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Pseudoalteromonas haloplanktis TAC125 produces 4-hydroxybenzoic acid that induces pyroptosis in human A459 lung adenocarcinoma cells.

ABSTRACT: In order to exploit the rich reservoir of marine cold-adapted bacteria as a source of bioactive metabolites, ethyl acetate crude extracts of thirteen polar marine bacteria were tested for their antiproliferative activity on A549 lung epithelial cancer cells. The crude extract from Pseudoalteromonas haloplanktis TAC125 was the most active in inhibiting cell proliferation. Extensive bioassay-guided purification and mass spectrometric characterization allowed the identification of 4-hydroxybenzoic acid (4-HBA) as the molecule responsible for this bioactivity. We further demonstrate that 4-HBA inhibits A549 cancer cell proliferation with an IC50 value???1??g?ml-1, and that the effect is specific, since the other two HBA isomers (i.e. 2-HBA and 3-HBA) were unable to inhibit cell proliferation. The effect of 4-HBA is also selective since treatment of normal lung epithelial cells (WI-38) with 4-HBA did not affect cell viability. Finally, we show that 4-HBA is able to activate, at the gene and protein levels, a specific cell death signaling pathway named pyroptosis. Accordingly, the treatment of A549 cells with 4-HBA induces the transcription of (amongst others) caspase-1, IL1?, and IL18 encoding genes. Studies needed for the elucidation of mode of action of 4-HBA will be instrumental in depicting novel details of pyroptosis.

SUBMITTER: Sannino F

PROVIDER: S-EPMC5775203 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Pseudoalteromonas haloplanktis TAC125 produces 4-hydroxybenzoic acid that induces pyroptosis in human A459 lung adenocarcinoma cells.

Sannino Filomena F Sansone Clementina C Galasso Christian C Kildgaard Sara S Tedesco Pietro P Fani Renato R Marino Gennaro G de Pascale Donatella D Ianora Adrianna A Parrilli Ermenegilda E Larsen Thomas Ostenfeld TO Romano Giovanna G Tutino Maria Luisa ML

Scientific reports 20180119 1

In order to exploit the rich reservoir of marine cold-adapted bacteria as a source of bioactive metabolites, ethyl acetate crude extracts of thirteen polar marine bacteria were tested for their antiproliferative activity on A549 lung epithelial cancer cells. The crude extract from Pseudoalteromonas haloplanktis TAC125 was the most active in inhibiting cell proliferation. Extensive bioassay-guided purification and mass spectrometric characterization allowed the identification of 4-hydroxybenzoic ...[more]

PMID: 29352134

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Project description:BackgroundA significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues.ResultsUsing a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology.ConclusionsCollectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural-functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce "intractable" proteins, especially those containing large intrinsically disordered regions.

Dataset Information

Pseudoalteromonas haloplanktis TAC125 produces 4-hydroxybenzoic acid that induces pyroptosis in human A459 lung adenocarcinoma cells.

Publications

Pseudoalteromonas haloplanktis TAC125 produces 4-hydroxybenzoic acid that induces pyroptosis in human A459 lung adenocarcinoma cells.

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