Biochemical and Biophysical Research Communications
Cleavage by MALT1 induces cytosolic release of A20
Research highlights
► Characterization of antibodies for selective detection of A20 proteolytic fragments after cleavage by MALT1. ► Detection of the N-terminal proteolytic fragment of endogenous A20. ► Sub-cellular localization analysis of A20 after cleavage by MALT1.
Introduction
A20 (also known as TNFaip3) is a key protein for termination of signaling cascades that activate nuclear factor-kappa B (NF-κB) [1]. NF-κB is a major transcription factor in innate and adaptive immunity and is also involved in the development and progression of lymphomas and other cancers [2]. A20 is believed to exert its NF-κB inhibitory function as a dual ubiquitin-editing enzyme [3]. While the N-terminal part of A20 bears an OTU motif that has de-ubiquitinating (DUB) activity, the C-terminal part contains seven zinc fingers of which zinc finger 4 has been shown to have E3 ubiquitin ligase activity (E3 ligase). Remarkably, A20 specifically removes lysine (K) 63-linked polyubiquitin chains but catalyzes K48-dependent polyubiquitination reactions. Whereas K63-linked ubiquitin chains regulate protein–protein interactions, K48-linked chains target their substrates for proteasomal degradation. The ubiquitin-editing function of A20 can, therefore, regulate the fate of its substrates. Recently, A20 was also shown to inhibit NF-κB signaling through disruption of E2/E3 ubiquitin enzyme complexes [4].
MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1), together with Caspase recruitment domain (CARD)-containing proteins such as BCL10 (B-cell lymphoma/leukemia 10) and CARMA1 (CARD-containing MAGUK protein 1), plays a critical role in the regulation of NF-κB pathways downstream of T- and B-cell receptors [5]. Besides an essential scaffolding function, a long-elusive catalytic function for MALT1 was recently revealed and two substrates for MALT1 proteolytic activity were identified. One substrate is BCL10 which is cleaved at the C-terminus [6]. This modification does not appear to impact on NF-kB signaling but may regulate integrin-dependent T cell adhesion [6]. The other identified substrate is A20 [7]. Cleavage of hA20 by MALT1 was shown to produce two fragments, namely hA20p50 which retains an intact OTU domain, and hA20p37 which retains 6 of the 7 zinc finger domains and therefore the E3-ligase domain [7]. The impact of A20 cleavage by MALT1 on its capacity to regulate NF-κB has only been partially elucidated. The p37 fragment retained NF-κB inhibitory activity but displayed increased sensitivity to proteolysis [7]. Thus, MALT1 proteolytic activity may regulate NF-κB activation through the release of an unstable hA20p37 fragment. The hA20p50 appeared more stable; however, it has not been characterized yet. To further understand the impact of MALT1-mediated cleavage of hA20, the present work has addressed the specific detection of the two fragments generated upon cleavage. Newly characterized antibodies, together with fluorescence microscopy, were also employed to examine the sub-cellular localization of full-length hA20 and the de novo produced fragments thereof.
Section snippets
Materials
OPTI-MEM-1 medium was from Gibco (#31985), FuGENE HD from Roche (#04709705001), the Western Blot (WB) and infrared detection system from Li-COR Biosciences, and the z-VRPR.fmk inhibitor compound was from Bachem (#N-2010). Rabbit polyclonal anti-hA20 (Ab1) was from abcam (#ab45366), mouse monoclonal anti-hA20 (Ab2) was from eBioscience (#60-6629-81), Goat anti-Rabbit IRDye800CW was from Li-COR Biosciences (#926-32211), Goat anti-mouse IgG (H + L) with Alexa Fluor 680-F(ab′)2 fragment was from
Cleavage of hA20 by MALT1: specific detection of hA20p50 and hA20p37
Human A20 was recently shown to be cleaved by MALT1 after R439 [7]. This cleavage generates a large N-terminal fragment of 439 amino acids (hA20p50) and a smaller C-terminal fragment of 351 amino acids (A20p37) (Fig. 1A). To produce and characterize both fragments, by comparison to full-length hA20, we used a constitutively active form of MALT1, which contains a helix-loop-helix (HLH) fused to its N-terminus. HLH-MALT1 can cleave A20 independently of BCL10 and other CARD-containing proteins,
Acknowledgments
A. Demeyer is a Ph.D. fellow with the “Instituut voor Innovatie door Wetenschap en Technologie” (IWT), J. Staal is a postdoctoral fellow with the “Fonds voor Wetenschappelijk Onderzoek-Vlaanderen” (FWO). We are grateful to S. Noppens (Microscopy Core Facility, Department for Molecular Biomedical Research, Ghent) for technical assistance and to M. Baens (KUL) for help with plasmids. Work in R. Beyaert’s lab is supported in part by the “Interuniversity Attraction Poles program” (IAP6/18), the
References (14)
- et al.
A20: central gatekeeper in inflammation and immunity
Journal of Biological Chemistry
(2009) - et al.
Localization of A20 to a lysosome-associated compartment and its role in NF-kappa B signaling
Biochimica et Biophysica Acta (BBA) – Molecular Cell Research
(2008) - et al.
Functional redundancy of the zinc fingers of A20 for inhibition of NF-kappa B activation and protein–protein interactions
FEBS Letters
(2001) - et al.
Nuclear factor-kappa B: from clone to clinic
Current Molecular Medicine
(2007) - et al.
De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappa B signalling
Nature
(2004) - et al.
Inhibition of NF-kappa B signaling by A20 through disruption of ubiquitin enzyme complexes
Science
(2010) Multifunctional roles for MALT1 in T-cell activation
Nature Reviews Immunology
(2008)
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