Regulation Of Polyamine Levels Research Articles

Insights into polyamine metabolism: homospermidine is double-oxidized in two discrete steps by a single copper-containing amine oxidase in pyrrolizidine alkaloid biosynthesis.

Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers Hspd over other amines. Nuclear magnetic resonance spectroscopy and mass spectrometry analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine (Hspd) to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.

Role of skeletal muscle homeostasis of functional food material

Functional food material, polyamines are considered to be essential for growth factors in virtually all cells. The polyamines putrescine, spermidine and spermine are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. The proposed functions of polyamines are the regulation of ion channels, nucleic acid packaging, signal transduction, cell proliferation, and differentiation, as well as gene expression. In skeletal muscle, regulation of polyamine levels is associated with muscle hypertrophy and atrophy, yet detailed studies are remained to be undergoing. Here, we studied how polyamines may affect the proliferation and/or differentiation of murine myoblast progenitor C2C12 cell line. Upon polyamine treatment of C2C12 cells during induction of myogenic differentiation, the number of myotubes significantly increased. Morphologically, polyamine-treated myotubes exhibited elongated cell body and contained larger amount of nuclei in the cell. On the other hand, the polyamine did not have influence on myoblasts proliferation. Furthermore, compensatory muscle hypertrophy of C57BL6 mice that underwent sciatic nerve transection of the left hindlimb was enhanced by administration of polyamines. Therefore, our study demonstrates that polyamines may play an important role in regulating myogenic differentiation rather than myoblasts proliferation.

Delving into the details: Investigation of the kinetic properties of SpeG Spermidine N‐acetyltransferases from Escherichia coli and Vibrio cholerae

Biofilms are communities of bacteria found in a variety of environments, including the intestinal mucosa of humans and on surfaces of medical devices. These biofilms protect bacteria from antibiotics and other adverse environmental stresses. Polyamines are polycationic small molecules that are involved in biofilm formation. When polyamines are acetylated, biofilm formation increases. One enzyme that acetylates polyamines in bacteria is the spermidine N‐acetyltransferase SpeG. This enzyme is important for regulating polyamine levels and responding to bacterial stress conditions, but its role in biofilm formation is still unknown. We previously determined the structures of the SpeG enzymes from Vibrio cholerae (VcSpeG) and Escherichia coli (EcSpeG), and found they exist as dodecamers and posses an allosteric site where polyamines bind. This site is not found in the human spermine/spermidine acetyltransferase (SSAT) enzyme, but they share a common core Gcn5‐related N‐acetyltransferase scaffold. These structural differences provide a unique opportunity to study the effect of the allosteric site on enzyme activity and regulation of polyamine levels in different organisms. As a first step toward elucidating the functional properties of SpeG, we present the kinetic characterization and comparison of VcSpeG and EcSpeG enzymes and show how they exhibit distinct properties between the two organisms and the human SSAT enzyme. Additionally, we present a structural comparison of the three enzymes to provide insight into their kinetic differences. Understanding the kinetic details of SpeG polyamine acetylation will ultimately provide important insight for designing drugs that specifically target SpeG over the human SSAT enzyme to prevent or reduce biofilm formation and pathogenesis.Support or Funding InformationStartup funds to MLK from San Francisco State University

Analysis by virus induced gene silencing of the expression of two proline biosynthetic pathway genes in Nicotiana benthamiana under stress conditions

Proline accumulation is responsible for stress adaptation in many plants. To distinguish the involvement of two proline synthetic pathways, the virus induced gene silencing (VIGS) system that silenced the expression of genes encoding Δ(1)-pyrroline-5-carboxylate synthetase (P5CS; EC:1.5.1.12) and ornithine-δ-aminotransferase (OAT; EC 2.6.1.13) was performed, separately or concomitantly, in four-week-old Nicotiana benthamiana. Leaf discs of VIGS-treated tobacco were subjected to the treatment of drought, abscisic acid (ABA), or polyethylene glycol (PEG). The treated leaf discs were then collected for the determination of mRNA, chlorophyll, proline and polyamine level. Under drought stress or PEG treatment, most proline accumulation was inhibited in P5CS-silenced plants and only a small portion was inhibited in OAT-silenced plants under drought stress and no inhibition was observed under PEG treatment. Under ABA treatment, proline accumulation was inhibited completely in P5CS-silenced plants but unaffected in OAT-silenced plants. The degradation of chlorophyll was enhanced in P5CS-silenced plants but retarded in OAT-silenced plants under PEG treatment. Under ABA treatment, the degradation of chlorophyll was unaffected in both P5CS-silenced and OAT-silenced plants. The increase of polyamine level was unaffected in P5CS-silenced plants but increased in OAT-silenced plants under PEG treatment. Under ABA treatment, the increase of polyamine level was unaffected in P5CS-silenced plants but the polyamine level was increased later in OAT-silenced plants. Therefore, P5CS plays a major role in proline accumulation under drought, PEG, or ABA treatment, while OAT plays a minor role in drought or PEG treatment and does not participate in ABA treatment. OAT appears to have a close relationship with the regulation of polyamine levels in PEG and ABA treatments.

