The polyamines (PAs) spermidine, spermine, putrescine and cadaverine are an essential class of metabolites found throughout all kingdoms of life. In this comprehensive review, we discuss their metabolism, their various intracellular functions and their unusual and conserved regulatory features. These include the regulation of translation via upstream open reading frames, the over-reading of stop codons via ribosomal frameshifting, the existence of an antizyme and an antizyme inhibitor, ubiquitin-independent proteasomal degradation, a complex bi-directional membrane transport system and a unique posttranslational modification—hypusination—that is believed to occur on a single protein only (eIF-5A). Many of these features are broadly conserved indicating that PA metabolism is both concentration critical and evolutionary ancient. When PA metabolism is disrupted, a plethora of cellular processes are affected, including transcription, translation, gene expression regulation, autophagy and stress resistance. As a result, the role of PAs has been associated with cell growth, aging, memory performance, neurodegenerative diseases, metabolic disorders and cancer. Despite comprehensive studies addressing PAs, a unifying concept to interpret their molecular role is missing. The precise biochemical function of polyamines is thus one of the remaining mysteries of molecular cell biology.

 The enzymes in PA metabolism are controlled via highly specialized and unconventional mechanisms at the level of transcription, translation and protein degradation, involving several feedback loops controlled by PA concentrations. These aforementioned features are highly conserved throughout all kingdoms of life, indicating that the regulation of PA levels is highly critical for the cell. Transcriptional and translational control of ornithine decarboxylase (ODC) ODC is in many species the limiting factor for the biosynthesis of Put, Spd and Spm and its intracellular concentrations are tightly controlled. ODC can alter its activity in response to many different types of cellular perturbation. For example, ODC activity is induced in response to growth stimuli and has elevated activity in cells infected by viruses and under conditions of disease such as cancer [22–28]. The regulation of ODC starts at transcription. Best studied in mammals, the Odc promoter contains various elements that respond to several stimuli, such as growth factors, hormones and tumor promoters [29–32]. For ODC translation, regulation is highly dependent on PA concentration (Fig. 3a). An increase in intracellular PA levels leads to ODC translation repression, whereas a decrease causes translation activation. So far, it is not well understood how this translation repression and activation by PAs occurs. In mammals, ODC mRNAs have a long 5′ untranslated region (UTR) that contains a strong secondary structure (reviewed in Ref. [33]). The 5′ UTR contains two additional elements that cause a reduction in efficiency of ODC mRNA translation: a small functional upstream open reading frame (uORF) (Fig. 3a) and a GC-rich sequence [34–36].

 Mammalian ODC mRNA translation can also be mediated by an internal ribosome entry site (IRES). This allows for translation initiation even when cap-dependent translation is blocked, as in mitosis for instance [37]. The turnover of ODC is also controlled. ODC is short-lived with a half-life of less than 1 h [38, 39]; in eukaryotes, ODC degradation occurs via the 26S proteasome; however, this process is independent of ubiquitination (Fig. 3b) [40]. As most proteins are ubiquitinated prior to protein degradation, this feature of ODC degradation is relatively unusual [41]. Other proteins that are degraded in this ubiquitin-independent way in eukaryotes include thymidylate synthase, a human enzyme responsible for the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate and Rpn4, a Saccharomyces cerevisiae transcription factor that activates proteasomal genes (reviewed in Ref. [41]). One similarity between these proteins that may explain their degradation independent of ubiquitin is the presence of an unstructured domain recognizable by the proteasome [41]. The regulation of polyamine synthesis through antizyme (AZ) levels Apart from ODC, AZ facilitates the degradation of other proteins such as the human Aurora A kinase, via the proteasome in an ubiquitin-independent manner [49]. This enzyme is conserved from bacteria to humans but is not described in plants [50–52]. 

