Polyamine Biosynthetic Enzymes Research Articles

Abstract 7154: Ivospemin/doxorubicin combination modulates polyamine metabolism to improve survival in murine ovarian cancer models

Abstract Polyamines are small polycationic alkylamines that are absolutely required for the continual growth and proliferation of cancer cells. As cancer cells maintain elevated polyamine pools through dysregulated polyamine metabolism, its pharmacological modulation is a promising avenue in cancer therapeutics. The polyamine analogue ivospemin has shown efficacy in slowing pancreatic and ovarian tumor progression in vitro and in vivo and has demonstrated encouraging results in pancreatic cancer clinical trials. Considering nearly 75% of late-stage ovarian cancer patients develop resistant to platinum-based chemotherapies, limiting treatment options, the aim of our study is to determine the potential efficacy of ivospemin in combination with doxorubicin, a common chemotherapeutic used in platinum-resistant ovarian tumors. We have previously shown that ivospemin exposure decreases polyamine content in a variety of cancer cell lines through downregulation of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) and induction of the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT). Here we examine the potential of combining ivospemin with doxorubicin. Ivospemin treatment reduces cell viability in ovarian adenocarcinoma cell lines and increases the toxicity of doxorubicin regardless of cisplatin sensitivity. Cells treated with the combination exhibit a greater decrease in polyamine levels than cells treated with either single agent. This increased polyamine depletion and decreased survival is accomplished through modulation of polyamine metabolism, predominately through an additive induction of SSAT activity. Using the syngeneic VDID8+ ovarian murine model, we further evaluated the ability of ivospemin to improve response to doxorubicin at clinical and sub-clinical dosing. Ascites fluid was used as a marker for tumor burden and evaluated for polyamine content. We found that the combination treatment increases median survival, delays tumor onset, and decreases overall tumor burden compared to either clinical or subclinical doxorubicin dosing schemes. Combination treatment also decreases overall polyamine content in the ascites by 75%. N1-acetylated spermidine is enriched in ascites from combination-treated mice, consistent with an upregulation of SSAT in response to treatment. Recognizing the non-representative mutational status of the VDID8+ model as a limitation, we are currently evaluating the combination of ivospemin and doxorubicin in genetically defined murine models that better recapitulate human high-grade serous ovarian carcinomas. Ongoing studies will determine influences on the tumor microenvironment and will mechanistically evaluate the cooperativity of ivospemin and doxorubicin on pathways outside of polyamine metabolism. Citation Format: Cassandra E. Holbert, Jackson R. Foley, Ashley C. Nwafor, Ting-Ann Liu, Elizabeth Bruckheimer, Jennifer K. Simpson, Tracy Murray Stewart, Robert A. Casero. Ivospemin/doxorubicin combination modulates polyamine metabolism to improve survival in murine ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7154.

Neofunctionalization of S-adenosylmethionine decarboxylase into pyruvoyl-dependent L-ornithine and L-arginine decarboxylases is widespread in bacteria and archaea

S-adenosylmethionine decarboxylase (AdoMetDC/SpeD) is a key polyamine biosynthetic enzyme required for conversion of putrescine to spermidine. Autocatalytic self-processing of the AdoMetDC/SpeD proenzyme generates a pyruvoyl cofactor from an internal serine. Recently, we discovered that diverse bacteriophages encode AdoMetDC/SpeD homologs that lack AdoMetDC activity and instead decarboxylate L-ornithine or L-arginine. We reasoned that neofunctionalized AdoMetDC/SpeD homologs were unlikely to have emerged in bacteriophages and were probably acquired from ancestral bacterial hosts. To test this hypothesis, we sought to identify candidate AdoMetDC/SpeD homologs encoding L-ornithine and L-arginine decarboxylases in bacteria and archaea. We searched for the anomalous presence of AdoMetDC/SpeD homologs in the absence of its obligatory partner enzyme spermidine synthase, or the presence of two AdoMetDC/SpeD homologs encoded in the same genome. Biochemical characterization of candidate neofunctionalized genes confirmed lack of AdoMetDC activity, and functional presence of L-ornithine or L-arginine decarboxylase activity in proteins from phyla Actinomycetota, Armatimonadota, Planctomycetota, Melainabacteria, Perigrinibacteria, Atribacteria, Chloroflexota, Sumerlaeota, Omnitrophota, Lentisphaerota, and Euryarchaeota, the bacterial candidate phyla radiation and DPANN archaea, and the δ-Proteobacteria class. Phylogenetic analysis indicated that L-arginine decarboxylases emerged at least three times from AdoMetDC/SpeD, whereas L-ornithine decarboxylases arose only once, potentially from the AdoMetDC/SpeD-derived L-arginine decarboxylases, revealing unsuspected polyamine metabolic plasticity. Horizontal transfer of the neofunctionalized genes appears to be the more prevalent mode of dissemination. We identified fusion proteins of bona fide AdoMetDC/SpeD with homologous L-ornithine decarboxylases that possess two, unprecedented internal protein-derived pyruvoyl cofactors. These fusion proteins suggest a plausible model for the evolution of the eukaryotic AdoMetDC.