Antizyme, a mediator of ubiquitin-independent proteasomal degradation and its inhibitor localize to centrosomes and modulate centriole amplification

The potential tumor suppressor antizyme and its endogenous inhibitor (antizyme inhibitor, AZI) have been implicated in the ubiquitin-independent proteasomal degradation of proteins involved in cell proliferation as well as in the regulation of polyamine levels. We show here that both antizyme and AZI concentrate at centrosomes and that antizyme preferentially associates with the maternal centriole. Interestingly, alterations in the levels of these proteins have opposing effects on centrosomes. Depletion of antizyme in various cell lines and primary cells leads to centrosome overduplication, whereas overexpression of antizyme reduces numerical centrosome abnormalities. Conversely, silencing of the antizyme inhibitor, AZI, results in a decrease of numerical centrosome abnormalities, whereas overexpression of AZI leads to centrosome overduplication. We further show that the numerical centrosome abnormalities are due to daughter centriole amplification. In summary, our results demonstrate that alterations in the antizyme/AZI balance cause numerical centrosomal defects and suggest a role for ubiquitin-independent proteasomal degradation in centrosome duplication.

Characterization of antizyme function in a yeast complementation system

Ornithine decarboxylase (ODC) is an important enzyme in the synthesis of polyamines. Polyamines are charged organic molecules that are essential for cell growth. Antizyme is an enzyme that aids in the regulation of polyamine levels within the cell by targeting ODC for degradation. A yeast strain has been established that involves the complementation of a null mutant endogenous ODC gene with expression of human ODC. The ODC gene was constitutively expressed while antizyme expression was controlled by galactose induction. In the presence of galactose, induced antizyme expression resulted in eventual cell death due to ODC degradation and depletion of polyamines. Using this system we will transform the strain with a yeast cDNA library controlled by a GAL promoter. We will screen for overexpressed yeast proteins that may inhibit antizyme function. The inhibition of antizyme will allow the yeast to grow since ODC will no longer be degraded. Identifying yeast genes that functionally interact with antizyme will lead to an increased understanding of the activity and function of antizyme in polyamine synthesis. The ability to understand the function and mechanism of antizyme’s ability to target ODC for degradation could help in the control of cell growth. Polyamine regulation is highly conserved in all organisms thus the study of AZ function in yeast can have direct applications to higher vertebrates. Supported by BRIN/INBRE Grant # P20 RR016457 from the NIH.

Up-regulation of arginine decarboxylase gene expression and accumulation of polyamines in mustard (Brassica juncea)in response to stress.

Arginine decarboxylase (ADC) is a key enzyme involved in the synthesis of polyamines, which have been implicated in a wide range of plant responses, including stress. However, regulation of polyamine levels in relation to ADC in response to stress at the molecular level is not well understood. In an attempt to address this question, we first cloned two cDNAs in mustard (Brassica juncea[L.] Czern & Coss var. Indian Mustard), designated MADC2 and MADC3, encoding predicted ADC. MADC2 and MADC3 encode polypeptides of 692 and 680 amino acid residues, respectively. A comparison of deduced amino acid sequence revealed that both were highly homologous to MADC1 (77%), a mustard ADC, and other plant ADCs (63-84%). Northern analysis revealed that ADC transcripts in mustard were generally more abundant in stem and root but were barely detectable in leaf. However, ADC expression in the leaf was up-regulated differentially in response to stress such as chilling, salt and mannitol and to treatments with exogenous polyamines. While chilling induced expression of all three ADC genes, salt predominantly resulted in increased accumulation of MADC3 transcript. Leaves exhibited a similar response to exogenous putrescine, spermidine and spermine, all of which stimulated accumulation of MADC2 and MADC3 transcripts but not MADC1. Furthermore, exogenous putrescine also increased the endogenous levels of spermidine and spermine, while a higher endogenous putrescine and spermidine content was detected in leaf incubated with exogenous spermine. Leaves also responded to chilling, salt and mannitol by increasing the levels of the cellular polyamine content, in which the level of spermine in free and conjugated forms increased most profoundly.

