Programmed cell death control by SHARPIN, a critical subunit of LUBAC

Background: Ubiquitin (Ub) is a ubiquitously expressed small protein that is covalently attached to substrates. We have shown that SHARPIN is one of the critical subunits of LUBAC (linear Ubiquitin chain assembly complex), which generates a novel linkage type of linear-Ub chain and is essential for TNF-alpha-induced NF-kappaB activation. Different linkage forms of Ub chain generate very different topologies leading to the specific regulation of the downstream biological effects. Therefore, it is critical to clarify the mechanistic basis of ubiquitination in specific biological settings. SHARPIN deficiency in mice leads to a heavy inflammatory phenotype in multiple organs but the regulatory mechanism of this is unknown. Interestingly, apoptosis is also dramatically upregulated in keratinocytes of SHARPIN deficient mice, and our preliminary data suggest that SHARPIN may play a role also in necroptosis, another type of programmed cell death. However, the regulatory mechanisms of SHARPIN and SHARPIN-dependent linear ubiquitination in programmed cell death and inflammation are completely unknown. Aim: In the proposed project, we aim to elucidate the regulatory mechanisms of programmed cell death by SHARPIN and SHARPIN-dependent linear ubiquitination. Experimental approaches: We will analyze the role of SHARPIN in apoptosis (Aim 1) and necroptosis (Aim 2) signaling cascades using primary cells derived from SHARPIN deficient mice. We will evaluate cell death signaling by various known cell death stimuli, such as TNF-alpha, TRAIL and Fas-L in SHARPIN deficient cells and use specific biological markers for apoptosis and necroptosis. We plan to identify the linear ubiquitination targets of SHARPIN by using in vitro ubiquitination assays and mass spectrometry. Further, we aim to clarify whether SHARPIN deficiency has an effect on the cell death complex formation. Finally, we will clarify whether the skin inflammation phenotype of SHARPIN deficient mice is caused by apoptosis and necroptosis. This will be approached by genetically depleting TRADD or RIP3, two factors which are essential for apoptosis or necroptosis, respectively, in the background of SHARPIN deficiency (Aim 3). Inflammation phenotype in these mice will be analyzed histologically as well as keratinocyte proliferation markers. Primary keratinocytes from these mice will be challenged with TNF-alpha and we will clarify the effects on the signaling. Expected significance in the field: This study will elucidate the mechanistic role of SHARPIN-mediated programmed cell death via linear-ubiquitination and the implications for inflammation for the first time. Understanding this link between inflammation and linear ubiquitination will be important for various diseases.

Ubiquitin is a fundamental protein to control human health implicated in many diseases such as inflammation and cancer. Ubiquitin controls such diseases by modifying proteins critical for disease regulation. For ubiquitination, it is known that a set of enzymes plays a key role. We and others have previously identified a specific ubiquitin enzyme complex called Linear UBiquitin chain Assembly Complex (LUBAC) consisting of three proteins SHARPIN, HOIP and HOIL-1, regulating inflammatory responses. By support of this research grant, we discovered that SHARPIN-deficiency in mice leads to severe skin inflammation accompanied by significantly increased cell death in keratinocytes, and the skin phenotype in these mice is dependent on the Tumor Necrosis Factor (TNF)- and TRADD-mediated apoptosis pathway (Kumari et al, 2014). During the course of this study, we found that not only SHARPIN but also HOIL-1L and HOIP play a crucial role in the regulation of keratinocyte cell death. Thus, it is critical to understand how LUBAC as a complex regulate inflammatory responses. Since HOIL-1L is implicated in autoimmune diseases in humans, and latest studies have shown that HOIL-1L is essential for mouse development and cell death regulation (Peltzer et al. 2018, Fujita et al. 2018), it became important to understand more detailed mechanisms how HOIL-1L regulates inflammation. To address how the LUBAC proteins regulate inflammation and cell death more precisely, we newly established gene-mutation mouse lines of the LUBAC components by using the gene editing technology called the CRISPR-Cas9 technology. We are currently analyzing an impact of these mutations in mice by histological analysis, as well as an impact at the cellular level by using primary fibroblasts and bone marrow-derived macrophages. By using these newly established mouse lines, and by combining the knowledge on SHARPIN from this research (Kumari et al., 2014), we aim to further examine the role of the LUBAC components in the regulation of apoptosis and inflammation in vivo (a manuscript in preparation). It is expected that our studies will make a major impact on understanding human disease mechanisms especially in autoimmune diseases by focusing on the specific ubiquitin enzyme complex, LUBAC.