Identification of a non-classical three-dimensional nuclear localization signal in the intestinal fatty acid binding protein

The intestinal fatty acid binding protein (FABP) is a small protein expressed along the small intestine that bind long-chain fatty acids and other hydrophobic ligands. Several lines of evidence suggest that, once in the nucleus, it interacts with nuclear receptors, activating them and thus transferring the bound ligand into the nucleus. Previous work by our group suggests that FABP2 would participate in the cytoplasm-nucleus translocation of fatty acids. Because the consensus NLS is absent in the sequence of FABP2, we propose that a 3D signal could be responsible for its nuclear translocation. The results obtained by transfection assays of recombinant wild type and mutated forms of Danio rerio Fabp2 in Caco-2 cell cultures, showed that lysine 17, arginine 29 and lysine 30 residues, which are located in the helix-turn-helix region, would constitute a functional non-classical three-dimensional NL

Chemosensitizer effect of violacein on cisplatin-treated bladder cancer cells

Background: Bladder cancer is the tenth most common cancer worldwide. Considering its high prevalence (vul- nerability to multiple recurrences and progression despite local therapy), which leads to a substantial health service burden, it becomes necessary to develop new strategies to increase the effectiveness of bladder tumor therapy. Natural compounds with antiproliferative effect on cancer cells could be a good choice for co-adjuvant chemotherapy. Microorganisms are one of the main sources for natural compounds. Pigments extracted from the cold-adapted microorganisms can contribute to the development of a broader range of applications in biotechnology. Violacein is a purple pigment commonly produced by many bacterial strains. We have previously shown that very low concentrations of violacein extracted from Janthinobacterium sp. produced an antiproliferative effect on HeLa cells.Objective: With the aim to determine if violacein has an antiproliferative activity on bladder cancer cells, as well as to test if it has synergistic effects on cisplatin treated cells in vitro, T24 and 253J cell lines (derived bladder cancer cells from carcinoma in situ and retroperitoneal metastasis, respectively) were exposed to different concentrations of violacein in the presence or absence of cisplatin. Methods: i) Resazurin assay and flow cytometry were performed in two bladder cancer-derived cell lines, namely T24 and 253J, to see if violacein affects cell viability and induce cell death. ii) To find out whether violacein sensitizes bladder cancer cells to cisplatin, the drug interaction among different doses of cisplatin and violacein was analyzed, as well their combination index was determined. iii) The effect of violacein to induce primary genetic damage was determined through the analysis of induced micronuclei frequency and H2AX foci, as well as performing the comet assay. Results: The half-maximal inhibitory concentration of violacein at 24 h for both cell lines were around 500 nM, and decreased below 400 nM in combination with 10 μM of cisplatin, indicating antiproliferative and sensitizing effects of violacein to cisplatin in both cell lines tested. A clear cell cycle delay, as well as an increase in the percentage of cell death was observed by flow cytometry at 300 nM of violacein, either alone or in combination with cisplatin. On the other hand, the analysis of the micronucleus frequency did not evidence an increase in genetic damage. Moreover, in combined treatments with cisplatin there was a slight decrease on micronucleus induction. Besides, the induction of genetic damage was not observed through comet assay when cells were treated with violacein alone, however, when cells were treated with violacein in the presence of cisplatin (10 μM). The production of genetic damage was diminished in T24 or 253J cells. By the same token, increase in the frequency of H2AX foci by violacein was not observed at any tested dose in both cell lines. Conclusion: It was shown that violacein has an in vitro antiproliferative effect in bladder cancer cell lines, sensitizing them to cisplatin. Interestingly, at doses tested, violacein did not induce genotoxicity and reduce the genotoxic effect produced by cisplatin

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC12 but not BODIPY-FLC5 to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC12 to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC12 was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity

Neuroprotective effects of violacein in a model of inherited amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.IIBCE (MEC), PEDECIBA y ANII

La célula de Schwann como fuente alternativa de los ARNs axonales

Tribunal: Dr. Angel Caputti, Dra. Beatriz Garat, Dra. Silvia Chifle

Identification of a non-classical three-dimensional nuclear localization signal in the intestinal fatty acid binding protein.

The intestinal fatty acid binding protein (FABP) is a small protein expressed along the small intestine that bind long-chain fatty acids and other hydrophobic ligands. Several lines of evidence suggest that, once in the nucleus, it interacts with nuclear receptors, activating them and thus transferring the bound ligand into the nucleus. Previous work by our group suggests that FABP2 would participate in the cytoplasm-nucleus translocation of fatty acids. Because the consensus NLS is absent in the sequence of FABP2, we propose that a 3D signal could be responsible for its nuclear translocation. The results obtained by transfection assays of recombinant wild type and mutated forms of Danio rerio Fabp2 in Caco-2 cell cultures, showed that lysine 17, arginine 29 and lysine 30 residues, which are located in the helix-turn-helix region, would constitute a functional non-classical three-dimensional NLS

Association of microtubules and axonal RNA transferred from myelinating Schwann cells in rat sciatic nerve.

Transference of RNAs and ribosomes from Schwann cell-to-axon was demonstrated in normal and regenerating peripheral nerves. Previously, we have shown that RNAs transfer is dependent on F-actin cytoskeleton and Myosin Va. Here, we explored the contribution of microtubules to newly synthesized RNAs transport from Schwann cell nuclei up to nodal microvilli in sciatic nerves. Results using immunohistochemistry and quantitative confocal FRET analysis indicate that Schwann cell-derived RNAs co-localize with microtubules in Schwann cell cytoplasm. Additionally, transport of Schwann cell-derived RNAs is nocodazole and colchicine sensitive demonstrating its dependence on microtubule network integrity. Moreover, mRNAs codifying neuron-specific proteins are among Schwann cell newly synthesized RNAs population, and some of them are associated with KIF1B and KIF5B microtubules-based motors

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration