Effects of polyamines on protein synthesis and growth of Escherichia coli

Polyamines and indole are small regulatory molecules that are involved in the adaptation to stress in bacteria, including the regulation of gene expression. Genes, the translation of which is under the regulatory effects of polyamines, form the polyamine modulon. Previously, we showed that polyamines upregulated the transcription of genes encoding the ribosome hibernation factors RMF, RaiA, SRA, EttA and RsfS in Escherichia coli. At the same time, indole affected the expression at the transcriptional level of only the raiA and rmf genes. Ribosome hibernation factors reversibly inhibit translation under stress conditions, including exposure to antibiotics, to avoid resource waste and to conserve ribosomes for a quick restoration of their functions when favorable conditions occur. In this work, we have studied the influence of indole on the expression of the raiA and rmf genes at the translational level and regulatory effects of the polyamines putrescine, cadaverine and spermidine on the translation of the rmf, raiA, sra, ettA and rsfS genes. We have analyzed the mRNA primary structures of the studied genes and the predicted mRNA secondary structures obtained by using the RNAfold program for the availability of polyamine modulon features. We have found that all of the studied genes contain specific features typical of the polyamine modulon. Furthermore, to investigate the influence of polyamines and indole on the translation of the studied genes, we have constructed the translational reporter lacZ-fusions by using the pRS552/λRS45 system. According to the results obtained, polyamines upregulated the expression of the rmf, raiA and sra genes, the highest expression of which was observed at the stationary phase, but did not affect the translation of the ettA and rsfS genes, the highest expression of which took place during the exponential phase. The stimulatory effects were polyamine-specific and observed at the stationary phase, when bacteria are under multiple stresses. In addition, the data obtained demonstrated that indole significantly inhibited translation of the raiA and rmf genes, despite the stimulatory effect on their transcrip- tion. This can suggest the activity of a posttranscriptional regulatory mechanism of indole on gene expression.

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified in a polyamine-requiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate instead of 0.4% glucose as an energy source. Under these conditions, enhancement of cell growth by polyamines was almost the same as that in the presence of 0.4% glucose. It was found that synthesis of RpoN, Cra, and H-NS was enhanced by polyamines at the level of translation at the early logarithmic phase of growth (A(540) of 0.15). The effects of polyamines on synthesis of RpoN, H-NS, and Cra were due to the existence of unusual Shine-Dalgarno sequences (RpoN and H-NS) and an inefficient GUG initiation codon (Cra) in their mRNAs. Thus, rpoN, cra, and hns genes were identified as new members of the polyamine modulon. Because most of the polyamine modulon genes thus far identified encode transcription factors (RpoS [sigma(38)], Cya, FecI [sigma(18)], Fis, RpoN [sigma(54)], Cra, and H-NS), DNA microarray analysis of mRNA expressed in cells was performed. At the early logarithmic phase of growth, a total of 97 species of mRNAs that were up-regulated by polyamines more than twofold were under the control of seven polyamine modulon genes mentioned above.

Polyamine metabolism is essential for cancer cell growth, with enzymes like ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) playing key roles in polyamine (PA) biosynthesis. These polyamines (putrescine, spermidine, and spermine) regulate vital cellular processes, including DNA replication, protein synthesis, and cell cycle progression. Dysregulated polyamine metabolism is common in cancer, making ODC and AdoMetDC attractive therapeutic targets. This review highlights polyamines' role in cancer and explores combination therapies targeting polyamine metabolism and critical signaling pathways for improved clinical outcomes. A comprehensive analysis of both historical and recent literature on polyamine metabolism in cancer was performed using PubMed, which provides access to over 37 million citations from biomedical literature. Expression data for key polyamine biosynthetic enzymes, ODC and AdoMetDC, were obtained from the UALCAN portal - an interactive web resource for the analysis of cancer OMICS data. The IUPAC names of drugs and inhibitors targeting the polyamine pathway were retrieved from the PubChem database and used to generate molecular structures using the BIOVIA Draw 2025 program. Additionally, the ClinicalTrials.gov database was explored to identify ongoing and completed clinical research studies, as well as to gather detailed information on therapeutic agents targeting polyamine metabolism. Aberrant polyamine metabolism in cancer is driven by oncogenic pathways like MYC, Akt, and mTOR. MYC upregulates ODC1, promoting polyamine dysregulation. Defects in enzymes such as MTA phosphorylase (MTAP) enhance cancer cell sensitivity to inhibitors of purine/pyrimidine synthesis and the ubiquitin-proteasome pathway, suggesting alternative therapeutic strategies. Therapeutic strategies combining polyamine biosynthesis inhibition with targeting nucleotide synthesis or proteasome function have shown synergistic potential. However, the dual nature of polyamines - supporting both, tumor growth and ferroptotic cell death - poses a therapeutic challenge. Balancing these effects is key to designing effective interventions. Advancing this field requires not only selective inhibitors but also a deeper understanding of context-dependent polyamine functions in tumor biology. Developing more potent inhibitors with improved drug-like properties is crucial for advancing polyamine- targeted therapies and positioning this field at the forefront of cancer research.