AZ levels are controlled by an unusual and highly conserved translational mechanism. It is encoded by two adjacent open reading frames (ORFs) and ribosomal frameshifting is required to generate a functional product. This frameshifting consists of ribosomes reaching the last codon of the first ORF (a stop codon), shifting one nucleotide and continuing to read the second ORF in the + 1 frame. In this way, the UGA stop codon is over-read (Fig. 3c). The + 1 ribosomal frameshifting at the stop codon is remarkably well conserved in many eukaryotes, while other features of the sequence evolved independently [51]. It is still unclear how this frameshift occurs mechanistically, how it is induced and, further, why such costly mechanisms are required to control AZ abundance. The efficiency of ribosomal frameshifting is controlled by the levels of PAs and thus AZ is the center of a homeostatic feedback loop. When PA levels are high, the efficiency of ribosomal frameshifting increases, resulting in higher levels of AZ, an increased rate of ODC degradation and, finally, reduced biosynthesis of PAs [53–55]. In mammals, this feedback loop also leads to a lower PA uptake from the extracellular environment [53–55]. Whether PAs regulate the AZ ribosomal frameshift directly or indirectly is still not clear. A recent study in S. cerevisiae revealed that, in the absence of PAs, the nascent AZ polypeptide inhibits its own synthesis; furthermore, when PAs are present, they can interact with the nascent peptide and prevent AZ synthesis inhibition (Fig. 3c) [56]. PAs also bind to the mammalian AZ, suggesting that this is a conserved mechanism [56] Furthermore, AZ is regulated by an AZ inhibitor (AZi) (Fig. 3b). AZi is similar to ODC but is catalytically inactive [57].

 AZ has more affinity to the AZi than to ODC; their subsequent interaction allows ODC to dimerize, be activated and escape degradation (Fig. 3b). Both AZ and AZi are degraded by ubiquitin-dependent proteasome degradation; thus, ODC activity can be controlled by ubiquitination. Interestingly, in mammals, one of the three AZ present, AZ 1, has an additional characteristic: it possesses two alternative start codons. These two start codons result in two isoforms of varying length. Notably, the longer isoform which is expressed at low levels is targeted to the mitochondria [58] and the nucleus, where it concerts a yet unknown role [59, 60]. Polyamines and S-adenosylmethionine decarboxylase (AdoMetDC) AdoMetDC catalyzes the formation of dcAdoMet [61] that acts as a donor of aminopropyl groups for the synthesis of Spd and Spm from Put. This enzyme is expressed at low levels and, in this way, does not deplete the concentration of S-adenosylmethionine (AdoMet) that is essential for methyl transfer reactions [61]. AdoMetDC is synthesized as an inactive proenzyme. To become active, the proenzyme undergoes an internal serinolysis, which causes cleavage of the proenzyme into two subunits (α and β) and also results in the formation of a pyruvoyl group at the N-terminus of the α-subunit (Fig. 4a) [62]. In mammals and yeast, Put stimulates AdoMetDC self-processing and activation [63–65]. The major regulators of AdoMetDC are PA levels. While Put positively regulates AdoMetDC, Spd and Spm negatively regulate the enzyme (reviewed in Ref. [61]).

 Increases in AdoMetDC levels have shown to be associated with growth stimulation, for example, during hormone treatment, tissue regeneration and cellular differentiation [66–68]. Although regulation of AdoMetDC transcription is not yet clear, translation is known to be mediated by uORFs. The mammalian uORF that precedes the ORF for AdoMetDC is located 14 nucleotides downstream of the 5′ cap and encodes a hexapeptide: MAGDIS [69]. During translation of MAGDIS, when the final tRNA (for serine) encounters the ribosome, it causes a ribosomal stall close to the uORF termination site, making the AdoMetDC start codon inaccessible (Fig. 4b) [70]. The stability of the bound tRNApeptide is specific for the peptide sequence, and additionally, this stability increases with higher levels of Spd and Spm [70, 71]. In plants, there are two uORFs that overlap by one nucleotide, the 5′ “tiny” and the 3′ “small”. The 3′ small uORF represses translation of the AdoMetDC ORF and is also affected by PA levels. It is hypothesized that, in low concentrations of PAs, the 5′ tiny uORF is translated and is able to inhibit translation of the 3′ small uORF. This inhibition would allow ribosomes to reach the initiation codon of AdoMetDC and initiate its translation [72]. In high concentrations of PAs, the 3′ small uORF is translated and subsequently blocks access to the AdoMetDC ORF. This is because the 5′ tiny uORF is bypassed by either the translation initiation complex or a ribosome -1 frameshifting that enables translation of the two ORFs [72]. Similar to ODC, AdoMetDC half-life is short (less than 1 h), and in extreme cases such as in Crithidia fasciculata, the turnover occurs in 3 min [73]. AdoMetDC turnover accelerates when concentrations of Spd and Spm are high [67]. 

