bims-polyam 2019-05-05 papers

on Polyamines

Issue of 2019–05–05
seven papers selected by
Sebastian J. Hofer, University of Graz and Alexander Ivanov, Engelhardt Institute of Molecular Biology

Polyamine depletion inhibits bunyavirus infection via generation of noninfectious interfering virions.
Coxsackievirus B3 Responds to Polyamine Depletion via Enhancement of 2A and 3C Protease Activity.
Synthesis and in vitro antitumor activity of novel acylspermidine derivative N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide (AAHD) against HepG2 cells.
Effects of spermidine supplementation on cognition and biomarkers in older adults with subjective cognitive decline (SmartAge)-study protocol for a randomized controlled trial.
Metabolic perturbations caused by depletion of nephronophthisis factor Anks6 in mIMCD3 cells.
Amino acid derangements in adults with severe falciparum malaria.
Evidence by GC-MS that lysine is an arginase-catalyzed metabolite of homoarginine in vitro and in vivo in humans.

J Virol. 2019 May 01. pii: JVI.00530-19. [Epub ahead of print]

Polyamine depletion inhibits bunyavirus infection via generation of noninfectious interfering virions.

Vincent Mastrodomenico, Jeremy J Esin, Marion L Graham, Patrick M Tate, Grant M Hawkins, Zachary J Sandler, David J Rademacher, Thomas M Kicmal, Courtney N Dial, Bryan C Mounce.

Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated polyamines' role in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, Coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as vaccine strategies.IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively-charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.

DOI: https://doi.org/10.1128/JVI.00530-19
Viruses. 2019 Apr 30. pii: E403. [Epub ahead of print]11(5):

Coxsackievirus B3 Responds to Polyamine Depletion via Enhancement of 2A and 3C Protease Activity.

Courtney N Dial, Patrick M Tate, Thomas M Kicmal, Bryan C Mounce.

  Polyamines are small positively-charged molecules abundant in eukaryotic cells that are crucial to RNA virus replication. In eukaryotic cells, polyamines facilitate processes such as transcription, translation, and DNA replication, and viruses similarly rely on polyamines to facilitate transcription and translation. Whether polyamines function at additional stages in viral replication remains poorly understood. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to polyamine depletion both in vitro and in vivo; however, precisely how polyamine function in picornavirus infection has not been described. Here, we describe CVB3 mutants that arise with passage in polyamine-depleted conditions. We observe mutations in the 2A and 3C proteases, and we find that these mutant proteases confer resistance to polyamine depletion. Using a split luciferase reporter system to measure protease activity, we determined that polyamines facilitate viral protease activity. We further observe that the 2A and 3C protease mutations enhance reporter protease activity in polyamine-depleted conditions. Finally, we find that these mutations promote cleavage of cellular eIF4G during infection of polyamine-depleted cells. In sum, our results suggest that polyamines are crucial to protease function during picornavirus infection. Further, these data highlight viral proteases as potential antiviral targets and highlight how CVB3 may overcome polyamine-depleting antiviral therapies.

Keywords:  Coxsackievirus B3; polyamines; protease

DOI:  https://doi.org/10.3390/v11050403
Bioorg Chem. 2019 Apr 16. pii: S0045-2068(19)30001-X. [Epub ahead of print]88 102937

Synthesis and in vitro antitumor activity of novel acylspermidine derivative N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide (AAHD) against HepG2 cells.

Abdulrahman L Al-Malki, Syed Shoeb Razvi, Furkhan Ahmed Mohammed, Mazin A Zamzami, Hani Choudhry, Taha A Kumosani, Khadijah S Balamash, Fawzia A Alshubaily, Shareefa A ALGhamdi, Khalid O Abualnaja, Wesam H Abdulaal, Mustafa A Zeyadi, Maryam H Al-Zahrani, Mahmoud Alhosin, Tadao Asami, Said S Moselhy.

  Naturally occurring polyamines like Putrescine, Spermidine, and Spermine are polycations which bind to the DNA, hence stabilizing it and promoting the essential cellular processes. Many synthetic polyamine analogues have been synthesized in the past few years, which have shown cytotoxic effects on different tumours. In the present study, we evaluated the antiproliferative effect of a novel, acylspermidine derivative, (N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide) (AAHD) on HepG2 cells. Fluorescence staining was performed with nuclear stain (Hoechst 33342) and acridine orange/ethidium bromide double staining. Dose and the time-dependent antiproliferative effect were observed by WST-1 assays, and radical scavenging activity was measured by ROS. Morphological changes such as cell shrinkage & blebbing were analyzed by fluorescent microscopy. It was found that AAHD markedly suppressed the growth of HepG2 cells in a dose- and time-dependent manner. It was also noted that the modulation of ROS levels confirmed the radical scavenging activity. In the near future, AAHD can be a promising drug candidate in chalking out a neoplastic strategy to control the proliferation of tumour cells. This study indicated that AAHD induced anti-proliferative and pro-apoptotic activities on HCC. Since AAHD was active at micromolar concentrations without any adverse effects on the healthy cells (Fibroblasts), it is worthy of further clinical investigations.

