|Topic:||Polyamine Biosynthesis .|
|Details:|| The polyamines are highly cationic molecules that tend to bind nucleic acids with high affinity. Because of this interaction activity, it is believed that the polyamines are important participants in DNA synthesis, or in the regulation of that process. In addition to their function in DNA replication, polyamines and polyamine derivatives are involved in the processes of protein synthesis, ion channel function, regulation of gene expression, cell proliferation, and apoptosis (programmed cell death).
One of the earliest signals that cells have entered their replication cycle is the appearance of elevated levels of mRNA for ornithine decarboxylase 1 (ODC1), and then increased levels of the enzyme. Ornithine decarboxylase is the first enzyme in the pathway to synthesis of the polyamines. The human genome contains two ODC sequences with only the ODC1 gene being functional. The other locus, identified as ODCP, is a pseudogene. The ODC1 gene is located on chromosome 2p25 and is composed of 13 exons that generate four alternatively spliced mRNAs. Three of the ODC1 splice variants encode the same enzyme identified as isoform 1 (461 amino acids). ODC isoform 2 is composed of 332 amino acids. Functional ODC is a homodimer and requires pyridoxal phosphate (PLP), derived from vitamin B6, as a cofactor. The catalytic activity of ODC produces the 4-carbon saturated diamine, putrescine.
An important intermediate required for the synthesis of spermidine and spermine from putrescine is derived from S-adenosylmethionine (SAM). The enzyme, adenosylmethionine decarboxylase 1 (AMD1; also known as S-adenosylmethionine decarboxylase or SAM-decarboxylase) cleaves the SAM carboxyl residue, producing decarboxylated SAM (S-adenosylmethylthiopropylamine; also known as decarboxylated S-adenosylmethionine, dcAdoMet or dcSAM), which retains the methyl group usually involved in SAM methyltransferase activity. Adenosylmethionine decarboxylase 1 activity is regulated by product inhibition and allosterically stimulated by putrescine. The AMD1 gene is located on chromosome 6p21 and is composed of 10 exons that generate four alternatively spliced mRNAs that encode four distinct isoforms of the enzyme. Spermidine synthase catalyzes a condensation reaction between dcAdoMet and putrescine, producing spermidine and 5'-methylthioadenosine. Spermidine synthase is encoded by the SRM gene (also known as SPDSY) located on chromosome 1p36–p22. The SRM gene is composed of 8 exons that encode a protein of 302 amino acids. A second propylamine residue from dcAdoMet is added to spermidine producing spermine by the enzyme spermine synthase encoded by the SMS gene (also known as SPMSY). The SMS gene is located on the X chromosome (Xp22.1) and is composed of 13 exons that generate two alternatively spliced mRNAs encoding SMS isoform 1 (366 amino acids) and SMS isoform 2 (313 amino acids).
The signals for regulating the production of ODC are complex. The promoter of the ODC1 gene contains elements that are regulated by growth factors, hormones, and tumor promoters. Production of ODC protein is also regulated at the level of translation by the product of the reaction, putrescine. The ODC1 mRNA contains a long 5'-untranslated region (5'-UTR) that folds into a complex secondary structure. Within the 5'-UTR there are two domains that reduce translational efficiency. One domain is a GC-rich sequence, the other is a small functional upstream open reading frame (uORF). Another significant mechanism for regulating ODC1 mRNA translation is particularly important during mitosis. Cap-dependent translation is frequently repressed during mitosis and the ODC1 mRNA contains an internal ribosome entry site (IRES) that allows the mRNA to be translated under these conditions.
