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Dynein function and protein clearance changes in tumor cells induced by a Kunitz-type molecule, Amblyomin-X.

Pacheco MT, Berra CM, Morais KL, Sciani JM, Branco VG, Bosch RV, Chudzinski-Tavassi AM - PLoS ONE (2014)

Bottom Line: Therefore, our study describes a Kunitz-type protein that acts on the proteasome to trigger distinct intracellular events compared to classic known proteasome inhibitors that are small-cell-permeable molecules.In investigating the experiments and literature on Amblyomin-X and the known proteasome inhibitors, we also found differences in the structures of the molecules, intracellular events, dynein involvement and tumor cell type effects.These findings also reveal a possible new target for Amblyomin-X, i.e., dynein, and may serve as a tool for investigating tumor cell death associated with proteasome inhibition.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, Brazil.

ABSTRACT
Amblyomin-X is a Kunitz-type recombinant protein identified from the transcriptome of the salivary glands of the tick Amblyomma cajennense and has anti-coagulant and antitumoral activity. The supposed primary target of this molecule is the proteasome system. Herein, we elucidated intracellular events that are triggered by Amblyomin-X treatment in an attempt to provide new insight into how this serine protease inhibitor, acting on the proteasome, could be comparable with known proteasome inhibitors. The collective results showed aggresome formation after proteasome inhibition that appeared to occur via the non-exclusive ubiquitin pathway. Additionally, Amblyomin-X increased the expression of various chains of the molecular motor dynein in tumor cells, modulated specific ubiquitin linkage signaling and inhibited autophagy activation by modulating mTOR, LC3 and AMBRA1 with probable dynein involvement. Interestingly, one possible role for dynein in the mechanism of action of Amblyomin-X was in the apoptotic response and its crosstalk with autophagy, which involved the factor Bim; however, we observed no changes in the apoptotic response related to dynein in the experiments performed. The characteristics shared among Amblyomin-X and known proteasome inhibitors included NF-κB blockage and nascent polypeptide-dependent aggresome formation. Therefore, our study describes a Kunitz-type protein that acts on the proteasome to trigger distinct intracellular events compared to classic known proteasome inhibitors that are small-cell-permeable molecules. In investigating the experiments and literature on Amblyomin-X and the known proteasome inhibitors, we also found differences in the structures of the molecules, intracellular events, dynein involvement and tumor cell type effects. These findings also reveal a possible new target for Amblyomin-X, i.e., dynein, and may serve as a tool for investigating tumor cell death associated with proteasome inhibition.

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Aggresome formation induced by Amblyomin-X.Cultured cells were treated with vehicle (PBS), 0.5 µM Ambly for 24 h, 5 µM MG-132 for 24 h, 3.5 µM CHX for 2 h or 0.5 µM Ambly for 24 h after pretreatment with 3.5 µM CHX for 2 h (CHX/Ambly). (A) Fluorescence microscopy analysis of aggresomes. Aggresomes were labeled with a commercial kit in red and nuclei were stained with Hoechst 33342 in blue. Images are representative of five fields from each experiment (n = 3). (B) Mean fluorescence intensity obtained from histograms of flow cytometry analysis of aggresomes. Results are reported as the means ± standard error of agressome propensity factor (APF) in arbitrary units calculated according to the manufacturer's instructions. Three independent experiments were performed. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant). (C) Cell viability assay of tumor cells treated with the compounds used in the aggresome analysis. Results are reported as the means ± standard error of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).
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pone-0111907-g003: Aggresome formation induced by Amblyomin-X.Cultured cells were treated with vehicle (PBS), 0.5 µM Ambly for 24 h, 5 µM MG-132 for 24 h, 3.5 µM CHX for 2 h or 0.5 µM Ambly for 24 h after pretreatment with 3.5 µM CHX for 2 h (CHX/Ambly). (A) Fluorescence microscopy analysis of aggresomes. Aggresomes were labeled with a commercial kit in red and nuclei were stained with Hoechst 33342 in blue. Images are representative of five fields from each experiment (n = 3). (B) Mean fluorescence intensity obtained from histograms of flow cytometry analysis of aggresomes. Results are reported as the means ± standard error of agressome propensity factor (APF) in arbitrary units calculated according to the manufacturer's instructions. Three independent experiments were performed. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant). (C) Cell viability assay of tumor cells treated with the compounds used in the aggresome analysis. Results are reported as the means ± standard error of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).

Mentions: Our data showed aggresome formation after proteasome inhibition induced by the recombinant protein treatment in both tumor cells lines (Fig. 3A and 3B). Aggresome formation was also blocked when the cells were pretreated with CHX (Fig. 3A and 3B). Cell viability was decreased in both tumor cell lines only when treated with Amblyomin-X alone or with MG-132 (Fig. 3C).


