Transglutaminase 2 regulates NF-κB activity through polyamination of COMMD1

Abstract

Transglutaminase 2 (TG2) is a calcium-dependent enzyme that catalyzes polyamination of glutamine residues in protein substrates. COMMD (copper metabolism MURR1 domain-containing) family is associated with many biological processes. COMMD1 is the best studied isotype and is known to regulate NF-κB activity via promoting ubiquitin-dependent proteasomal degradation of p65. In numerous previous reports, TG2 has been reported to be involved in various pathophysiologies through NF-κB activation including tumorigenesis, inflammation, and fibrosis. However, the exact regulation mechanism of TG2 in NF-κB activation has not been fully elucidated. In this study, we found that all human COMMD paralogues, COMMD1 to COMMD10, except COMMD6 are polyaminated by TG2. Among them, we identified two modifiable glutamine (Q) residues in COMMD1 by TG2, using site-direct mutagenesis and mass spectrometry analysis, which were Q71 and Q112. We showed that 6xNF-κB-luc reporter activity was more down-regulated by overexpression of polyamination-defective mutants of COMMD1 (Q71N and/or Q112N) and up-regulated by overexpression of its polyamination-mimic mutants (Q71R and/or Q112R) in 293 cells. In addition, overexpression of Q71N and/or Q112N COMMD1 in 293 cells and HeLa cells decreased the expression levels of CXCL1, CXCL5, CXCL8 and CXCL10 compared to overexpression of wild-type COMMD1. Interestingly, cells overexpressing Q71R and/or Q112R COMMD1, which are considered to mimic COMMD1 polyamination, showed higher mRNA expression levels of CXCL1, CXCL5, CXCL8 and CXCL10 than cells overexpressing wild-type COMMD1. Furthermore, ubiquitination assay revealed that Q71N and/or Q112N COMMD1 enhanced poly-ubiquitination of p65 in cells when compared to wild-type COMMD1. Therefore, these results indicate that polyamination of Q71 and Q112 of COMMD1 is responsible for TG2-mediated NF-κB activation.