N-Carbamoylputrescine Amidohydrolase of Bacteroides thetaiotaomicron, a Dominant Species of the Human Gut Microbiota

Polyamines are bioactive amines that play a variety of roles, such as promoting cell proliferation and protein synthesis, and the intestinal lumen contains up to several mM polyamines derived from the gut microbiota. In the present study, we conducted genetic and biochemical analyses of the polyamine biosynthetic enzyme N-carbamoylputrescine amidohydrolase (NCPAH) that converts N-carbamoylputrescine to putrescine, a precursor of spermidine in Bacteroides thetaiotaomicron, which is one of the most dominant species in the human gut microbiota. First, ncpah gene deletion and complemented strains were generated, and the intracellular polyamines of these strains cultured in a polyamine-free minimal medium were analyzed using high-performance liquid chromatography. The results showed that spermidine detected in the parental and complemented strains was depleted in the gene deletion strain. Next, purified NCPAH-(His)6 was analyzed for enzymatic activity and found to be capable of converting N-carbamoylputrescine to putrescine, with a Michaelis constant (Km) and turnover number (kcat) of 730 µM and 0.8 s-1, respectively. Furthermore, the NCPAH activity was strongly (>80%) inhibited by agmatine and spermidine, and moderately (≈50%) inhibited by putrescine. This feedback inhibition regulates the reaction catalyzed by NCPAH and may play a role in intracellular polyamine homeostasis in B. thetaiotaomicron.

Abstract 295: Targeting spermine synthase triggers lipid metabolism reprogramming as a new therapeutic option to combat colorectal cancer

Abstract Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC). Our recent work demonstrates that spermine synthase (SMS), a polyamine biosynthetic enzyme that converts spermidine to spermine, is overexpressed in CRC, which is required for balancing cellular spermidine levels to facilitate CRC tumorigenesis (Nat Commun 11:3243, 2020). Our findings reveal SMS as an attractive therapeutic target in CRC; yet, genetic depletion of SMS expression only shows a limited antitumor effect. Using unbiased metabolomics and transcriptomics analyses, we identified a lipid metabolism reprograming as among the most impacted metabolic change by SMS depletion in CRC cells. Specifically, SMS depletion significantly altered long-chain fatty acid, triacylglycerol and phospholipid metabolism. Furthermore, targeted inhibition of SMS significantly increased the number of lipid droplets and the levels of long-chain fatty acid acylcarnitines for oxidative phosphorylation in mitochondria, and upregulated expression of genes associated with increased mobilization of polyunsaturated fatty acids and the genes associated with lipid peroxidation for induction of ferroptosis, an iron-dependent form of nonapoptotic cell death. The glutathione peroxidase 4 (GPX4) is a key negative regulator of ferroptosis by neutralizing lipid peroxides. Notably, pharmacological inhibition of GPX4 or its upstream regulator system xc − in combination with genetic depletion or pharmacologic inhibition of SMS synergistically caused lipid peroxidation leading to ferroptosis induction and marked suppression of CRC cell growth. Collectively, these findings highlight lipid metabolism reprograming as an adaptive response to targeted inhibition of SMS to enable CRC cell survival, which represents an Achilles’ heel that can be exploited for potential effective therapy for CRC. Citation Format: Murong Ma, Yubin Guo, Qing Ye, Pan Deng, Daheng He, Chi Wang, Qing-Bai She. Targeting spermine synthase triggers lipid metabolism reprogramming as a new therapeutic option to combat colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 295.

