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  • br Conclusions br ISG is an ubiquitin like protein that

    2020-10-23


    Conclusions
    ISG15 is an ubiquitin‐like protein that is induced by treating mammalian h89 with interferon‐α/β. UbE1L was identified as the E1‐activating enzyme for ISG15 in the course of experiments that demonstrated that the nonstructural or NS1 protein of influenza B virus inhibits ISG15 conjugation. UbE1L is a monomeric protein of 120 kDa. Its most distinctive property is its selectivity in transferring its ISG15 substrate to a single E2 enzyme, UbcH8, an E2 that has been reported to also function in Ub conjugation. This chapter describes the methods for expressing and purifying UbE1L using a baculovirus vector and for assaying UbE1L activity using a P‐labeled ISG15 substrate.
    Labeling of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) small protein modifiers is important for regulating key cellular processes such as protein degradation, subcellular localization, and progression through the cell cycle., , Among nine known Ub/Ubl modifiers, including SUMO and NEDD8, the recently discovered Ub-fold modifier 1 (UFM1), , , is implicated in the promotion of erythroid differentiation in hematopoiesis, the regulation of the endoplasmic reticulum (ER) stress response in pancreatic cells and more recently, has been implicated in the progression of breast cancer as a result of its role in upregulating the expression of pro-proliferative gene products such as c-Myc and cyclin D1. Although the UFM1 pathway has been linked to an important subset of cellular functions, the full extent of its biological significance, particularly in disease progression, has yet to be elucidated. Like other Ub/Ubl modifiers, the process of protein UFMylation is controlled by an E1–E2–E3 enzymatic cascade (). First, the UFM1 activating E1 enzyme (UBA5) catalyzes the ATP-dependent adenylation of the UFM1 C-terminal carboxylate., The subsequent intermolecular nucleophilic attack of the UFM1-adenylate by a cysteine residue (Cys250) on UBA5 results in the formation of a covalent UBA5-UFM1 thioester intermediate., The UFM1 E2 conjugating enzyme (UFC1) then binds to the UBA5-UFM1 complex and performs a transthioesterification, or transthiolation, reaction with UBA5 ()., This results in the transfer of UFM1 from Cys250 to an acceptor cysteine on UFC1, which attaches UFM1 to protein targets in concert with the UFM1 E3 ligase (UFL1). To date, only one inhibitor has been developed for UBA5. The Takeda Oncology Company: Millennium identified an ATP-competitive small molecule, adenosine 5′ sulfamate (ADS), which abrogated UBA5 activity through a substrate-assisted mechanism. When evaluated in HCT116 human colorectal carcinoma cells, ADS reduced UFM1 protein conjugation through intracellular UBA5 inhibition. However, ADS demonstrated activity against the other E1 enzymes, and will likely not be used as a molecular tool to selectively investigate the cellular roles of UFM1 conjugation. As demonstrated previously by Millennium in the development of MLN4924 for the selective inhibition of the NEDD8 E1 (NAE),, E1-specific inhibition can be achieved by exploiting discriminant features of their ATP pockets. Through the design of a novel inhibitor scaffold that targets distinct features of the UBA5 ATP pocket, we herein report the development of a selective UBA5 inhibitor with promising intracellular activity. In this study, the structural features proximal to the ATP pocket were investigated in order to design an inhibitor selective for UBA5 over other E1 enzymes. Based on the structural overlay and sequence alignment of the known human E1s (), a unique Glu209 was identified that lined the edge of the ATP pocket on UBA5 and was located adjacent to the conserved Asp183, which has been shown to be important in Ub/Ubl adenylation (A)., In contrast, other E1 enzymes contain a small hydrophobic or neutral side chain in place of the negatively charged Glu209 (). To target these two sites simultaneously, a focused library of ditopic inhibitors was prepared, featuring an adenosine molecule coupled to a zinc(II)cyclen coordination complex. To investigate the optimal distance between the respective sites, the adenosine was appended to the zinc(II)cyclen through alkyl linkers of varying length (1–5 methylene groups) (B).