Keywords:  Anticancer; Antiproliferative; HepG2; Hepatocellular carcinoma; Polyamine analogues; ROS

DOI:  https://doi.org/10.1016/j.bioorg.2019.102937
Alzheimers Res Ther. 2019 May 01. 11(1): 36

Effects of spermidine supplementation on cognition and biomarkers in older adults with subjective cognitive decline (SmartAge)-study protocol for a randomized controlled trial.

Miranka Wirth, Claudia Schwarz, Gloria Benson, Nora Horn, Ralph Buchert, Catharina Lange, Theresa Köbe, Stefan Hetzer, Marta Maglione, Eva Michael, Stefanie Märschenz, Knut Mai, Ute Kopp, Dietmar Schmitz, Ulrike Grittner, Stephan J Sigrist, Slaven Stekovic, Frank Madeo, Agnes Flöel.

   BACKGROUND: Given the global increase in the aging population and age-related diseases, the promotion of healthy aging is one of the most crucial public health issues. This trial aims to contribute to the establishment of effective approaches to promote cognitive and brain health in older individuals with subjective cognitive decline (SCD). Presence of SCD is known to increase the risk of objective cognitive decline and progression to dementia due to Alzheimer's disease. Therefore, it is our primary goal to determine whether spermidine supplementation has a positive impact on memory performance in this at-risk group, as compared with placebo. The secondary goal is to examine the effects of spermidine intake on other neuropsychological, behavioral, and physiological parameters.
METHODS: The SmartAge trial is a monocentric, randomized, double-blind, placebo-controlled phase IIb trial. The study will investigate 12 months of intervention with spermidine-based nutritional supplementation (target intervention) compared with 12 months of placebo intake (control intervention). We plan to recruit 100 cognitively normal older individuals with SCD from memory clinics, neurologists and general practitioners in private practice, and the general population. Participants will be allocated to one of the two study arms using blockwise randomization stratified by age and sex with a 1:1 allocation ratio. The primary outcome is the change in memory performance between baseline and post-intervention visits (12 months after baseline). Secondary outcomes include the change in memory performance from baseline to follow-up assessment (18 months after baseline), as well as changes in neurocognitive, behavioral, and physiological parameters (including blood and neuroimaging biomarkers), assessed at baseline and post-intervention.
DISCUSSION: The SmartAge trial aims to provide evidence of the impact of spermidine supplementation on memory performance in older individuals with SCD. In addition, we will identify possible neurophysiological mechanisms of action underlying the anticipated cognitive benefits. Overall, this trial will contribute to the establishment of nutrition intervention in the prevention of Alzheimer's disease.
TRIAL REGISTRATION: ClinicalTrials.gov, NCT03094546 . Registered 29 March 2017-retrospectively registered.
PROTOCOL VERSION: Based on EA1/250/16 version 1.5.

Keywords:  Aging; Autophagy; Dietary supplementation; Health; Memory; Nutrition; Polyamines

DOI:  https://doi.org/10.1186/s13195-019-0484-1
Metabolomics. 2019 Apr 30. 15(5): 71

Metabolic perturbations caused by depletion of nephronophthisis factor Anks6 in mIMCD3 cells.

Manuel Schlimpert, Simon Lagies, Barbara Müller, Vadym Budnyk, Kelly Daryll Blanz, Gerd Walz, Bernd Kammerer.

   INTRODUCTION: Nephronophthisis (NPH) is an inherited form of cystic kidney disease with various extrarenal manifestations accounting for the largest amount of endstage renal disease in childhood. Patient mutations of Anks6 have also been found to cause NPH like phenotypes in animal models. However, little is known about functionality of Anks6.
OBJECTIVES/METHODS: We investigated the impact of Anks6 depletion on cellular metabolism of inner medullary collecting duct cells by GC-MS profiling and targeted LC-MS/MS analysis using two different shRNA cell lines for tetracycline-inducible Anks6 downregulation, namely mIMCD3 krab shANKS6 i52 and mIMCD3 krab shANKS6 i12.
RESULTS: In combination, we could successfully identify 158 metabolites of which 20 compounds showed similar alterations in both knockdown systems. Especially, large neutral amino acids, such as phenylalanine, where found to be significantly downregulated indicating disturbances in amino acid metabolism. Arginine, lysine and spermidine, which play an important role in cell survival and proliferation, were found to be downregulated. Accordingly, cell growth was diminished in tet treated mIMCD3 krab shANKS6 i52 knockdown cells. Deoxynucleosides were significantly downregulated in both knockdown systems. Hence, PARP1 levels were increased in tet treated mIMCD3 krab shANKS6 i52 cells, but not in tet treated mIMCD3 krab shANKS6 i12 cells. However, yH2AX was found to be increased in the latter.
CONCLUSION: In combination, we hypothesise that Anks6 affects DNA damage responses and proliferation and plays a crucial role in physiological amino acid and purine/pyrimidine metabolism.

