Lars van Steeg

Introduction:
OTULIN is known to play a key role in various immune response pathways such as the nuclear factor kappa B (NF-κB) signalling pathway. Being a deubiquitinase (DUB) it removes linear M1 linked ubiquitin chains created by the E3 ligase Linear Ubiquitin Chain Assembly Complex (LUBAC) and is crucial for defence against infections. Genetic deficiencies in the catalytic domain of OTULIN are known causes of inborn errors of immunity. Notably, haploinsufficiency of OTULIN leads increases susceptibility to infection triggered necrosis in the skin and lungs by Staphylococcus aureus, whilst its complete loss results in OTULIN-related autoinflammatory syndrome (ORAS). However, less is known about the PUB-interacting motif (PIM) domain of OTULIN and how it contributes to disease. It has been shown that the PIM domain is essential for binding and regulating the activity of LUBAC. There are several naturally occurring mutations identified in the general population in databases like GNOMAD. We are interested in characterizing these mutations and identify potential disease mechanisms.

Methods:
The mutations were brought to exogenous expression in Human Embryonic Kidney (HEK) 293T cells. The ability of OTULIN variants to bind to LUBAC were determined using co-immunoprecipitation of cell lysates. Phosphorylation of OTULIN was investigated under stimulation with tumor necrosis factor (TNF) and MG132.

Results:
There was normal expression of the variants of OTULIN in the cell lysates. The immunoblots of precipitated OTULIN showed diminished binding to LUBAC for several mutants. When probed for their phosphorylation status, the mutants displayed both hyper- and hypo phosphorylation phenotypes.

Conclusion:
We demonstrated that several naturally occurring mutations in the PIM of OTULIN disrupt its binding to LUBAC. We also found that most of these mutations underlie a spectrum of phosphorylation states. With more evidence on their NF-κB signalling capacity we can characterize these variants and their underlying immunological and infectious phenotypes.