to DENSPM in a Chinese hamster ovary cell line. It was subsequently shown that the analog was unable to effectively sustain high levels of SSAT because of its inability to prevent the ubiquitination and rapid turnover of the mutant SSAT protein (McCloskey and Pegg, 2003). Taking an alternative tack, Vujcic et al (2000) showed that conditional overexpression of SSAT in MCF-7 cells gave rise to a steady depletion of Spd and Spm pools and a gradual inhibition of cell growth.…”

Histone Deacetylase Inhibition Overcomes Drug Resistance through a miRNA-Dependent Mechanism

“…It is likely that the well-studied, post-translational regulatory abilities of BENSpm on the SSAT protein, e.g., enzyme stabilization, are more effective than those of PG-11047, thereby accounting for the more dramatic increase in enzyme activity (42, 46). These results also suggest that the main contribution of MS-275 in the observed SSAT induction is at the level of enhanced transcription.…”