Dynamic arrangement of linear ubiquitination
Ubiquitin is an important regulatory protein used for post-translational modification and plays a crucial role in several biological functions, including inflammation, cell death, autophagy, cancer, and cell cycle. By an enzymatic reaction, ubiquitin modifies substrates with different linkage types of ubiquitin chains. We are particularly interested in understanding the roles of novel type of ubiquitin chains known as linear ubiquitin chains in the regulation of various stress-induced cellular responses.
Linear ubiquitination is induced by the LUBAC E3 ligase complex
Linear ubiquitin chain is a unique linkage type of ubiquitin polymer, linked through an intrinsic residue Met 1 instead of commonly used 7 Lys residues (Figure 1A). Ubiquitination is induced by a three-step enzymatic reaction of E1 activating enzyme, E2 conjugating enzyme and E3 ligase. For the linear ubiquitination, E3 ligase complex called Linear Ubiquitin Assembly Complex (LUBAC) plays a crucial role (Figure 1B). LUBAC consists of a catalytic RBR E3 ligase HOIP, and two regulatory subunits Sharpin and HOIL-1L (Figure 1B). We have previously demonstrated that the LUBAC plays a critical role in the regulation of Tumor Necrosis Factor (TNF) -induced NF-kB signalling (Ikeda et al., 2011). However, it is not known whether the enzymatic activity of HOIP is regulated in a similar manner as that of other known E3 ligases such as Parkin.
To elucidate the molecular mechanisms that regulate the HOIP activity we first established an in vitro ubiquitination assay. This assay monitors ubiquitin chain formation by using purified proteins, HOIP, Sharpin and HOIL-1L. Similar to the HHARI E3 ligase, which was shown to be the first example of the ‘HECT-RING hybrid’ type of E3 ligase, we identified a conserved Cys885 residue in the HOIP catalytic domain (Figure 1C). Our current research has shown that this is crucial for the enzymatic activity (Figure 1D). This suggests that the Cys885 residue is used for the ubiquitin loading site at the intermediate status of the ubiquitin transfer to substrates like HHARI, in line with the data reported by Stieglitz et al., 2013. Moreover, the HOIP Cys885 mutant no longer activated the NF-kB in cells determined by the gene reporter assay (Figure 1E).
Collectively these observations suggest that HOIP functions as a HECT-RING type of E3 ligase, and that the process of linear ubiquitination is critical for the NF-kB activation. We are further analyzing how the catalytic activity of HOIP is regulated upon TNF stimulation by focusing on the formation of the specific signalling complex and protein modifications.
LUBAC plays a crucial role in the regulation of apoptosis
We have shown that systemic inflammation, including skin inflammation is observed in Sharpin deficient (Cpdm) mice (Ikeda et al., 2011). Based on histological analysis using an apoptosis marker cleaved-caspase 3, we found that apoptosis is significantly upregulated in the Cpdm skin tissue (Figure 2A). In order to establish whether this is a cell intrinsic effect of keratinocytes, we generated a Sharpin-knockdown keratinocyte line (HaCaT) by stably expressing shRNA against Sharpin. Identical to the in vivo situation, shShapin HaCaT cells were sensitized to TNF-induced apoptosis determined by caspase-8 activity assay (Figure 2B). Upregulation of apoptosis signal (caspase-8 activity) in Sharpin-knockdown HaCaT was rescued by additional depletion of FADD (Figure 2B), which is a critical factor for apoptosis induction. These observations suggest that Sharpin regulates apoptosis signalling through FADD-containing complex called TNF-receptor complex II. Interestingly, we found that the knockdown of other LUBAC components, HOIP in HaCaT, also sensitized cells to apoptosis (Figure 2C).
This result strongly suggests that the LUBAC as a complex plays a role in the regulation of apoptosis. Currently, we are trying to identify the targets of LUBAC-induced linear ubiquitination in the apoptosis signalling pathway, and to understand how linear ubiquitination regulates apoptosis at the molecular level.