Monoubiquitination and Activity of the Paracaspase MALT1 Requires Glutamate 549 in the Dimerization Interface

Katrin Cabalzar,Christiane Pelzer,Stephan Hailfinger,Vincent Zoete,Margot Thome,Georg Lenz,Justyna Iwaszkiewicz,Annette Wolf,Marek Cebecauer

doi:10.1371/journal.pone.0072051

Abstract

The mucosa-associated lymphoid tissue protein-1 (MALT1, also known as paracaspase) is a protease whose activity is essential for the activation of lymphocytes and the growth of cells derived from human diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL). Crystallographic approaches have shown that MALT1 can form dimers via its protease domain, but why dimerization is relevant for the biological activity of MALT1 remains largely unknown. Using a molecular modeling approach, we predicted Glu 549 (E549) to be localized within the MALT1 dimer interface and thus potentially relevant. Experimental mutation of this residue into alanine (E549A) led to a complete impairment of MALT1 proteolytic activity. This correlated with an impaired capacity of the mutant to form dimers of the protease domain in vitro, and a reduced capacity to promote NF-κB activation and transcription of the growth-promoting cytokine interleukin-2 in antigen receptor-stimulated lymphocytes. Moreover, this mutant could not rescue the growth of ABC DLBCL cell lines upon MALT1 silencing. Interestingly, the MALT1 mutant E549A was unable to undergo monoubiquitination, which we identified previously as a critical step in MALT1 activation. Collectively, these findings suggest a model in which E549 at the dimerization interface is required for the formation of the enzymatically active, monoubiquitinated form of MALT1.

Highlights

The protease MALT1 plays a central role in the antigen receptor-mediated activation of lymphocytes and the pathogenesis of human diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype [1,2]
These residues are invariant across species [33], and we hypothesized that mutation of these into uncharged alanine residues (E549A and R551A, respectively) might affect MALT1 dimerization and activity
We have provided several lines of evidence that support a model in which E549 within the dimerization interface of MALT1 is required to promote the formation of a monoubiquitinated, catalytically active MALT1 conformation (Fig. 5)

Summary

Introduction

The protease MALT1 ( known as paracaspase) plays a central role in the antigen receptor-mediated activation of lymphocytes and the pathogenesis of human diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype [1,2]. MALT1 is present in its catalytically inactive form, constitutively associated with the adaptor protein BCL10 [3,4]. MALT1 and BCL10 form a complex with the scaffold protein CARMA1 ( known as CARD11) [5,6] that promotes the activation of the transcription factor nuclear factor kappa B (NF-kB). NF-kB complexes are present mainly as p50-RelA and p50-cRel heterodimers [8] These are kept inactive by inhibitor of kappa B (IkB) proteins, which retain NF-kB heterodimers in the cytoplasm [9], and by the NF-kB family member RelB, which acts as an NF-kB inhibitor in lymphocytes by binding RelA and cRel and preventing their DNA binding [10,11,12,13]. As an enzyme with protease activity, MALT1 promotes NF-kB activation by cleaving the inhibitor RelB, which is subsequently degraded by the proteasome [10]. Inhibition of MALT1 with the arginine-based peptide inhibitor z-VRPR-fmk leads to a significant reduction in antigenreceptor mediated lymphocyte activation [21]

Methods

Results

Conclusion