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Ubiquitin is a small modifier protein that is highly conserved during evolution from yeast to human. Ubiquitin modification is one of the most sophisticated and versatile post-translational modifications that regulates numerous biological functions, including inflammation, apoptosis, cancer, cell cycle, DNA repair, Parkinson's disease and endocytosis. We are especially interested in understanding the role of a specific type of ubiquitination known as linear ubiquitination.

• Linear ubiquitin chain is a unique linkage type of chain which is linked via a Met residue instead of commonly used Lys residues. As this atypical type of ubiquitin chain was discovered quite recently, little is known about its regulation. The only known linear ubiquitination-specific E3 ligase is LUBAC (linear ubiquitin assembly complex), which consists of HOIP, SHARPIN and HOIL-1L (Figure 1A).

  We showed that the E3 ligase complex LUBAC plays a critical role in the regulation of TNF-a-induced NF-_kB signaling. We aim to understand a) how LUBAC generates linear ubiquitin chains, and b) what the target substrates are. In the laboratory we established an in vitro ubiquitination assay to monitor ubiquitin chain formation by using purified proteins HOIP, SHARPIN and HOIL-1L. We identified a critical residue Cys885 in the HOIP catalytic domain in the regulation of linear ubiquitin chain formation. Cys885 is conserved in different species and is located at the second RING domain (Figure 1A).

  In HHARI E3 ligase, the Cys is a ubiquitin-loading residue for the intermediate status of ubiquitin transfer to the substrates, which was the first example of the 'HECT-RING hybrid' type of E3 ligase. Accordingly, the purified HOIP-C885S mutant no longer generated linear ubiquitin chains in vitro (Figure 1B) or no longer activated NF-_kB (Figure 1C), suggesting HOIP as the HECT-RING type of E3 ligase. However, the manner in which HOIP is specific for generating linear ubiquitin chains is entirely unclear. By further analyzing the catalytic activity of HOIP, we aim to elucidate the enzymatic regulation of LUBAC in the generation of linear ubiquitin chains. Moreover, we are currently setting up a system to screen new targets of LUBAC E3 ligase by combining the in vitro ubiquitin assay with protein chip array.

  Role of LUBAC E3 ligase complex in vivo

  SHARPIN, one of the non-catalytic subunits of LUBAC, plays a critical role in the regulation of inflammatory responses in vivo. In SHARPIN-deficient (cpdm) mice, inflammation is significantly upregulated in several organs, including the skin. Histological analysis of skin tissue of cpdm mice clearly shows that a SHARPIN deficiency causes inflammation of the skin and apoptosis, as determined by immunohistochemistry using anti-cleaved caspase 3 antibody (Figure 2A). We identified FADD as a new substrate of LUBAC that regulates the TNF-induced apoptosis pathway. FADD is ubiquitinated by LUBAC E3 ligase in vitro (Figure 2B), and this ubiquitination event is required for the anti-apoptosis signaling cascade. We plan to clarify whether apoptosis in keratinocytes plays a role in the regulation of skin inflammation of cpdm, mice using genetically modified mouse models.

  To further understand the linear ubiquitination signal in biological functions in vivo, we are establishing a mutant mouse line of HOIP. Depletion of HOIP in mice leads to embryonic lethality before the age of E11-E12 due to developmental defects. This is similar to the phenomena identified in many NF-_kB-deficient mice, including NEMO, IKK2, and p65. We aim to understand how HOIP functions during development. Based on the results of screening, we will focus on the role of HOIP in different diseases, using genetically modified mouse lines, in order to discover new biological functions of the linear ubiquitination signal. In summary, we will elucidate novel functions of the linear ubiquitin signal by combining the biochemical screening method to identify new targets of LUBAC and genetically modified animal models.

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