Polyamine-Regulated Translation of Spermidine/Spermine-N¹-Acetyltransferase

Abstract

Rapid synthesis of the polyamine catabolic enzyme spermidine/spermine-N 1 -acetyltransferase (SSAT) in response to increased polyamines is an important polyamine homeostatic mechanism. Indirect evidence has suggested that there is an important control mechanism involving the release of a translational repressor protein that allows the immediate initiation of SSAT protein synthesis without RNA transcription, maturation, or translocation. To identify a repressor protein, we used a mass spectroscopy-based RNA-protein interaction system and found six proteins that bind to the coding region of SSAT mRNA. Individual small interfering RNA (siRNA) experiments showed that nucleolin knockdown enhances SSAT translation. Nucleolin exists in several isoforms, and we report that the isoform that binds to SSAT mRNA undergoes autocatalysis in the presence of polyamines, a result suggesting that there is a negative feedback system that helps control the cellular content of polyamines. Preliminary molecular interaction data show that a nucleolin isoform binds to a 5= stem-loop of the coding region of SSAT mRNA. The glycine/arginine-rich C terminus of nucleolin is required for binding, and the four RNA recognition motif domains are included in the isoform that blocks SSAT translation. Understanding SSAT translational control mechanisms has the potential for the development of therapeutic strategies against cancer and obesity. P olyamines are small positively charged molecules present in all cells. The common polyamines putrescine, spermidine, and spermine are essential for cell growth. The rate of synthesis and content both increase with increased cell proliferation (47). Polyamine functions include stabilization of polynucleotides, regulation of transcription and translation, control of enzyme activities, modulation of ion channels, and response to oxidative stress (42, 61). Because so many processes are affected, levels are maintained within a relatively narrow range by shifts in anabolism/catabolism and import/export (1, 46). Manipulation of polyamine metabolism has been an anticancer strategy, with pool depletion in tumor cells used as a surrogate marker of efficacy (21,34,38,48).Many mechanisms contribute to control of eukaryotic polyamine metabolic enzymes (15,41,43,50). Ornithine decarboxylase (ODC), the rate-limiting anabolic enzyme, is regulated allosterically, at transcription, at translation, and by "ODC antizyme," a protein that binds to ODC monomers, thereby blocking homodimerization required for activity and accelerating monomer degradation (41). Antizyme itself is regulated by a translation control mechanism involving polyamine-induced ribosomal frameshifting (15, 35). Spermidine/spermine-N 1 -acetyltransferase (SSAT) is the principal catabolic regulator. SSAT acetylates spermidine and spermine using acetyl-coenzyme A (CoA), thereby altering their charge and facilitating excretion. SSAT basal activity is very low but increases quickly when polyamines are in excess (11). There is evidence for transcription, translati...

Elimination of polyamine N-acetylation and regulatory engineering improved putrescine production by Corynebacterium glutamicum

“…Similar polyamine N-acetyltransferases have been described in bacteria, e.g. SpeG acetylates spermidine in E. coli (Limsuwun and Jones, 2000), BltD (Woolridge et al, 1999) and PaiA (Forouhar et al, 2005) acetylate spermidine and spermine in Bacillus subtilis, spermidine and diamine acetyltransferase in the yeast Candida boidinii (Haywood and Large, 1985), and spermidine/spermine-N1-acetyltransferase in human (Perez-Leal et al, 2012). It is believed that cells typically adjust intracellular polyamine concentrations by acetylation in adaptation to stress (Carper et al, 1991;Casero and Pegg, 1993).…”

Cryptosporidium parvum Induces an Endoplasmic Stress Response in the Intestinal Adenocarcinoma HCT-8 Cell Line

“…hSSAT-1 is a dimer or tetramer of ϳ65-80 kDa, with a subunit size of 20 kDa; contains 171 amino acids; and expresses a single 1.3-kb transcript (18). SSAT genes from mouse, rat, and hamster share regions of extensive similarity, including extensive 5Ј-and 3Ј-untranslated regions and a 200 -300-nucleotide poly(A) tail (19). The catalytic active site contains eight arginine residues, five of which have been implicated in acetyl-CoA binding (18).…”

“…Polyamine binding at this site may prevent protease attack and stabilize the protein (21), making it a good candidate for the amine substrate binding of the active site. Evidence from both human and mouse SSATs indicates a TATA-less promoter, a consensus sequences for a cAMP-responsive element at Ϫ487 bp, and several putative cisacting transcriptional elements (19). CpSSAT exhibits different kinetic characteristics compared with the host enzyme but is also found to be a homotetramer of 18 kDa, with arginine-rich motifs that are responsible for acetyl-CoA binding (4).…”

Remaining Mysteries of Molecular Biology: The Role of Polyamines in the Cell

“…Recently, a study using a human embryonic kidney cell line revealed these uORFs were able to repress SSAT translation [99]. In addition, the initiation region of SSAT mRNA contains a stem loop which is stabilized by a specific isoform of a protein called nucleolin [99].…”

“…In addition, the initiation region of SSAT mRNA contains a stem loop which is stabilized by a specific isoform of a protein called nucleolin [99]. This interaction subsequently results in SSAT translation repression ( Fig 5A).…”