Abstract 4944: Evaluating the efficacy of spermine analogue ivospemin (SBP-101) in combination with chemotherapy in ovarian cancer

Abstract Polyamines are small cationic alkylamines that play critical roles in essential cellular processes governing growth and proliferation. As such, cancers are fully reliant on increased polyamine pools maintained through dysregulation of polyamine metabolism. Pharmaceutical modulation of polyamine metabolism is a promising avenue in cancer therapeutics and has been attempted with enzyme inhibitors, including DFMO (difluoromethylornithine), and polyamine analogues. Ivospemin is a spermine analogue that has shown efficacy in slowing pancreatic and ovarian tumor progression both in vitro and in vivo and demonstrated encouraging results in pancreatic cancer clinical trials. We have shown that ivospemin decreases polyamine content through depression of the activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) in a variety of cancer cell lines. Treatment of the VDID8+ murine ovarian cancer model with ivospemin resulted in a marked increase in survival. Here we examine the potential of combining ivospemin and chemotherapeutic agents that are used to treat cisplatin-resistant ovarian cancer. Treatment with gemcitabine, topotecan, and doxorubicin increased the in vitro toxicity of ivospemin, while paclitaxel and docetaxel did not have any added benefit over ivospemin alone. Using the VDID8+ model, we further evaluated the efficacy of ivospemin in combination with gemcitabine, topotecan, and doxorubicin in vivo. Ascites fluid was used as a marker of tumor burden and evaluated for polyamine content. Addition of ivospemin improved the survival of mice treated with any of the three chemotherapeutics. The ivospemin and doxorubicin combination mice had the greatest median survival time; this combination is being further evaluated in mechanistic studies and additional murine studies. Ovarian cancers have extremely immunosuppressive tumor microenvironments (TME) and metabolic reprogramming of the TME to reduce immunosuppressive phenotypes is a promising approach for treatment. Sustained elevation of polyamine levels supports an immunosuppressive TME, and evidence suggests that pharmacologic depletion of polyamines may reduce immunosuppressive phenotypes. DFMO treatment in the immunosuppressive VDID8+ model influences the immune cells of the TME, and we therefore are investigating the combination of ivospemin and DFMO in ovarian cancer. In addition to the cooperativity of ivospemin and chemotherapeutic agents, we have observed a cooperative antiproliferative response in ovarian cancer cells following DFMO and ivospemin cotreatment. Together, these studies suggest the potential of polyamine modulation by ivospemin and DFMO in combination with standard of care chemotherapy. Future studies will determine influences on the immune microenvironment and will evaluate cooperativity between ivospemin, DFMO, and chemotherapy. Citation Format: Cassandra E. Holbert, Jackson R. Foley, Tracy Murray Stewart, Michael J. Walker, Elizabeth Bruckheimer, Jennifer K. Simpson, Robert A. Casero. Evaluating the efficacy of spermine analogue ivospemin (SBP-101) in combination with chemotherapy in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4944.

The humidity level matters during the desiccation of Norway spruce somatic embryos.

In Norway spruce, as in many other conifers, the germination capacity of somatic embryos is strongly influenced by the desiccation phase inserted after maturation. The intensity of drying during desiccation eminently affected the formation of emblings (i.e., seedlings developed from somatic embryos). Compared to non-desiccated embryos, the germination capacity of embryos desiccated at 100% relative humidity was about three times higher, but the reduction of relative humidity to 95 and 90% had a negative effect on the subsequent embryo development. The water loss observed in these embryos did not lead to an increase in lipid peroxidation, as shown by malondialdehyde levels. Another metabolic pathway in plants that mediates a response to abiotic stresses is directed toward the biosynthesis of polyamines (PAs). The activities of PA biosynthetic enzymes increased steadily in embryos during desiccation at 100% relative humidity, whereas they decreased at lower humidity. The total content of free PAs in the embryos gradually decreased throughout desiccation. The increase in free putrescine (Put) and perchloric acid-insoluble Put conjugates was observed in embryos desiccated at lower humidity. These changes were accompanied to some extent by the transcription of the genes for the PA biosynthesis enzymes. Desiccation at 100% relative humidity increased the activity of the cell wall-modifying enzymes β-1,3-glucanases and chitinases; the activities of these enzymes were also significantly suppressed at reduced humidity. The same pattern was observed in the transcription of some β-1,3-glucanase and chitinase genes. Desiccation treatments triggered metabolic processes that responded to water availability, suggesting an active response of the embryo to the reduction in humidity. A positive effect was demonstrated only for desiccation at high relative humidity. Some of the physiological characteristics described can be used as markers of inappropriate relative humidity during somatic embryo desiccation.