The stability of the ODC1 protein also contributes to the level of enzyme activity in cells. The half-life of ODC protein is short (< 1hr) and this half-life is controlled by proteosomal degradation. However, entry into the proteosome, in the case of ODC, does not require prior ubiquitylation. The ability of the proteosome to recognize, and degrade, non-ubiquitylated ODC involves a family of proteins called antizymes. The antizyme proteins interact with ODC monomers preventing formation of functional homodimers while simultaneously altering the conformation of the monomers so that they are recognized by the proteosome. Humans express three distinct ODC antizymes identified as antizyme 1, 2, and 3. Antizyme 1 is encoded by the OAZ1 (ornithine decarboxylase antizyme 1) gene which is located on chromosome 19p13.3 and is composed of 5 exons that generate two alternatively spliced mRNAs. These two mRNAs encode antizyme 1 isoform 1 (228 amino acids) and antizyme 1 isoform 2 (226 amino acids). The translation of the functional antizyme 1 proteins is regulated by a polyamine-regulated ribosome frameshifting mechanism. The antizyme 1 mRNAs contain two overlapping open reading frames (ORFs). These ORFs are identified as ORF1 which is the shorter of the two, and ORF2. The reading frame of ORF2 is in the +1 frame relative to reading frame of ORF1. The ORF2 reading frame does not contain an AUG translation initiation codon and as such translation through ORF2 requires the failure of the ribosome to terminate at the end of ORF1 and instead shift to the +1 reading frame. This ribosomal frameshifting event is stimulated by polyamines. In the presence of high concentrations of polyamines the ribosome frameshifting occurs resulting in the synthesis of functional antizyme 1 which comprises amino acids from both ORF1 and ORF2. In addition to the two different isoforms, the antizyme 1 mRNA contains two in-frame AUG codons that have been shown, in rodents, to be alternatively utilized resulting in differentially localized antizyme 1 isoforms.
Antizyme 2 is encoded by the OAZ2 gene which is located on chromosome 15q22.31 and is composed of 5 exons that generate two alternatively spliced mRNAs that encode two distinct protein isoforms. Although widely expressed like the OAZ1 gene, the level of OAZ2 expression is much lower. Antizyme 3 is encoded by the OAZ3 gene which is located on chromosome 1q21.3 and is composed of 7 exons that generate three alternatively spliced mRNAs encoding three distinct protein isoforms. Antizyme 3 isoform 1 is the longest protein encoded by the OAZ3 gene and the translation of this form of the enzyme begins from a CUG codon and not an AUG codon. Expression of the OAZ3 gene is restricted to haploid germ cells in the testis.
As discussed, the synthesis of the polyamines is regulated by several independent mechanisms. In addition to these mechanism of control, intracellular polyamine levels are also regulated via their catabolism. Putrescine is catabolized by amine oxidase, copper containing 1 encoded by the AOC1 gene, while spermidine and spermine are catabolized by spermidine/spermine N1-acetyltransferase 1 (also known as diamine acetyltansferase 1) encoded by the SAT1 gene (also known as SSAT). The SAT1 gene is located on the X chromosome (Xp22.1) and is composed of 7 exons that generate two alternatively spliced mRNAs. Only one of the mRNAs encodes a functional SAT1 enzyme as the other mRNA is subjected to nonsense-mediated mRNA decay. The functional SAT1 enzyme is a 171 amino acid protein. In addition to putrescine, the AOC1 encoded enzyme is responsible for the catabolism of histamine and other related biogenic amines.
The butylamino group of spermidine is used in a posttranslational modification reaction important to the process of translation. A specific lysine residue in the translational initiation factor eIF-5A is modified. Following the modification the residue is hydroxylated yielding a residue in the protein termed hypusine. The term hypusine is derived from hydroxyputrescine and lysine. The hypusination of eIF-5A is necessary for the promotion of translational elongation. The hypusination of eIF-5A involves a two-step reaction process. The first reaction involves the enzyme deoxyhypusine synthase which transfers the butylamine moiety from spermidine to the specific eIF-5A lysine residue. The second reaction involves the enzyme deoxyhypusine hydroxylase which hydroxylates the deoxyhypusine to hypusine. Deoxyhypusine synthase is encoded by the DHPS gene located on chromosome 19p13.2 which is composed of 13 exons that generate three alternatively spliced mRNAs, each of which encode a distinct isoform of the enzyme. Deoxyhypusine hydroxylase is encoded by the DOHH (deoxyhypusine hydroxylase/monooxygenase) gene located on chromosome 19p13.3 which is composed of 7 exons that generate two alternatively spliced mRNAs that both encode the same 302 amino acid protein
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