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  • What would be the possible off target effects of enhancing


    What would be the possible off-target effects of enhancing Ndfip1 to control T cell responses? Increasing Ndfip1 activity could limit cytokine production in macrophages, where it has been shown to function in mouse BMDMs to degrade Tak1 and downregulate NFκB signaling [108]. Whether Ndfip1 regulates Itch in any other cell types remains to be determined. Notably, enhancing Ndfip1 would not be expected to change NFκB signaling in T cells, because this signaling pathway is regulated by a separate Itch-activator-substrate combination; Tax1bp1-mediated activation of Itch promoted degradation of Rip1 to limit NFκB [15]. Targeting JNK to promote Itch activation in T ARQ 621 is predicted to have liver toxicity because JNK-activated Itch promoted degradation of cFLIP and enhanced cell death in hepatocytes [43], [125]. Together, the data from primary T cells support that targeted enhancement of Ndfip1 would be the best approach to activating Itch to regulate T cell lineage commitment to Th2 (JunB), Th17 (RORgt), Treg cell cytokine production, and cytokine signaling (Jak1). These findings show the promise and challenge of identifying and targeting cell-type and substrate specific modulators of Itch to modify specific functional outcomes for immune therapies.
    Introduction The protein Cereblon (CRBN) can recognize and bind specific substrates as a component of CRL4 E3 ubiquitin ligase (Ito et al., 2010). The complex, which includes CRBN, DDB1 (DNA damage-binding protein 1), Cul4 (Cullin-4A), and Roc1 (GTP-binding protein ROC1), mediates the substrate degradation processed through the ubiquitin proteasome system (UPS) pathway. CRBN is a multifunctional protein related to cellular activities, such as energy metabolism and ion-channel activation through substrate degradation (Lee et al., 2014, Bavley et al., 2018, Choi et al., 2018). CRBN KO mice are resistant to a high fat diet by exhibiting a constantly phosphorylated AMP-activated protein kinase (AMPK), which stimulates glucose uptake through the AMPK pathway responsible for energy homeostasis (Lee et al., 2011, Lee et al., 2013). CRBN also reacts to cellular stresses including UPS impairment, endoplasmic reticulum (ER) stress, and oxidative stress (Lee et al., 2015, Sawamura et al., 2015, Kataoka et al., 2016). Furthermore, mutations of CRBN cause Intellectual Disability (ID) (Higgins et al., 2004, Sheereen et al., 2017). Since CRBN is common in eukaryotes, it may have retained its roles through positive/purifying selection. Thalidomide and related drugs are immunomodulatory drugs (IMiDs) that are effective for multiple diseases, such as multiple myeloma (MM) and lymphocytic leukemia (Giannopoulos et al., 2011, Zhu et al., 2011, Zhu et al., 2013). In 2010, CRBN was detected as a direct target of thalidomide. Therefore, studies explored the role of CRBN, including experiments on zebrafish and chickens. These studies indicated that CRBN-thalidomide binding leads to alteration of CRBN activity, resulting in teratogenicity including limb malformation (Ito et al., 2010). It was reported that IMiDs can recruit an E3 ubiquitin ligase complex to different substrates for degradation (Fischer et al., 2014). Recently, we reported that thalidomide augments nerve cell death through inhibition of AMPK phosphorylation by binding to CRBN (Sawamura et al., 2018). Therefore, CRBN, which appears to play significant role as a binding partner of thalidomide, is an important research topic. Human CRBN has 442 amino acid residues in three regions, an N-terminal extension, Lon domain and cereblon domain of unknown activity, binding cellular ligands and thalidomide (CULT) domain. The domains have 79, 238, and 125 residues respectively. The N-terminus is disordered for the first 30 residues with increasing conservation closer to the Lon domain (Lupas et al., 2015). The N-terminal 1-119 residues in human CRBN are required for localization to the nucleus and binding transcription factors, such as Ikaros (Wada et al., ARQ 621 2016). The Lon domain, which contains conserved regions derived from the Lon protease, has a region essential for DDB1 binding. We previously showed that mitochondrial CRBN has Lon-type protease activity, which exhibits a cytoprotective effect under oxidative stress (Kataoka et al., 2016). The CULT domain was defined as the binding site of substrates and IMiDs and was revealed experimentally and by crystal structure construction (Fischer et al., 2014, Chamberlain et al., 2014, Mori et al., 2018).