Altered Brain Arginine Metabolism and Polyamine System in a P301S Tauopathy Mouse Model: A Time-Course Study.

Altered arginine metabolism (including the polyamine system) has recently been implicated in the pathogenesis of tauopathies, characterised by hyperphosphorylated and aggregated microtubule-associated protein tau (MAPT) accumulation in the brain. The present study, for the first time, systematically determined the time-course of arginine metabolism changes in the MAPT P301S (PS19) mouse brain at 2, 4, 6, 8 and 12 months of age. The polyamines putrescine, spermidine and spermine are critically involved in microtubule assembly and stabilization. This study, therefore, further investigated how polyamine biosynthetic and catabolic enzymes changed in PS19 mice. There were general age-dependent increases of L-arginine, L-ornithine, putrescine and spermidine in the PS19 brain (particularly in the hippocampus and parahippocampal region). While this profile change clearly indicates a shift of arginine metabolism to favor polyamine production (a polyamine stress response), spermine levels were decreased or unchanged due to the upregulation of polyamine retro-conversion pathways. Our results further implicate altered arginine metabolism (particularly the polyamine system) in the pathogenesis of tauopathies. Given the role of the polyamines in microtubule assembly and stabilization, future research is required to understand the functional significance of the polyamine stress response and explore the preventive and/or therapeutic opportunities for tauopathies by targeting the polyamine system.

Diabetic Triple Negative Breast Cancer: Polyamine Enzymes, Ornithine decarboxylase and Spermine oxidase as Potential Targets

Breast cancer (BC) is the second leading cause of death in women. Co‐existence of diabetes and BC, termed diabetic BC, can be lethal, leading to higher mortality (15‐40%) than BC alone. In triple negative breast cancer (TNBC) patients, chance of TNBC recurrence is about 3 months, and chance of mortality can be as high as 75%. TNBC is highly refractive, heterogenous, and resistant to most chemotherapies. Polyamines, which are ubiquitous in most cells, and play a role in cell growth, have been shown to be elevated in cancer, though their role in diabetic BC has not been explored. We hypothesized that high glucose/diabetic conditions increase TNBC cell proliferation through modification of the enzymes (ornithine decarboxylase and spermine oxidase) in the polyamine pathway. The objective was to determine potential targets in the polyamine pathway to mitigate TNBC advancement in diabetic conditions. For this purpose, MDA‐MB‐231 (TNBC) and MCF10‐A (normal breast epithelial) cell lines were used. Both were treated with low (5mM) and high glucose (25mM) concentrations and effects on polyamine levels, polyamine enzyme, and cell proliferation were monitored. Polyamine levels were assayed using reverse phase high performance liquid chromatography. Cell proliferation was assessed using a fluorescence‐based assay, and polyamine enzyme expression was monitored at the mRNA and protein level using RT‐PCR and Western Blots. Samples were sent to Johns Hopkins University for polyamine enzyme activity analysis. Further, an approved inhibitor of polyamine synthesis, alpha‐difluoromethylornithine (a‐DFMO, 5mM) was used in combination with glucose treatments. Under diabetic/high glucose conditions, putrescine levels were elevated in MDA‐MB‐231 cells (135%), which correlated with higher cell proliferation (123%). Polyamine enzyme activity of ODC increased (from ~75 pMolCO2/hr/mg protein to ~150 pMolCO2/hr/mg protein) and SMOX decreased (from ~0.4 pMolH202/min/mg protein to ~0.06 pMolH202/min/mg protein) in concert with changes observed at protein levels with high glucose exposure. DFMO was effective in reducing polyamine levels and cell proliferation, however re‐supplementation of polyamines (spermidine and spermine) restored cell proliferation to that, indicating that DFMO is only cytostatic. MCF‐10A cells showed marked elevation in cell proliferation with high glucose. DFMO prevented this increase; however no apparent change was observed in polyamine levels. The polyamine biosynthetic enzymes (ornithine decarboxylase and spermine oxidase) play an active role in increasing cell proliferation under high glucose conditions in TNBC. Since DFMO caused only cytostatic effects, there is a need for a more targeted approach to regulate polyamine levels and mitigate TNBC growth under diabetic conditions.