Dynein function and protein clearance changes in tumor cells induced by a Kunitz-type molecule, Amblyomin-X.

Pacheco MT, Berra CM, Morais KL, Sciani JM, Branco VG, Bosch RV, Chudzinski-Tavassi AM - PLoS ONE (2014)

Aggresome formation induced by Amblyomin-X.Cultured cells were treated with vehicle (PBS), 0.5 µM Ambly for 24 h, 5 µM MG-132 for 24 h, 3.5 µM CHX for 2 h or 0.5 µM Ambly for 24 h after pretreatment with 3.5 µM CHX for 2 h (CHX/Ambly). (A) Fluorescence microscopy analysis of aggresomes. Aggresomes were labeled with a commercial kit in red and nuclei were stained with Hoechst 33342 in blue. Images are representative of five fields from each experiment (n = 3). (B) Mean fluorescence intensity obtained from histograms of flow cytometry analysis of aggresomes. Results are reported as the means ± standard error of agressome propensity factor (APF) in arbitrary units calculated according to the manufacturer's instructions. Three independent experiments were performed. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant). (C) Cell viability assay of tumor cells treated with the compounds used in the aggresome analysis. Results are reported as the means ± standard error of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4257547&req=5

pone-0111907-g003: Aggresome formation induced by Amblyomin-X.Cultured cells were treated with vehicle (PBS), 0.5 µM Ambly for 24 h, 5 µM MG-132 for 24 h, 3.5 µM CHX for 2 h or 0.5 µM Ambly for 24 h after pretreatment with 3.5 µM CHX for 2 h (CHX/Ambly). (A) Fluorescence microscopy analysis of aggresomes. Aggresomes were labeled with a commercial kit in red and nuclei were stained with Hoechst 33342 in blue. Images are representative of five fields from each experiment (n = 3). (B) Mean fluorescence intensity obtained from histograms of flow cytometry analysis of aggresomes. Results are reported as the means ± standard error of agressome propensity factor (APF) in arbitrary units calculated according to the manufacturer's instructions. Three independent experiments were performed. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant). (C) Cell viability assay of tumor cells treated with the compounds used in the aggresome analysis. Results are reported as the means ± standard error of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).
Mentions: Our data showed aggresome formation after proteasome inhibition induced by the recombinant protein treatment in both tumor cells lines (Fig. 3A and 3B). Aggresome formation was also blocked when the cells were pretreated with CHX (Fig. 3A and 3B). Cell viability was decreased in both tumor cell lines only when treated with Amblyomin-X alone or with MG-132 (Fig. 3C).

Bottom Line: Therefore, our study describes a Kunitz-type protein that acts on the proteasome to trigger distinct intracellular events compared to classic known proteasome inhibitors that are small-cell-permeable molecules.In investigating the experiments and literature on Amblyomin-X and the known proteasome inhibitors, we also found differences in the structures of the molecules, intracellular events, dynein involvement and tumor cell type effects.These findings also reveal a possible new target for Amblyomin-X, i.e., dynein, and may serve as a tool for investigating tumor cell death associated with proteasome inhibition.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, Brazil.

ABSTRACT
Amblyomin-X is a Kunitz-type recombinant protein identified from the transcriptome of the salivary glands of the tick Amblyomma cajennense and has anti-coagulant and antitumoral activity. The supposed primary target of this molecule is the proteasome system. Herein, we elucidated intracellular events that are triggered by Amblyomin-X treatment in an attempt to provide new insight into how this serine protease inhibitor, acting on the proteasome, could be comparable with known proteasome inhibitors. The collective results showed aggresome formation after proteasome inhibition that appeared to occur via the non-exclusive ubiquitin pathway. Additionally, Amblyomin-X increased the expression of various chains of the molecular motor dynein in tumor cells, modulated specific ubiquitin linkage signaling and inhibited autophagy activation by modulating mTOR, LC3 and AMBRA1 with probable dynein involvement. Interestingly, one possible role for dynein in the mechanism of action of Amblyomin-X was in the apoptotic response and its crosstalk with autophagy, which involved the factor Bim; however, we observed no changes in the apoptotic response related to dynein in the experiments performed. The characteristics shared among Amblyomin-X and known proteasome inhibitors included NF-κB blockage and nascent polypeptide-dependent aggresome formation. Therefore, our study describes a Kunitz-type protein that acts on the proteasome to trigger distinct intracellular events compared to classic known proteasome inhibitors that are small-cell-permeable molecules. In investigating the experiments and literature on Amblyomin-X and the known proteasome inhibitors, we also found differences in the structures of the molecules, intracellular events, dynein involvement and tumor cell type effects. These findings also reveal a possible new target for Amblyomin-X, i.e., dynein, and may serve as a tool for investigating tumor cell death associated with proteasome inhibition.

Show MeSH
Related in: MedlinePlus