Suppression of exogenous gene expression by spermidine/spermine N1-acetyltransferase 1 (SSAT1) cotransfection.
Spermidine/spermine N(1)-acetyltransferase 1 (SSAT1), which catalyzes the N(1)-acetylation of spermidine and spermine to form acetyl derivatives, is a rate-limiting enzyme in polyamine catabolism. We now report a novel activity of transiently transfected SSAT1 in suppressing the exogenous expression of other proteins, i.e. green fluorescent protein (GFP) or GFP-eIF5A. Spermidine/spermine N(1)-acetyltransferase 2 (SSAT2) or inactive SSAT1 mutant enzymes (R101A or R101K) were without effect. The loss of exogenous gene expression is not due to accelerated protein degradation, because various inhibitors of proteases, lysosome, or autophagy did not mitigate the effects. This SSAT1 effect cannot be attributed to the depletion of overall cellular polyamines or accumulation of N(1)-acetylspermidine (N(1)-AcSpd) because of the following: (i) addition of putrescine, spermidine, spermine, or N(1)-AcSpd did not restore the expression of GFP or GFP-eIF5A; (ii) depletion of cellular polyamines with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, did not inhibit exogenous gene expression; and (iii) N(1),N(11)-bis(ethyl)norspermine caused a drastic depletion of cellular polyamines through induction of endogenous SSAT1 but did not block exogenous gene expression. SSAT1 transient transfection did not affect stable expression of GFP, and stably expressed SSAT1 did not affect exogenous expression of GFP, suggesting that only transiently (episomally) expressed SSAT1 blocks exogenous (episomal) expression of other proteins. SSAT1 may regulate exogenous gene expression by blocking steps involved in transcription/translation from an episomal vector by targeting non-polyamine substrate(s) critical for this pathway.
Lee
Seung Bum
SB
Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892.
Park
Jong Hwan
JH
Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892.
Woster
Patrick M
PM
Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48201.
Casero
Robert A
RA
The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231.
Park
Myung Hee
MH
Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892. Electronic address: mhpark@nih.gov.
D000123:Acetyltransferases / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D014162:Transfection; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D000099228:Diamine N-Acetyltransferase