Polyamine Metabolism in Leishmania Parasites: A Promising Therapeutic Target.

Parasites of the genus Leishmania cause a variety of devastating and often fatal diseases in humans and domestic animals worldwide. The need for new therapeutic strategies is urgent because no vaccine is available, and treatment options are limited due to a lack of specificity and the emergence of drug resistance. Polyamines are metabolites that play a central role in rapidly proliferating cells, and recent studies have highlighted their critical nature in Leishmania. Numerous studies using a variety of inhibitors as well as gene deletion mutants have elucidated the pathway and routes of transport, revealing unique aspects of polyamine metabolism in Leishmania parasites. These studies have also shed light on the significance of polyamines for parasite proliferation, infectivity, and host–parasite interactions. This comprehensive review article focuses on the main polyamine biosynthetic enzymes: ornithine decarboxylase, S-adenosylmethionine decarboxylase, and spermidine synthase, and it emphasizes recent discoveries that advance these enzymes as potential therapeutic targets against Leishmania parasites.

Exogenous application of spermidine mitigates the adverse effects of drought stress in faba bean (

In Tunisia, drought stress is a major environmental factor limiting crop production and causing relatively low and unstable faba bean yields. In the present study, we explored the putative role of spermidine (0.5, 1, 1.5 and 2mM) in ameliorating the effects of drought stress induced by polyethylene glycol (PEG-6000, -0.58MPa) in faba bean seedlings. Drought stress reduced photosynthetic performance, chlorophyll and relative water content in leaves of faba bean variety Badii. Moreover, drought increased proline, electrolyte leakage and malondialdehyde content by inducing reactive oxygen species (hydrogen peroxide) generation in leaves. However, applying spermidine increased the activities of catalase, superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase. The results show that the application of spermidine especially at a rate of 1.5mM effectively reduces oxidative damage and alleviates negative effects caused by drought stress. In addition, exogenous spermidine increased the expression of polyamine biosynthetic enzymes' genes (VfADC , VfSAMDC and VfSPDS ), and reduced the expression of VfSPMS suggesting that exogenous spermidine can regulate polyamines' metabolic status under drought challenge, and consequently may enhance drought stress tolerance in faba bean. Real-time quantitative polymerase chain reaction analysis revealed that some drought responsive genes (VfNAC , VfHSP , VfNCED , VfLEA , VfCAT , VfAPX , VfRD22 , VfMYB , VfDHN , VfERF , VfSOD and VfWRKY ) from various metabolic pathways were differentially expressed under drought stress. Overall, these genes were more abundantly transcribed in the spermidine-treated plants compared to untreated suggesting an important role of spermidine in modulating faba bean drought stress response and tolerance.

Characterizing the homeostatic regulation of the polyamine pathway using the Drosophila melanogaster model system

Polyamines are small, naturally occurring polycations that are essential for normal cell growth and development. Maintaining a homeostatic level of both the polyamine pathway enzymes and the levels of the polyamines putrescine, spermidine and spermine is important because the dysregulation of the polyamine pathway has been associated with physical anomalies including cancer, Parkinson's disease, Snyder-Robinson syndrome and aging in mammals. To date, little is understood regarding how the polyamine pathway maintains polyamine homeostasis in the fly, an emerging model for polyamine studies. The aim of these studies was to better characterize how the downregulation or ablation of the polyamine biosynthetic enzymes ornithine decarboxylase, spermidine synthase and spermine synthase influence the expression of the genes coding for other polyamine pathway enzymes and the levels of the polyamines themselves utilizing the model organism, Drosophila melanogaster. We demonstrate the complexities of the polyamine metabolic pathway and show that downregulation of any of these polyamine pathway enzymes can lead to an array of changes in other parts of the pathway as well as alter the cellular levels of polyamines themselves. These data highlight the complex nature of regulation of this pathway and underscore the need to better understand its self-regulation in this and other model systems.

