| Streszczenie: | The linear ubiquitin chain assembly complex (LUBAC) regulates NF-κB activation and prevents cell death by conjugating M1 polyubiquitin on inflammatory complexes through its catalytic subunit HOIP. Although the conjugation of M1 chains is well described, less is known about the mechanism of LUBAC’s recruitment to inflammatory signaling complexes. This thesis elucidates the role of HOIP’s zinc finger region, which encompasses its B-box, Zf and NZF domains, in the binding and recruitment to ubiquitin chains, by employing biophysical techniques such as NMR and ITC to characterize the mechanism. The HOIP NZF1 domain is the driving force behind ubiquitin-binding and interacts with monoubiquitin with a higher affinity than previously studied NZF domains, while HOIP NZF2 employs a similar interface as NZF1 to bind to monoubiquitin weakly. HOIP’s specificity for K63-linked chains is determined by the tandem NZF domains, which cooperatively decrease the protein’s affinity to monoubiquitin and increase its affinity to polyubiquitin, as shown by ITC binding experiments. The interaction of HOIP NZF1 with the downstream signaling effector NEMO hinders, but does not abolish, ubiquitin binding due to an overlap in the binding interfaces for ubiquitin and NEMO. To widen the scope of the regulation of LUBAC’s recruitment, phosphorylation of S383 of the NZF1-NZF2 linker region and HOIP oligomerization via the B-box domain are studied as regulatory mechanisms for the affinity to K63-linked polyubiquitin and found to not affect it. It is therefore proposed that HOIP NZF1 and NZF2 jointly drive LUBAC’s recruitment to preformed K63-linked polyubiquitin in inflammatory signaling complexes. A novel binding mechanism for linkage-specific NZF domains is described, in which binding is driven by NZF1 while K63 diubiquitin specificity is established independently by each NZF domain.
|
|---|