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Regulation and function of linear ubiquitination by HOIP

Final Report Summary - LUBI (Regulation and function of linear ubiquitination by HOIP)

Ubiquitin (Ub) is a small protein modifier, regulating diverse biological functions such as inflammation, signal transduction, autophagy, cancer, proteasomal protein degradation, and endocytosis. The special feature of Ub modification (ubiquitination) is Ub can form chains via generating linkages at the Met 1 and at the 7 Lys residues of Ub itself. We are interested in a novel type of Ub polymer linked via Met-1, termed ‘linear Ub chain’ that regulates immune responses in human and mice. Because linear Ub is unique, and our understanding of it is still in infancy, the only E3 ligase known to catalyze linear Ub chain formation is Linear Ubiquitin chain Assembly Complex (LUBAC), a protein complex comprising a catalytic protein HOIP, and two regulatory subunits, SHARPIN and HOIL-1L. We are aiming to discover the regulatory mechanisms of HOIP catalytic activity of linear ubiquitination and the biological implications of HOIP-induced linear ubiquitination remain poorly understood. The specific aims and outcomes of each aim are the following.

(Aim 1) Elucidate the roles of HOIP in Drosophila
Because understanding of biological functions of linear ubiquitination is still in infancy, we aimed to discover its new roles using a newly established animal model. We first clarified that a bioinformatically predicted gene CG11321 has a ubiquitin E3 ligase activity, which is specific to linear ubiquitin chains. Thereby we named this gene Linear UBiquitin Ligase (LUBEL). By performing in vitro ubiquitination assay using recombinant proteins, we demonstrated that LUBEL generates linear ubiquitin chains using an identical RING-HECT hybrid mechanism as a known human protein HOIP. Interestingly, LUBEL does not require any subunits while human HOIP does for generating linear ubiquitin chains. In addition to the initial domain prediction of the NZF and UBA domains, we found the second UBA domain. Among those three domains, which were speculated to interact with ubiquitin chains only one of the UBA domains binds directly to different linkage types of ubiquitin chains, which is unique to LUBEL and it is not the case in human HOIP. Collectively, we identified the 1st Drosophila ubiquitin E3 ligase, which is capable of generating atypical M1-linked chains (published, Asaoka et al., 2016 EMBO Rep).

(Aim 2) Elucidate the roles of ubiquitination and ligase activities of mammalian HOIP in vivo.
One of the important open questions regarding linear ubiquitination remained to be how the enzymes involved are regulated in the context of cellular signaling. We focused on the posttranslational modifications that occur on the central enzyme, HOIP. We confirmed that HOIP ubiquitination is induced by TNF stimulation in cells. In addition, we found that HOIP is ubiquitinated in vitro using recombinant enzymes. We found one of the ubiquitination sites in HOIP is critical for the TNF-dependent NF-kB activation based on the gene reporter assay and by qPCR.
Therefore, we are focused on the role of this particular ubiquitination site in HOIP in the regulation of immune responses in mice and mammalian cells. We established the knockin mouse line of HOIP ubiquitination site mutant. Cells derived from these knockin mice show significantly reduced levels of NFkB activation. When they are crossed with SHARPIN deficient mice, homozygous mice become embryonic lethal.
The manuscript is under revision and preprint is on BioRxiv (Fennell et al.). We are currently performing experiments for the revision and partially this will be taken over to my new lab in Japan.

(Aim 3) Identify novel substrates of human HOIP and elucidate their roles.
To identify the substrates of ubiquitin E3 ligases is key to understand the biological functions of the E3 ligases. We performed an in vitro protein array-based screening for the identification of substrates for linear ubiquitination mediated by HOIP by which we obtained 15 hits. Thus far, we verified at least 3 proteins identified from the screen are ubiquitinated by the HOIP-containing LUBAC E3 ligase complex in cells. We set up the TR-TUBE system in the lab and currently we are aiming to identify further the substrates and ubiquitination sites.
In the last years, we established a protein purification method of LUBAC, which is suitable for the in vitro ubiquitination assay and may identify new types of ubiquitination. Moreover, a significant number of the hits is known immune regulators. This allows us to investigate the role of linear ubiquitination using established techniques in my lab.
Ub plays such a wide variety of pathological functions. Therefore, by achieving these aims, I expect a better understanding of the functional role of HOIP but will also identify novel aspects of linear ubiquitination in human disease. During the 1st half of the period, we were able to publish one paper on Aim 1 and established various tools and animal models to pursue the study.