Degradation of AdoMetDC by polyubiquitination via the proteasome is accelerated when its pyruvoyl group is transaminated and transformed into alanine by its substrate AdoMet [74]. This transformation is suggested to induce a conformational change in this enzyme, making its ubiquitination site more accessible and thus more prone for degradation [61, 74]. Additionally, besides transamination promoting AdoMetDC degradation, this reaction has also shown to inactivate its function [74]. Controlling Polyamine levels through catabolism In addition to their synthesis, PAs can be oxidized and/or acetylated to maintain their cellular activity or concentration (Fig. 2). Regulation of PA concentration is mediated by PA oxidases (PAOs) that can be classified depending on their cofactor, flavin adenine dinucleotide (FAD) or copper. Within the FADcontaining enzymes fall acetylated polyamine oxidase (APAO) [75–77], the PA oxidase PAO [7, 78, 79] and the spermine oxidase SpmO [7, 80, 81]. The substrates of PAO and APAO are nonacetylated or acetylated PAs, respectively, O2 and H2O. Most PAOs produce smaller PAs, an amino aldehyde and H2O2 (Fig. 2). An exception is diamine oxidase (DAO), a copper-containing enzyme that produces ammonia in addition to H2O2 (Fig. 2) [82–85]. The amino aldehydes produced can then be precursors for amino acids such as β-alanine and gammaaminobutyric acid (GABA) and for several different alkaloids (reviewed in Ref. [7]). The acetylation of Spm or Spd is catalyzed by spermidine/spermine acetyltransferase (SSAT) [86– 89]. 

When SSAT is activated, it leads to PA acetylation that subsequently reduces their positive charge, preventing their interaction with other molecules [90]. These acetylated metabolites are then excreted (reviewed in Ref. [90]) or oxidized by the abovementioned FAD-dependent APAO. These reactions thus form a cycle that allows the cell to regulate Spd and Spm cellular concentrations quickly [90]. In addition, cells have adapted other mechanisms to regulate PA concentration at this stage [86, 90]. In human cells, the SSAT gene contains a PA-responsive element (PRE) in the 5′ regulatory region that allows transcription to be regulated by PA concentration [91]. Moreover, it was found that Nrf-2 (nuclear factor erythroid 2–related factor 2) binds constitutively to PRE and when the levels of PAs are high this complex interacts with another protein, PA-modulated factor-1, to activate SSAT transcription [92, 93]. This mechanism was only found in tumor cells that are sensitive to synthetic PA analogues, that is, cells that respond to these compounds by upregulating SSAT and consequently increasing PA catabolism. This indicates that different cell types regulate SSAT dissimilarly [91–93]. SSAT expression can also be modulated during RNA processing by alternative splicing. During its splicing, the intron between exon 3 and exon 4 might be retained. This intron contains multiple codon stops, making this splice variant prone for degradation by nonsense-mediated mRNA decay [94]. There is evidence that this splice variant can give rise to a truncated SSAT protein. 

In the presence of high levels of PAs or its analogues, the formation of this alternative splice variant is decreased, leading to increased SSAT activity and therefore PA acetylation [86, 95]. SSAT translation can increase drastically in the presence of PAs or synthetic PA analogues [96, 97]. Contrary to other enzymes of the PA pathway, the 5′ or 3′ UTRs do not seem to have the same prominent role in the translational regulation of SSAT [96, 97]. Nonetheless, one or two uORFs can be found depending on the species [98]. Recently, a study using a human embryonic kidney cell line revealed that these uORFs were able to repress SSAT translation [99]. In addition, the initiation region of SSAT mRNA contains a stem–loop that is stabilized by a specific isoform of a protein called nucleolin [99]. This interaction subsequently results in SSAT translation repression (Fig. 5a). PAs at high levels promote the autocatalysis of nucleolin, which likely causes a reduction in the stability of the stem–loop of SSAT and in turn alleviates translation repression (Fig. 5b) [99]. This would then lead to an increase in PA catabolism and re-establishment of PA levels. Finally, for degradation, SSAT is polyubiquitinated and degraded by the 26S proteasome [100]. SSAT has a very short half-life of approximately 20 min [99], which can be extended in the presence of an Spm analogue, such as N1 N12-bis(ethyl)spermine [99].Controlling Polyamine levels through transport PA transport has been detected in almost every model organism. In bacteria and single cellular eukaryotes, membrane transport systems are well described (Fig. 2). E. coli imports PAs mainly by two ABC (ATP binding cassettes) type transporters, PotABCD and PotFGHI, that are Spd preferential and Put specific, respectively [101, 102].