Developmental aspects of polyamine-oxidizing enzyme activities in the mouse kidney. Effects of testosterone

In the present study developmental patterns of renal polyamine-oxidizing enzymes polyamine oxidase (PAO) and diamine oxidase (DAO) in male and female ICR mice were demonstrated. The effects of testosterone (10 micrograms/100g body weight) on renal PAO and DAO activities were also studied. The differences between sexes in both PAO and DAO activities were most clearly expressed in the immature kidney. At the age of 20 days PAO and DAO activities were 1.52 fold (p < 0.01) and 1.75 (p < 0.02) respectively higher in male mouse kidney than in female. Maturational processes reflected in significant increases in polyamine-oxidizing enzyme activities mainly in female mouse kidney, comparable with the gain in the kidney wet weight. Our data show that testosterone is able to influence renal PAO and DAO activities in addition to the well-known stimulation of polyamine biosynthesis. The hormonal effects were sex and age dependent. The influence of testosterone on renal PAO activity was mainly age dependent. The slight stimulation of renal PAO activity observed in 20- and 50-day old mice, 24 h after testosterone administration, change with a decrease in the enzyme activity at the age of 70 days. The effects of testosterone on renal DAO activity were mainly sex dependent. Testosterone caused stimulation of DAO activity with a very close magnitude (nearly twice) in female mouse kidney, independently of the age of mice. In contrast, in male mice the hormone treatment resulted in a statistically significant increase in renal DAO activity at the age of 70 days (1.3 fold, p < 0.05) only. It could be suggested that our data indicate the different contribution of renal PAO and DAO in androgen regulation of polyamine levels, depending on sex and the stage of the postnatal development.

Proteasomal Degradation of Spermidine/Spermine N1-Acetyltransferase Requires the Carboxyl-terminal Glutamic Acid Residues

The rapid turnover of spermidine/spermine N1-acetyltransferase (SSAT), a key enzyme in the regulation of polyamine levels, was found to be mediated via ubiquitination and the proteasomal system. SSAT degradation was blocked by the binding of polyamines or of the polyamine analog, N1,N12-bis(ethyl)spermine (BE-3-4-3), to the protein, providing a mechanism for the increase of SSAT activity in response to these agents. Site-directed mutagenesis indicated that a number of residues including arginine 19, cysteine 122, histidine 126, glutamic acid 152, arginine 155, and methionine 167 were needed for protection of SSAT by BE-3-4-3. These residues have previously been shown to reduce the affinity for the binding of polyamines to the SSAT protein, and these results indicate that the change in protein configuration brought about by this binding renders the protein resistant to proteasomal degradation. Mutations to alanines of residues arginine 7, cysteine 14, and lysine 141 also prevented the protection by BE-3-4-3, and these residues may be required for the formation of the protected conformation. The rapid degradation of SSAT required the carboxyl-terminal region of the protein, and the two terminal glutamic acid residues at positions 170 and 171 were found to be of critical importance. Truncation of the protein to remove these residues or the mutation of either of these acidic residues to glutamine completely abolished the rapid degradation of SSAT. The addition of two extra lysine residues at the carboxyl terminus or the conversion of the glutamic acids at positions 170 and 171 to lysines also prevented SSAT degradation by the proteasome. These results show the key role of the acidic residues at the carboxyl terminus of the protein in reacting with the proteasome. In contrast, mutation of lysine 166 to alanine, which extends the length of the acidic region in the carboxyl-terminal fragment of SSAT, actually increased the rate of degradation of SSAT without affecting its stabilization by BE-3-4-3. The binding of BE-3-4-3 or polyamines is therefore likely to change the configuration of the SSAT protein in a way that prevents the exposure of the carboxyl-terminal region of the ubiquitinated protein to the proteasome.

