Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05
  • br Acknowledgements E B was supported by grant mol

    2019-08-12


    Acknowledgements E.B. was supported by grant 16-34-60213 mol_a_dk from the Russian Foundation for Basic Research (RFBR). R.S. and A.V. were supported by grant of the President of Russian FederationMK-4253.2018.4. The work was performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.
    Introduction Ubiquitination is a post-translational modification that is required for virtually all cellular processes (Hershko and Ciechanover, 1998). Ubiquitination involves the covalent attachment of the small protein ubiquitin (Ub) to N termini or lysines of proteins by the E1-E2-E3 enzyme cascade, and different Ub chains confer distinct functions on protein substrates (Komander and Rape, 2012, Pickart and Eddins, 2004). Ub ligases (E3s) mediate the final step of the process and control specificity, efficiency, and patterns of ubiquitination. Consequently, dysregulation of E3s occurs in many diseases, including diabetes, neurodegeneration, atherosclerosis, and cancer (Goru et al., 2016, Popovic et al., 2014), and E3s are attractive therapeutic targets (Nalepa et al., 2006, Petroski, 2008). E3s are classified into three families: RING (really interesting new gene) and U-box E3s, HECT (homologous to E6AP C terminus) E3s, and RBR (RING between RING) E3s (Buetow and Huang, 2016). With ∼600 members, the RING/U-box E3s form the largest family (Deshaies and Joazeiro, 2009). This family uses a RING or U-box domain to recruit E2 5224 thioesterified with Ub (E2∼Ub) and facilitates transfer of Ub directly to substrate. RING/U-box E3s can be further categorized as multisubunit complexes, which contain scaffold, adaptor, receptor, and RING subunits on distinct polypeptide chains (Deshaies and Joazeiro, 2009), or simple, which contain E2∼Ub and substrate-binding domains within the same polypeptide chain. Some simple RING/U-box E3s are active as monomers, like CBL and UBE4B, whereas others, like XIAP (X-linked inhibitor of apoptosis), only function when dimerized via their E2∼Ub binding domains (Buetow and Huang, 2016). Despite the plethora of RING/U-box family members, small molecule modulators are limited to a few E3-substrate interactions (Bulatov and Ciulli, 2015, Landré et al., 2014). Substrate-binding domains vary among E3s; hence, new screening strategies are required to establish a general targeting platform. Although a RING or U-box domain is a common feature, these domains lack binding pockets easily targeted by inhibitors. Thus, we sought to develop a general platform to systematically modulate activities of RING and U-box E3s. RING/U-box domains promote Ub transfer by shifting the equilibrium of E2∼Ub into a closed conformation (Dou et al., 2012b, Plechanovová et al., 2012, Pruneda et al., 2012). These domains comprise ∼75–100 aa that form two loops stabilized by two Zn2+ ions or hydrogen bonds in RING or U-box domains, respectively. These loops and intervening region form the E2∼Ub-binding surface in RING-E2∼Ub complexes, and the C-terminal tail and Ile36 patch of Ub form interactions with the RING while the Ile44 hydrophobic patch abuts the E2 (Buetow and Huang, 2016). Typically, a small surface area of only ∼450 Å2 from the RING domain contacts Ub. Previously, we used phage display to select for ubiquitin variants (UbVs) with enhanced affinities for proteins that naturally interact weakly with Ub (Ernst et al., 2013), including deubiquitinases, HECT E3s, multi-subunit E3s, and small Ub-interacting motifs (Brown et al., 2016, Ernst et al., 2013, Gorelik et al., 2016, Manczyk et al., 2017, Zhang et al., 2016, Zhang et al., 2017a, Zhang et al., 2017b). Here, we tested whether UbV modulators can be selected for simple RING E3s. We targeted three RING/U-box E3s from three important classes: (1) UBE4B, a monomeric U-box E3 (Wu et al., 2011); (2) CBL, a monomeric RING E3 activated by phosphorylation (Dou et al., 2012a); and (3) XIAP, a RING E3 that is activated upon dimerization (Nakatani et al., 2013). We generated selective UbVs for each RING/U-box domain and used biochemical assays and structural studies to identify two types of UbVs: competitive inhibitors of E2∼Ub binding sites on UBE4B or phosphorylated CBL and an activator of XIAP. Our work demonstrates the versatility of the UbV technology and provides a resource for the rapid development of inhibitors and activators across the large RING/U-box E3 family.