**3. RNF186**

A study of the distribution of RNF186 has indicated that its expression is highest in the lower gastrointestinal tract in both human and mouse tissues [10]. Initially, the RNF186 gene was identified as a locus associated with the risk of UC by several genome-wide association studies (GWAS) [27–30]. However, its function has only recently been clarified. Recently, deep resequencing of GWAS loci identified a causal variant in RNF186 that encodes an alanine-to-threonine substitution at position 64 [31]. This variant confers risk for the development of UC. The A64T variant is located in the RING-finger domain of RNF186. Since the RING-finger domain possesses E3 ubiquitin-protein ligase activity, there is the possibility that the A64T variant exhibits a reduction or complete loss of enzyme activity. A report indicates that RNF186 is thus a candidate for an association with the development of chronic inflammation in the intestine of UC. In addition, a recent study focusing on protein-truncating variants identified a novel genetic variant in RNF186, R179X [32]. This variant contributes to protection against UC. The R179X truncation is expressed at reduced levels and is di ffusely localized, not in the ER, but preferentially in the plasma membrane [32]. Studying the specific e ffects of this variant, which has not ye<sup>t</sup> been performed, should be useful for understanding the mechanism by which it protects against UC. In humans, RNF186 is also expressed in the kidney [10]. Interestingly, R179X truncation, which confers protection against UC, has also been identified as a factor associated with an increased risk of chronic kidney disease (CKD) [34].

Several RNF186 substrates have been identified, for example, Bcl2/adenovirus E1B 19-kDa interacting protein 1 (BNip1), occludin, and Sestrin-2 [23–25]. BNip1, a Bcl-2 family protein, co-localizes with and binds to RNF186 in the ER. RNF186 conjugates polyubiquitin through K29 and K63 linkage to BNip1 and the ubiquitylated BNip1 is transferred from the ER to the mitochondria [23]. Because BNip1 can induce a moderate level of apoptosis [61], RNF186 may regulate ER stress-induced apoptosis by the ubiquitylation of BNip1. The overexpression of RNF186 upregulates the critical regulators in the unfolded protein response (UPR), such as chaperone protein BiP/GRP78 and pro-apoptotic transcription factor CHOP/GADD153. Additionally, RNF186 triggers caspase-12 activation, which plays a role in inducing apoptosis in ER stress. Ca2+ is a secondary messenger of ER stress, and RNF186 actually promotes Ca2+ leakage from the ER [23]. Moreover, the expression of Rnf186 has been shown to be induced in the livers of mice with diabetes, obesity, and diet-induced obesity. In mouse primary hepatocytes, the overexpression of Rnf186 increases the protein levels of the ER stress markers, IRE1 and CHOP, as well as the level of eIF2 α phosphorylation, and the treatment with tauroursodeoxycholic acid, the inhibitor of ER stress, decreases the expression of ER stress markers. It has been indicated that the overexpression of Rnf186 induces ER stress. These e ffects interfere with insulin action through the phosphorylation of insulin receptor substrate 1 (IRS1) by c-Jun N-terminal kinase (JNK). Furthermore, it has also been shown that the expression levels of proinflammatory cytokines, including TNFα, IL-6, and MCP1, are increased by overexpressing Rnf186. Thus, Rnf186 may be a novel therapeutic target for the treatment of metabolic diseases associated with insulin resistance [26].

Occludin is one of the tight junction proteins, which control gastrointestinal tract permeability. In the colonic epithelia of mice with Rnf186 knockout, the expression of occludin protein is increased and its distribution is changed, resulting in increased permeability of small organic molecules. Rnf186 ubiquitylates occludin through K48-linked chains. Because K48-linked polyubiquitylation contributes to regulating protein turnover through proteasomal degradation, it is consistent with the increased expression of occludin in colonic epithelia of Rnf186 knockout mice. Disordered protein homeostasis in Rnf186 knockout mice correlates with enhanced ER stress in colonic epithelium and increased susceptibility to intestinal inflammation by DSS treatment. Therefore, it has been suggested that RNF186 is an E3 ubiquitin ligase controlling the homeostasis of occludin and ER stress in the colon [25].

Sestrin-2 is one of the intracellular amino acid sensors and functions as an inhibitor of mTORC1 signaling by interacting with GAP activity toward Rags 2 (GATOR2) [62]. This function occurs in a cytosolic leucine-dependent manner. When leucine is abundant in the cytosol, interference of the interaction between Sestrin-2 and GATOR2 occurs, allowing the activation of GATOR2. Because GATOR2 negatively regulates the GAP (GTPase-activating protein) activity of GATOR1, which inhibits mTORC1 activity, the decrease of Sestrin-2 indirectly activates mTOR1. Although Sestrin-2 has been shown to be transcriptionally controlled by several mechanisms, the regulation of the post-translational degradation of Sestrin-2 remains unclear. A recent study has shown that RNF186 and Sestrin-2 bind to each other via C-terminal motifs and RNF186 polyubiquitylated lysine residue at position 13 in Sestrin-2 with K48 linkage [24]. The ubiquitylated Sestrin-2 is degraded through the proteasome system. This is a new mechanism regulating mTORC1 activity through E3 ligases. In addition, Sestrin-2 also contains polyubiquitin chains with K63 linkages. Although this type has been shown to be involved in various processes, such as cellular trafficking, the role of the K63-linked ubiquitylation of Sestrin-2 remains unknown.