Cereblon harnesses Myc-dependent bioenergetics and activity of CD8+ T lymphocytes

AbstractImmunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known “neosubstrates,” such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.

Spermine synthase and MYC cooperate to maintain colorectal cancer cell survival by repressing Bim expression

Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC), but the underlying mechanism is incompletely characterized. Here, we report that spermine synthase (SMS), a polyamine biosynthetic enzyme, is overexpressed in CRC. Targeted disruption of SMS in CRC cells results in spermidine accumulation, which inhibits FOXO3a acetylation and allows subsequent translocation to the nucleus to transcriptionally induce expression of the proapoptotic protein Bim. However, this induction is blunted by MYC-driven expression of miR-19a and miR-19b that repress Bim production. Pharmacological or genetic inhibition of MYC activity in SMS-depleted CRC cells dramatically induces Bim expression and apoptosis and causes tumor regression, but these effects are profoundly attenuated by silencing Bim. These findings uncover a key survival signal in CRC through convergent repression of Bim expression by distinct SMS- and MYC-mediated signaling pathways. Thus, combined inhibition of SMS and MYC signaling may be an effective therapy for CRC.

Inhibition of the polyamine synthesis enzyme ornithine decarboxylase sensitizes triple-negative breast cancer cells to cytotoxic chemotherapy

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

Understanding the Cellular Functions Polyamines Play in Proliferation, Replication, and Survival of Leishmania donovani

Leishmaniasis, a neglected tropical disease, affects an estimated billion people worldwide. A better understanding of parasite biology is vital for the development of much needed new therapeutic strategies. Our research addresses this critical need by focusing on the role of polyamines for growth and survival of Leishmania parasites. The main polyamine biosynthetic enzymes in Leishmania are ornithine decarboxylase (ODC) and spermidine synthase (SPDSYN), which sequentially convert ornithine to the polyamines, putrescine and spermidine. With generated gene deletion mutants, Δodc and Δspdsyn, we have demonstrated in vitro that the metabolite putrescine is essential beyond its function as the precursor for spermidine formation. Additionally, we observed that putrescine‐depleted Δodc parasites immediately ceased proliferation and died within two weeks. In contrast, putrescine‐rich Δspdsyn mutants showed an intermediate growth phenotype and entered a quiescent‐like state with cell death occurring after six weeks. Furthermore, putrescine‐depleted cells were unable to synthesize DNA and stopped replicating. We also observed two hallmarks of programmed cell death in the putrescine‐depleted cells: cell‐rounding and phosphatidylserine exposure. To reduce the variability associated with working with two cell lines and to further understand the functions polyamines play, we are in the process of generating a double gene deletion mutant, ΔodcΔspdsyn.Support or Funding InformationPacific University School of Pharmacy Research Incentive Grant

Ornithine capture by a translating ribosome controls bacterial polyamine synthesis.

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation, and microbial virulence1–3. In order to survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryo-EM structure of an Escherichia coli (E. coli) ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor RF1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

Chloroviruses.

Chloroviruses are large dsDNA, plaque-forming viruses that infect certain chlorella-like green algae; the algae are normally mutualistic endosymbionts of protists and metazoans and are often referred to as zoochlorellae. The viruses are ubiquitous in inland aqueous environments throughout the world and occasionally single types reach titers of thousands of plaque-forming units per ml of native water. The viruses are icosahedral in shape with a spike structure located at one of the vertices. They contain an internal membrane that is required for infectivity. The viral genomes are 290 to 370 kb in size, which encode up to 16 tRNAs and 330 to ~415 proteins, including many not previously seen in viruses. Examples include genes encoding DNA restriction and modification enzymes, hyaluronan and chitin biosynthetic enzymes, polyamine biosynthetic enzymes, ion channel and transport proteins, and enzymes involved in the glycan synthesis of the virus major capsid glycoproteins. The proteins encoded by many of these viruses are often the smallest or among the smallest proteins of their class. Consequently, some of the viral proteins are the subject of intensive biochemical and structural investigation.

