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  • AZD 3965 As an endogenous negative modulator CRBN inhibits


    As an endogenous negative modulator, CRBN inhibits AMPK’s activation (phosphorylation of Thr172) by directly binding to the α-subunit of AMPK, disrupting γ-subunit recruitment to the AMPK complex (Lee et al., 2011, Lee et al., 2013). In our study, we did not focus on AMPK activation, since only a single mutation was introduced. Further species-specific structural changes may be crucial for producing visibly different functions of CRBN considering the coevolution of CRBN and other binding proteins. Although only AMPKα was statistically indicative of coevolution, other CRBN-binding proteins tended to have higher correlation coefficients. Thus, modification of CRBN and other binding proteins may have also occurred during evolution. A previous study indicated that several residues in the thalidomide binding pocket are conserved in both conformational and functional properties (Akuffo et al., 2018). Since residue 366 is considered a non-related residue for binding in the pocket, it may be the surrounding residues that impart the species-specific functions of CRBN, especially for protein-protein interaction that occur on the surface area of each protein.
    Acknowledgements This work was supported by the Center of Innovation Science and Technology based Radical Innovation and Entrepreneurship Program (COI STREAM) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) in Japan. We would like to thank Editage ( for English language editing.
    Covalent, reversible, post-translational modification of cellular proteins with the small modifier, ubiquitin (Ub), regulates virtually every known cellular process in eukaryotes. A protein may be modified with a single Ub (mono-ubiquitination), several single Ubs, or multiple Ubs that are covalently linked together via any of seven lysine (Lys) residues (poly-ubiquitination) or the N-terminus (N-term) of Ub (linear chains). The nature of the modification specifies the biological outcome. Regardless of what type of protein ubiquitination ensues, the process is carried out by a trio of enzymes: a Ub-activating (E1) enzyme, a Ub-conjugating (E2) enzyme, and a Ub ligase (E3) enzyme. E3 ligases orchestrate the final Ub transfer step to a substrate by binding the latter and an E2–ubiquitin intermediate known as a conjugate (E2~Ub). E3 ligases are categorized into three AZD 3965 based on the type of transfer mechanism utilized: (1) eally nteresting ew ene (RING)-type E3s, (2) omologous to the 6AP arboxyl erminus (HECT)-type E3, and (3) ING-in-etween-ING (RBR) E3s. RING-type E3s promote Ub transfer from E2~Ub onto substrate directly, whereas HECT-type and RBR-type E3s require a covalent E3–ubiquitin conjugate (E3~Ub) thioester intermediate to transfer Ub to substrate. While RING and HECT domains are structurally and mechanistically distinct, the more recently discovered RBR E3s contain a RING domain and an active-site cysteine (Cys), leading to their designation as RING–HECT hybrids. This review focuses on emerging mechanistic and structural understanding in the still young field of RBR E3 ligases. Meet the RBR E3s The 14 RBR E3s [1] in humans regulate diverse cellular processes that are still being defined. The most noted member is Parkin, a highly studied enzyme whose E3 ligase activity is associated with mechanisms that clear damaged mitochondria via a process called mitophagy [2], [3]. Early studies linked mutations in the gene that encodes Parkin (PARK2) to autosomal-recessive juvenile Parkinson's disease [4], [5]. Linear ubiquitin chain assembly complex (LUBAC), the only E3 enzyme in any class known to generate linear Ub chains, contains two RBR family members, heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L) and HOIL1-interacting protein (HOIP) [6]. Originally, LUBAC was found to regulate inflammation by activating NF-κB pathways [7], [8], but later studies have established a broader biological role. Malfunction of the LUBAC complex is now associated with B-cell function, regulation of apoptosis, oncogenesis, and diverse autoimmune diseases [9], [10], [11], [12], [13], [14]. uman homolog of Ariadne (HHARI) and its homologs in Drosophila and Caenorhabditis elegans (C. elegans) are implicated in the regulation of translation, cellular proliferation, and developmental processes [15], [16], [17], [18]. In humans, two RING fingers and DRIL (TRIAD1) has been associated with regulating the proliferation of myeloid progenitors, NF-κB signaling, and membrane trafficking [19], [20], [21]. HHARI and TRIAD1 have both been shown to associate physically with Cullin (CUL) RING ligases (CRLs), potentially extending their known biological roles [22], [23], [24]. Although currently understudied, the RBR E3s RNF144A and RNF19A are thought to promote apoptosis in a p53-dependent manner and to confer neuronal protection, respectively [25], [26]. In this article, AZD 3965 we focus mainly on the non-Parkin members of the family. A useful list of all human RBR gene names and nicknames is available [1], [27].