bims-polyam 2020-05-24 papers

J Biol Chem. 2020 May 19. pii: jbc.RA120.013867. [Epub ahead of print]

Autophagy induction by exogenous polyamines is an artifact of bovine serum amine oxidase activity in culture serum.

Cassandra E Holbert, Matthew Dunworth, Jackson R Foley, Tiffany T Dunston, Tracy Murray Stewart, Robert A Casero.

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including proliferation, nucleic acid synthesis, apoptosis, and protection from oxidative damage. It has been proposed that in addition to these functions, elevated levels of polyamines promote longevity in various biological systems, including yeast, drosophila, and murine models. A series of in vitro mechanistic studies by multiple investigators has led to the conclusion that addition of exogenous spermidine promotes longevity through autophagy induction; however, these experiments were confounded by the use of mammalian cell culture systems supplemented with fetal bovine serum (FBS). Using cell viability assays, LC3B immunoblots, and live-cell fluorescence microscopy, we report here that in the presence of ruminant serum, exogenously added polyamines are quickly oxidized by the copper-containing bovine serum amine oxidase (BSAO). This polyamine oxidation resulted in the production of harmful byproducts including hydrogen peroxide, ammonia, and reactive aldehydes. Our data demonstrate that it is critically important to prevent confounding BSAO-induced cytotoxicity in mechanistic studies of the roles of polyamines in autophagy.

Keywords: amine oxidase; autophagy; beta-oxidation; bovine serum amine oxidase; cytotoxicity; hydrogen peroxide; oxidative stress; polyamine; spermidine; spermine

DOI: https://doi.org/10.1074/jbc.RA120.013867

Nucleic Acids Res. 2020 May 20. pii: gkaa340. [Epub ahead of print]

Dengue virus targets RBM10 deregulating host cell splicing and innate immune response.

Berta Pozzi, Laureano Bragado, Pablo Mammi, María Florencia Torti, Nicolás Gaioli, Leopoldo G Gebhard, Martín E García Solá, Rita Vaz-Drago, Néstor G Iglesias, Cybele C García, Andrea V Gamarnik, Anabella Srebrow.

RNA-seq experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.

DOI: https://doi.org/10.1093/nar/gkaa340

Phys Chem Chem Phys. 2020 May 20.

Polyamines stimulate RecA-mediated recombination by condensing duplex DNA and stabilizing intermediates.

Naciye Esma Tirtom, Yang Hsu, Hung-Wen Li.

Polyamines are naturally occurring cationic molecules in cells. In addition to their roles in modulating gene expression and cell proliferation, they have been shown to stimulate DNA recombination. The molecular mechanism for stimulation is not clear. We utilized single-molecule tethered particle motion (TPM) experiments to investigate how polyamines stimulate RecA-mediated recombination. We showed that natural polyamines, spermine and spermidine, condense duplex DNA, but with different efficiencies. While ∼300 μM of spermine condenses 50% of duplex DNA, 2.0 mM of spermidine is required to achieve the same level of condensation. The condensation takes place in a stepwise manner, and is reversible upon removal of polyamines. We also showed that addition of polyamines stimulates the duplex capture activity of RecA filament and stabilizes the intermediates with longer dwell time. Through condensing duplex DNA and stabilizing the complex of RecA filaments and duplex DNA, polyamines stimulate the formation of functional intermediates by ∼20-fold, and promote recombination progression.

DOI: https://doi.org/10.1039/d0cp01061h

Acta Biochim Pol. 2020 May 21.

Antioxidative action of polyamines in protection of phospholipid membranes exposed to ozone stress.

Elżbieta Rudolphi-Szydło, Maria Filek, Barbara Dyba, Zbigniew Miszalski, Maria Zembala.

In the present work, Langmuir monolayers were used to study the interaction of putrescine (a cationic antioxidant) with anionic charged membranes (1,2-dioleoyl-sn-glycerol-3-phosphate) under oxidative stress caused by the presence of ozone in the water phase. Calcium ions and acidic environment were used to compare the electrostatic and antioxidant effects of putrescine with those of an inorganic cation. It has been shown that the main role of putrescine in protecting systems against oxidation is its rapid reaction with ROS. The initial rate of ROS neutralization rose as the concentration of putrescine increased. No such reaction was observed for calcium ions. The consequence of putrescine's ozone removal was lesser lipid destruction that depended on the pH conditions.

DOI: https://doi.org/10.18388/abp.2020_5230