Effect of polyamines on polypeptide synthesis in rat liver cell-free system

Polyamines (putrescine, spermidine, and spermine) are essential for normal cell growth. The polyamine level in cells is regulated by biosynthesis, degradation, and transport. The role of antizyme on polyamine biosynthesis and transport in mammalian cells and characteristics of polyamine transport in Escherichia coli and yeast are described briefly in this review. In addition, the effects of polyamines on protein synthesis and the NMDA receptor are outlined. Finally, the correlation between acrolein produced from polyamines by polyamine oxidase and chronic renal failure and brain stroke is summarized. Increased levels of polyamine oxidase and acrolein are good markers of chronic renal failure and brain stroke.

In Vivo X-Ray Footprinting of Pre-30S Ribosomes Reveals Chaperone-Dependent Remodeling of Late Assembly Intermediates

This chapter deals with the effects of polyamines on the translation machinery, and more specifically on the structure and function of ribosomal particles mainly in bacteria. It describes effects of several antibiotics on protein synthesis of wild-type bacteria or polyamine-deficient mutants. In order to demonstrate the involvement of polyamines in the equilibrium occurring between ribosomal particles and in the formation or assembly of ribosomal subunits, several experiments were performed with Escherichia coli mutants blocked in the biosynthesis of polyamines and grown in the absence and presence of these substances. The analyses of ribosomal profiles in cell extracts obtained after slow cooling of growing cultures have shown that polyamine-starved bacteria contained a low amount of 70S monomers with most of the ribosomal particles accumulated as subunits. The addition of polyamines might presumably contribute to stabilize the U-A base pair in the recognition of the asparagine codon AAU involved, with a concomitant increase of fidelity.

The polyamines putrescine (1,4-diaminobutane), spermidine (1,8-diamino-4-azaoctane, 2), and spermine (1,12-diamino-4,9-diazadodecane, 3) (Fig. 1) are ubiquitous polycationic compounds that are found in significant amounts in nearly every prokaryotic and eukaryotic cell type. Spermidine and spermine primarily exist in aqueous solution at pH 7.4 as fully protonated polycations and possess the pKa values indicated in Fig. 1 (). This high degree of positive charge is an important factor in the biological functions of these molecules, and, as will be discussed later in this chapter, alterations in the pKa of polyamine nitrogens can affect and disrupt their cellular function. Polyamines are widely distributed in nature and are known to be required in micromolar to millimolar concentrations to support a wide variety of cellular functions. However, data that establish the precise role of the polyamines and their analogs in cellular processes are incomplete. The ongoing identification of new functions for the polyamines ensures that new avenues for research are arising continuously in an extremely diverse set of disciplines. The human and mammalian pathways for polyamine metabolism have been extensively studied, and analogous pathways have been elucidated for a relatively small number of organisms. There are important interspecies differences in polyamine metabolism, especially among eukaryotic cells, plants, and some bacteria and protozoa. In some prokaryotes, only putrescine and spermidine are synthesized, whereas in other cases, such as certain thermophilic bacteria, polyamines with chains longer than spermine are found. In some parasitic organisms, there are additional enzymes that are not present in the host cell, and, as such, provide a target for the design of specific antiparasitic agents. The enzymes involved in human and mammalian polyamine metabolism are reasonably similar, and inhibitors targeted to these enzymes rely on the observation that polyamine metabolism is accelerated, and polyamines are required in higher quantities, in target cell types. The diversity of biological research in the polyamine field is the subject of an excellent book (2). Keeping in mind the diverse nature of polyamine distribution and function, it is reasonable to assume that carefully designed polyamine analogs could have the potential to disrupt polyamine metabolism, and thus such agents have been investigated as potential therapeutic agents in vitro and in vivo. The polyamine pathway represents an important target for chemotherapeutic intervention because depletion of polyamines results in the disruption of a variety of cellular functions and may, in specific cases, result in cytotoxicity (,). This chapter will summarize the development of synthetic derivatives of the polyamines, and describe their use as potential chemotherapeutic agents. A comprehensive review of polyamine biosynthesis inhibitors () and a review of the role of polyamines in normal and tumor cell metabolism () have recently been published.

Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5' domain of the 16S rRNA. In vitro, transformation of initial S4-rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4-rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein-RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4-RNA interactions guide rRNA folding and impact late steps of 30S assembly.

Effect of polyamines on perfused rat heart contractility

Voltage-dependent inhibition of thermo-TRPV channels by intracellular spermine.

Effects of polyamines on tyrosine hydroxylase activity in adrenals

Mitochondria from Vigna sinensis (L.) Savi cv. Pitiuba contain the polyamines spermine, spermidine, and putrescine. The membrane-bound F1-ATPase from mitochondria of Vigna sinensis is activated by these polyamines at physiological concentrations. The effect of polyamines on the membrane-bound of F1-ATPase is dependent on the concentrations of Na+, K+, MgATP, and Mg2+. Excess Na+ or K+ prevents the activation of the membrane-bound F1-ATPase by spermine and spermidine, but not by putrescine. The most pronounced effects were observed at low MgATP concentrations in the absence of Na+ and K+. At [MgATP] = 0.08 mM, spermine activation of the membrane-bound F1-ATPase was 130%. The membrane-bound F1-ATPase is slightly activated by Mg2+ at lower concentrations and strongly inhibited by Mg2+ at higher concentrations. Activation as well as inhibition is dependent on the substrate MgATP concentration. Although there is competition between Mg2+ and MgATP, the binding sites for these two ligands are different (pseudocompetitive inhibition). The inhibition of the membrane-bound F1-ATPase can be reversed by polyamines. There is evidence that the binding sites for Mg2+ and polyamines are identical. The F1-ATPase detached from the membrane is neither activated by polyamines nor inhibited by Mg2+. Therefore, the binding sites for Mg2+ and polyamines seem to be localized on the membrane.

The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of human embryonic kidney (HEK) 293T cells with an adenovirus encoding a key polyamine catabolic enzyme, spermidine N¹-acetyltransferase 1 (SSAT1)/SAT1 (AdSAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/propidium iodide FACS analyses, terminal uridine nucleotide end-labelling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1-transduced cells (at 24-72 h), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in the polyamine-depleted cells occurs by the mitochondrial intrinsic pathway, as evidenced by loss of mitochondrial membrane potential, increase in pro-apoptotic Bax, decrease in anti-apoptotic Bcl-xl, Bcl2 and Mcl-1 and release of cytochrome c from mitochondria, upon transduction with AdSAT1. Moreover, TEM images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial alteration, vacuolization and membrane blebbing. The apoptosis appears to result largely from depletion of the polyamines spermidine and spermine, as the polyamine analogues α-methylspermidine (α-MeSpd) and N¹,N¹²-dimethylspermine (Me₂Spm) that are not substrates for SAT1 could partially restore growth and prevent apoptosis of AdSAT1-transduced cells. Inhibition of polyamine oxidases did not restore the growth of AdSAT1-transduced cells or block apoptosis, suggesting that the growth arrest and apoptosis were not induced by oxidative stress resulting from accelerated polyamine catabolism. Taken together, these data provide strong evidence that the depletion of the polyamines spermidine and spermine leads to mitochondria-mediated apoptosis.

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.