A polyamine-independent role for S-adenosylmethionine decarboxylase.

The only known function of S-adenosylmethionine decarboxylase (AdoMetDC) is to supply, with its partner aminopropyltransferase enzymes such as spermidine synthase (SpdSyn), the aminopropyl donor for polyamine biosynthesis. Polyamine spermidine is probably essential for the growth of all eukaryotes, most archaea and many bacteria. Two classes of AdoMetDC exist, the prokaryotic class 1a and 1b forms, and the eukaryotic class 2 enzyme, which is derived from an ancient fusion of two prokaryotic class 1b genes. Herein, we show that 'eukaryotic' class 2 AdoMetDCs are found in bacteria and are enzymatically functional. However, the bacterial AdoMetDC class 2 genes are phylogenetically limited and were likely acquired from a eukaryotic source via transdomain horizontal gene transfer, consistent with the class 2 form of AdoMetDC being a eukaryotic invention. We found that some class 2 and thousands of class 1b AdoMetDC homologues are present in bacterial genomes that also encode a gene fusion of an N-terminal membrane protein of the Major Facilitator Superfamily (MFS) class of transporters and a C-terminal SpdSyn-like domain. Although these AdoMetDCs are enzymatically functional, spermidine is absent, and an entire fusion protein or its SpdSyn-like domain only, does not biochemically complement a SpdSyn deletion strain of E. coli This suggests that the fusion protein aminopropylates a substrate other than putrescine, and has a role outside of polyamine biosynthesis. Another integral membrane protein found clustered with these genes is DUF350, which is also found in other gene clusters containing a homologue of the glutathionylspermidine synthetase family and occasionally other polyamine biosynthetic enzymes.

Nitric oxide modulates polyamine homeostasis in sunflower seedling cotyledons under salt stress

ABSTRACTFree polyamine (PA) titers in plants may be regulated through reversible conjugate formation and/or through modulation of their synthesis, transport and degradation. PA signaling involves the well-acknowledged signaling molecule, nitric oxide (NO), which functions in diverse biological processes. Present investigations demonstrate the influence of salt stress (120 mM NaCl) and exogenous NO donor (250 µM Diethylenetriamine, DETA) on PA homeostasis of 2 d old, etiolated sunflower (Helianthus annuus L.) seedling cotyledons as a long-distance signaling response. Significantly enhanced intracellular spermine (Spm) accumulation was observed in seedling cotyledons under salt stress and in response to NO donor, the increase being more pronounced in seedlings treated with NO, evidently as a result of upregulation of the PA biosynthetic enzymes – arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) – as revealed by Western blot and confocal imaging (CLSM). Moreover, salt stress induced the activity of polyamine oxidase (PAO), a PA catabolic enzyme, while NO lowered its activity in salt-stressed seedling cotyledons. NO, thus, appears to assist the seedlings in adapting to salt stress by positively regulating PA homeostasis through regulation of PA distribution between free, conjugated and bound forms, increased accumulation of PA biosynthetic enzymes and lowering the rate of PA catabolism.

P53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis.

Cancer cells exhibit altered and usually increased metabolic processes to meet their high biogenetic demands1,2. Under these conditions, ammonia is concomitantly produced by the increased metabolic processing. However, it is unclear how tumour cells dispose of excess ammonia and what outcomes might be caused by the accumulation of ammonia. Here we report that the tumour suppressor p53, the most frequently mutated gene in human tumours, regulates ammonia metabolism by repressing the urea cycle. Through transcriptional downregulation of CPS1, OTC and ARG1, p53 suppresses ureagenesis and elimination of ammonia in vitro and in vivo, leading to the inhibition of tumour growth. Conversely, downregulation of these genes reciprocally activates p53 by MDM2-mediated mechanism(s). Furthermore, the accumulation of ammonia causes a significant decline in mRNA translation of the polyamine biosynthetic rate-limiting enzyme ODC, thereby inhibiting the biosynthesis of polyamine and cell proliferation. Together, these findings link p53 to ureagenesis and ammonia metabolism, and further reveal a role for ammonia in controlling polyamine biosynthesis and cell proliferation.