Irreversible inhibition of S-adenosylmethionine decarboxylase of trypanosoma bru cei bru cei by S-adenosylmethionine analogues

S-Adenosylmethionine analogues designed as active-site directed inhibitors were tested in vitro for their effects on S-adenosylmethionine decarboxylase (AdoMetDC) of Trypanosoma brucei brucei. These analogues contained a tertiary nitrogen atom in place of the sulfonium and had a side chain of variable length ending in a reactive group (hydrazino-, aminooxy-, hydrazido- or a methylnitrosourea). The hydrazino- derivatives were the most potent inhibitors with ic 50 values in the range of 40–100 nM. The most active compound ( ic 50 of 0.04 μM) was 5'-deoxy-5'-[(2-hydrazinoethyl)-methylamino] adenosine (MHZEA). Addition of MHZEA produced a time-dependent inactivation with an apparent K i, of 0.4 μM, and the enzyme half-life at a saturating concentration of MHZEA was 0.4 min. Increasing the length of the side chain or changing the methyl group attached to the nitrogen to an ethyl group reduced the potency. Replacement of the hydrazino moiety with an aminooxy group resulted in about a 30- to 35-fold decrease in inhibition potency. However, the relative order of activities of these aminooxy analogues was similar to that found in the hydrazino series with 5'-deoxy-5'-[(2-arninooxyethyl) methylamino]adenosine (MAOEA), which had an ic 50 of 1.3 μM, being the most active. The hydrazido analogs were even less effective with 5'-deoxy-5'-[(3-hydrazino-3-oxopropyl)-methylamino] adenosine, the best inhibitor, having an ic 50, value of 8.7 μM. The methylnitrosourea derivatives were inactive. The inactivation of trypanosomal AdoMetDC with MHZEA or MAOEA was irreversible and was greatly stimulated by putrescine, a known activator of the enzyme, indicating that the compounds bind to the active site and form a covalent bond with the enzyme. These inhibitors may have considerable potential as chemotherapeutic agents against trypanosomiasis and other protozoal infections and may also be useful in studying the role of AdoMetDC in the regulation of polyamine levels in these organisms.

Comparison of S-adenosylmethionine decarboxylases from rat liver and muscle.

S-Adenosylmethionine decarboxylase was purified to homogeneity from rat liver and from rat psoas. The major step involved affinity chromatography on methylglyoxal bis-(guanylhydrazone) linked to Sepharose. The muscle enzyme was more tightly retained to this absorbent, and the enzymes from the two sources could readily be separated by chromatography on this material. The psoas and liver enzymes could not be distinguished by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, both giving a single band corresponding to an Mr of 32 500, but were separated by electrophoresis under nondenaturing conditions and by isoelectric focusing (the isoelectric points were 5.3 for psoas and 5.7 for liver enzyme). The liver and psoas enzymes also differed in respect to Km for S-adenosylmethionine, the degree to which they were activated by putrescine, and their sensitivity to inhibition by methylglyoxal bis(guanylhydrazone) and related compounds. These results indicate that there are two forms of S-adenosylmethionine decarboxylase. The presence of a particular form could, therefore, be important both in the regulation of polyamine levels and also in the pharmacology involving inhibitors of polyamine synthesis.

The relationship between levels and rates of synthesis of polyamines during mammalian cell cycle

The objective of this study was to examine the rate of synthesis and the intracellular levels of polyamines as a function of the HeLa cell cycle. The intracellular levels of ornithine, which were high during mitosis and early G1 phase, decreased rapidly during late G1 phase when the ornithine decarboxylase activity was at its peak. The activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase reached a peak during G1 and decreased rapidly during the S phase. The levels of polyamines were maximum in mitosis and S phase. In constrast, the rate of polyamine synthesis during S phase was 5–10 fold lower than that in mitosis or G1 phase. We have also observed fluctuations in diamine-oxidase activity during the cell cycle. The enzyme activity was high during mitosis and late G1 and low during S phase. Thus, the results of this study suggest an important role for the catabolic enzymes in the regulation of polyamine levels during the mammalian cell cycle.