Keywords:  Anks6; Cystic kidney disease; GC–MS; LC–MS/MS; Metabolomics; Nephronophthisis; SamCystin

DOI:  https://doi.org/10.1007/s11306-019-1535-0
Sci Rep. 2019 Apr 29. 9(1): 6602

Amino acid derangements in adults with severe falciparum malaria.

Stije J Leopold, Siribha Apinan, Aniruddha Ghose, Hugh W Kingston, Katherine A Plewes, Amir Hossain, Asok Kumar Dutta, Sujat Paul, Anupam Barua, Abdus Sattar, Nicholas P J Day, Joel Tarning, Markus Winterberg, Nicholas J White, Arjen M Dondorp.

Amino acid derangements are common in severe falciparum malaria and have been associated with endothelial dysfunction (L-arginine), metabolic acidosis (alanine and lactate), and disease severity (phenylalanine and tryptophan metabolites). Whether these amino acid perturbations reflect isolated pathogenic mechanisms or if they are part of overall changes in amino acid metabolism is unclear. To investigate this, we prospectively simultaneously quantified a broad range of plasma free amino acids (PFAA) using HPLC-MRM-Mass spectrometry in relation to presenting symptoms in adults with severe malaria (n = 88), septicaemia (n = 88), uncomplicated malaria (n = 71), and healthy controls (n = 48) from Bangladesh. The total plasma concentration of measured amino acids was significantly reduced in each of the patient groups when compared to normal levels observed in healthy local controls: uncomplicated malaria -54%, severe malaria -23%, and sepsis -32%, (p = <0.001). Inspection of amino acid profiles revealed that in each group the majority of amino acids were below normal levels, except for phenylalanine. Among patients with severe malaria, L-lactate was strongly associated with an increase of the total amino acid concentration, likely because this reflects tissue hypoxia. Our data confirm previously described amino acid abnormalities, likely resulting from overall changes in the concentration of PFAA.

DOI: https://doi.org/10.1038/s41598-019-43044-6
Anal Biochem. 2019 Apr 24. pii: S0003-2697(19)30280-5. [Epub ahead of print]577 59-66

Evidence by GC-MS that lysine is an arginase-catalyzed metabolite of homoarginine in vitro and in vivo in humans.

Alexander Bollenbach, Kathrin Cordts, Erik Hanff, Dorothee Atzler, Chi-Un Choe, Edzard Schwedhelm, Dimitrios Tsikas.

  l-Homoarginine (hArg) is biosynthesized from l-arginine (Arg) and l-lysine (Lys) by arginine:glycine amidinotransferase (AGAT). AGAT also catalyzes the formation of guanidinoacetate (GAA) from Arg and glycine (Gly). GAA is converted to creatine (N-methyl guanidinoacetate) by guanidinoacetate N-methyl-transferase (GAMT). Low circulating and excretory concentrations of hArg are associated with worse cardiovascular outcome and mortality. hArg is a poor substrate of nitric oxide synthase (NOS) and a weak inhibitor of arginase. The metabolism of hArg in humans is little investigated. Previously, we found that orally administered hArg (125 mg/day) increased the plasma concentration of hArg, but not of Arg, the substrate of NOS, in healthy subjects. We newly analyzed the plasma samples collected in that study for Lys and other amino acids. Repeated measures ANOVA revealed statistically significant differences between the groups (P = 0.008) with respect to plasma Lys concentration which increased by about 8% after a 4-week hArg supplementation. In vitro, recombinant human arginase and bovine liver arginase I were demonstrated by a specific and sensitive stable-isotope GC-MS assay to hydrolyze hArg to Lys. Our results suggest that Lys is a metabolite of hArg produced by the hydrolytic activity of arginase. Arginase may play a key role in hArg homeostasis in humans.

Keywords:  Arginase; L‐Arginine; N(G)-Hydroxy-l-arginine; Nitric oxide; l‐Homoarginine

DOI:  https://doi.org/10.1016/j.ab.2019.04.019