 Moreover, there are two uptake/antiporters known as PotE (Orn or Lys/Put) and CadB (Lys/Cad) that uptake Orn or Lys and excrete Put or Cad, respectively [103–105]. These transporters are induced under acidic conditions and also have an important role in the cell's response to acidic stress [104, 105]. In E. coli, studies have also shown a Spd exporter (MdtJI) to be important for PA homeostasis since it is capable of rescuing a SSAT knockout strain from the toxicity caused by high levels of Spd [106]. Yeast, a model organism for single cell eukaryotes, has at least 10 transmembrane proteins capable of PA transport. There are four transporters in the plasma membrane involved in the PA uptake: Dur3, Sam3, Agp2 and Gap1 [107–109]. Dur3 and Sam3 are predominantly responsible for PA import [108, 110], while Uga4 dominates PA vacuolar transport [111]. Conversely, yeast has four transporters (Tpo1–Tpo4) that function as PA efflux pumps [7, 112–114]. Tpo1 and Tpo4 are responsible for the transport of Spd, Spm [115] and Put [116], while Tpo2 and Tpo3 only recognize Spm [115]. Deletion of Tpo1, the best-studied PA transporter, shows sensitivity toward high levels of PAs, while its overexpression increases tolerance to excess PA supplementation [117, 118]. 

Finally, there is also a fifth transporter, Tpo5, that is localized in the Golgi or post-Golgi secretory vesicles and is responsible for the excretion of Put and less effectively of Spd [114]. PA transporters have also been studied in T. cruzi and Leishmania major. Here, they transport Put, Cad and Spd and share 41.3% identity [9, 10, 119, 120]. As T. cruzi lacks ODC, import of PA from external sources is the only way T. cruzi can obtain Put or Spd, highlighting the essentiality of PA transport for such organisms. PA-specific transport systems in mammals and plants are less well understood. In mammals, no PA transporter has yet been identified. It has been suggested that such a PA transport system would involve an endocytic mechanism (reviewed in Refs. [103] and [121]). Moreover, it has been proposed that transport systems with other functions such as SLC7 (Lys/Arg/Orn permeases), CCC9 (an inorganic ion transporter) and OCT6 (cation/anion/zwitterion transporter) could also be responsible for PA uptake (reviewed in Ref. [121]). In Arabidopsis, PA transport has been attributed to the LAT (L-type amino acid transporter) family [122]. These LAT transporters, particularly RMV1/LAT1/AtPUT3, PAR1/AtLAT4/ AtPUT2 and AtLAT3/AtPUT1, are 68–76% similar to each other and are localized in the plasma membrane, Golgi apparatus and endoplasmic reticulum, respectively [122].The Role of Polyamines in the Cell Polyamines in gene expression PAs are important for gene expression due to their ability to bind to nucleic acids and proteins; thus, these molecules can stabilize and remodel the chromatin structure (reviewed in Ref. [123]). 

For example, the analysis of chromatin structure of nuclei isolated from U-87 MG human brain tumor cells revealed upon PA depletion by treatment with an ODC inhibitor [α-difluoromethylornithine (DFMO)] chromatin condensation is impaired [124]. DNA structural changes mediated by PAs have also shown to modulate the rate of transcription. For example, PAs enhance the affinity of the mammalian estrogenic receptor (ER) for its response elements (ERE) by promoting the transition of the B-DNA to the Z-DNA conformation [125]. PAs can further mediate the activation of c-MYC transcription by modifying the quadruplex structure (present in the regulatory sequence of this gene) into an active conformation [126]. PAs additionally promote the binding between proteins and DNA. In vitro studies using the herpes simplex virus I DNA-binding protein ICP-4 demonstrated that physiological concentrations of Spd and Spm increase the rate of association of this protein to DNA [127]. Most PAs are bound to RNA as shown in bovine lymphocytes, rat liver and E. coli (57.2%, 78.3% and 89.7% respectively for Spd bound RNA; Spm: 65.2% and 85.2%, for rat liver and E. coli; Put: 47.9% for E. coli), suggesting that one of the major roles of PAs is related to structural changes in RNA [128]. It is therefore highly probable that PAs have an essential role in translation. The “PA modulon” is comprised of a group of genes that increase their translation in the presence of PAs, which in certain cases is cased by an increase of their transcription factors [129, 130]. Members of this modulon have been identified in E. coli and eukaryotes in which intracellular PA concentrations have been modified [129–131].