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Functional characterization of a BCL10 isoform in the rainbow trout Oncorhynchus mykiss.

Mazzone P, Scudiero I, Coccia E, Ferravante A, Paolucci M, D'Andrea EL, Varricchio E, Pizzulo M, Reale C, Zotti T, Vito P, Stilo R - FEBS Open Bio (2015)

Bottom Line: Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20.Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein.This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

View Article: PubMed Central - PubMed

Affiliation: Biogem, Via Camporeale, Ariano Irpino (AV), Italy.

ABSTRACT
The complexes formed by BCL10, MALT1 and members of the family of CARMA proteins have recently been the focus of much attention because they represent a key mechanism for regulating activation of the transcription factor NF-κB. Here, we report the functional characterization of a novel isoform of BCL10 in the trout Oncorhynchus mykiss, which we named tBCL10. tBCL10 dimerizes, binds to components of the CBM complex and forms cytoplasmic filaments. Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20. Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein. This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

No MeSH data available.


tBCL10 replaces hBCL10 (A) Cell lysates from HEK293 cells infected with retroviruses encoding for shRNAs targeting hBCL10 were monitored for hBCL10 expression by immunoblot assay. (B) NF-κB-driven luciferase activity in HEK-293 cells silenced for hBCL10 and stimulated with PMA. (C) NF-κB-driven luciferase activity in HEK-293 cells silenced for hBCL10 and transfected with tBCL10. Data shown represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate.
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f0030: tBCL10 replaces hBCL10 (A) Cell lysates from HEK293 cells infected with retroviruses encoding for shRNAs targeting hBCL10 were monitored for hBCL10 expression by immunoblot assay. (B) NF-κB-driven luciferase activity in HEK-293 cells silenced for hBCL10 and stimulated with PMA. (C) NF-κB-driven luciferase activity in HEK-293 cells silenced for hBCL10 and transfected with tBCL10. Data shown represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate.

Mentions: To exclude the possibility that NF-κB activation mediated by tBCL10 was due to its interaction and subsequent oligomerization of hBCL10, we abolished expression of hBCL10 in the human cell line HEK293 through retrovirus-mediated expression of short hairpin RNAs (shRNA) targeting hBCL10. As shown in Fig. 6A, introduction of hBCL10sh#3 and hBCL10sh#5 in HEK293 cells results in a great reduction of BCL10 expression. Depletion of hBCL10 in these cells abrogates their ability to activate NF-κB following exposure to phorbol-12-myristate-13-acetate (PMA) (Fig. 6B). However, introduction of tBCL10 in these hBCL10-depleted cells fully recovers their ability to activate NF-κB (Fig. 6C). Thus, tBCL10 can functionally replace hBCL10.


Functional characterization of a BCL10 isoform in the rainbow trout Oncorhynchus mykiss.

Mazzone P, Scudiero I, Coccia E, Ferravante A, Paolucci M, D'Andrea EL, Varricchio E, Pizzulo M, Reale C, Zotti T, Vito P, Stilo R - FEBS Open Bio (2015)

tBCL10 replaces hBCL10 (A) Cell lysates from HEK293 cells infected with retroviruses encoding for shRNAs targeting hBCL10 were monitored for hBCL10 expression by immunoblot assay. (B) NF-κB-driven luciferase activity in HEK-293 cells silenced for hBCL10 and stimulated with PMA. (C) NF-κB-driven luciferase activity in HEK-293 cells silenced for hBCL10 and transfected with tBCL10. Data shown represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372615&req=5

f0030: tBCL10 replaces hBCL10 (A) Cell lysates from HEK293 cells infected with retroviruses encoding for shRNAs targeting hBCL10 were monitored for hBCL10 expression by immunoblot assay. (B) NF-κB-driven luciferase activity in HEK-293 cells silenced for hBCL10 and stimulated with PMA. (C) NF-κB-driven luciferase activity in HEK-293 cells silenced for hBCL10 and transfected with tBCL10. Data shown represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate.
Mentions: To exclude the possibility that NF-κB activation mediated by tBCL10 was due to its interaction and subsequent oligomerization of hBCL10, we abolished expression of hBCL10 in the human cell line HEK293 through retrovirus-mediated expression of short hairpin RNAs (shRNA) targeting hBCL10. As shown in Fig. 6A, introduction of hBCL10sh#3 and hBCL10sh#5 in HEK293 cells results in a great reduction of BCL10 expression. Depletion of hBCL10 in these cells abrogates their ability to activate NF-κB following exposure to phorbol-12-myristate-13-acetate (PMA) (Fig. 6B). However, introduction of tBCL10 in these hBCL10-depleted cells fully recovers their ability to activate NF-κB (Fig. 6C). Thus, tBCL10 can functionally replace hBCL10.

Bottom Line: Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20.Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein.This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

View Article: PubMed Central - PubMed

Affiliation: Biogem, Via Camporeale, Ariano Irpino (AV), Italy.

ABSTRACT
The complexes formed by BCL10, MALT1 and members of the family of CARMA proteins have recently been the focus of much attention because they represent a key mechanism for regulating activation of the transcription factor NF-κB. Here, we report the functional characterization of a novel isoform of BCL10 in the trout Oncorhynchus mykiss, which we named tBCL10. tBCL10 dimerizes, binds to components of the CBM complex and forms cytoplasmic filaments. Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20. Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein. This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

No MeSH data available.