| الملخص: | <p>Activation of nuclear factor kappaB (NF-kB) transcription factor-mediated gene expression is key for coordinating innate immune response and cell survival. A feature commonly shared amongst most inflammatory response pathways is their dependence on ubiquitin, a small modifier that can be post-translationally conjugated to target proteins. The formation of (non-)degradative ubiquitin chains is an essential signal in inflammatory response and subject to tight regulation by ubiquitin editing enzymes. Amongst them are two deubiquitinases: the zinc finger protein A20 and the ubiquitin interacting motif (UIM)- containing protein OTUD4. Both deubiquitinases (DUBs) have been shown to play a pivotal role during NF-kB negative feedback regulation, but previous studies lack fundamental experiments on the biochemical level. Using biochemical and biophysical techniques this study investigates how phosphorylation and ubiquitin binding domains regulate activity and specificity of A20 and OTUD4 and how A20 is regulated by A20-binding inhibitors of NF- kB (ABINs).</p>
<p>This study reveals that IKKb-mediated phosphorylation in the catalytic domain of A20 is required for full DUB activity. Two novel phosphorylation sites, Thr161 and Ser220 were identified as being required for this activity enhancement, likely through enhanced binding to the distal ubiquitin binding site. I propose that A20 catalytic domain is phosphorylated by IKKb in response to activation of NF-kB signalling and that phosphorylated A20 readily cleaves Lys11, Lys48, and with lower specificity Lys63-linked chains to counteract a pro-inflammatory signalling outcome. The biophysical characterization of all A20 zinc finger domains showed that only ZnF4 and 7 bind ubiquitin and that A20 linkage type specificity for Lys48-linked chains is encoded in the catalytic domain. A mapping of the A20/ABIN interaction indicated that the interaction requires ii multiple interfaces on both proteins and no contribution of ABIN binding to A20’s activity could be observed when using full-length ABIN2 and truncated A20.</p>
<p>This study and others show that OTUD4 is a chain type specific DUB that preferably cleaves Lys48- and Lys63-linked chains. A biochemical analysis from this study reveals that specificity for both chain types resides within the catalytic domain and that a UIM outside the catalytic domain enhances Lys63 specificity while leaving Lys48 specificity unchanged. To validate molecular details of the specificity mechanism, orthologs were screened for a suitable candidate but a low overall yield prevented further biophysical or structural insights. A previously reported phosphoswitch for OTUD4 could not be observed in in vitro studies using the recombinant version of the kinase (CKII) or insect cell expression of OTUD4.</p>
<p>In summary, this work provides detailed insights into how phosphorylation governs A20 activity and how selective ubiquitin chain binding orchestrates OTUD4 specificity. This study increases the current understanding of allosteric regulation of deubiquitinases and may together with further mechanistic and structural studies allow for improved therapeutic intervention strategies.</p>
|
|---|