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  • Initially the E uses ATP to

    2021-03-04

    Initially, the E1 uses ATP to activate the C-terminal glycine residue of ubiquitin prior to ligation. In the first step of E1 activation, the E1 catalyzes the adenylation of ubiquitin and pyrophosphate (PPi) release. In the second step, the E1 releases adenosine monophosphate (AMP) and a thioester bond is formed between the C-terminal glycine of ubiquitin and sulfhydryl residue of the E1. In the final E1 activation step, E1 carries two ubiquitins by their thioester bond and adenylate bond of C-terminal ubiquitin (Haas & Siepmann, 1997). Next, the activated ubiquitin is transferred from E1 to an E2 active site cysteine by thioester bond. The formation of an isopeptide linkage between the C-terminal glycine of ubiquitin and the lysine ε-amino group of the target is catalyzed by ubiquitin-ligating enzyme (E3) (Haas and Siepmann, 1997, Huibregtse et al., 1995). Lastly, the polyubiquitinated proteins are recruited by a 26S proteasome for degradation (Pickart, 1997). Proteins are degraded by the core particle in a progressive manner, generating peptides of 3–25 CBiPES hydrochloride in length (Nussbaum et al., 1998). Several classes of proteasome inhibitors block ubiquitylated protein degradation. Currently, only a few classes of compounds have been discovered that inhibit the ubiquitin-activating enzyme (E1), such as PYR-41 and Largazole. PYR-41 and related pyrazones provide a proof of principle for drugs with the capacity to differentially kill transformed cells, inhibit NF-κB activation and increase p53 levels and activity (Yang et al., 2007). Thus, PYR-41 serves as a therapeutic actor and potential E1 inhibitor in cancer. Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro (Ungermannova et al., 2012b). NSC624206 could be useful for the control of excessive ubiquitin-mediated proteolysis in vivo (Ungermannova et al., 2012a, Ungermannova et al., 2012b). MLN4924 inhibits the E1 enzyme responsible for NEDDylation and forms a covalent bond with NEDD, a ubiquitin-like protein, to specifically target proteins, including SCFSkp2 (Chen et al., 2008, Nawrocki et al., 2012). MLN4924 is currently in Phase II clinical trial for hematologic cancers (Mattern, Wu, & Nicholson, 2012). Unlike proteasome inhibitor Bortezomib (PS341), there is no E1 inhibitor that is currently approved to treat patients with multiple myeloma. Chinese herbal medicines, including P. ginseng, have been used for more than 1000years in the treatment and possible prevention of malignancy by inhibiting ubiquitin-activating enzyme E1. In previous work, we showed that ginsenoside Rd serves as a 26S proteasome inhibitor (Chang et al., 2008). In this study, we focused on the inhibition mechanism of ginseng on E1-ubiquitin activation for cancer prevention. Here, we report in vitro mechanistic studies that reveal a potential role for ginsenoside Rg1 as an antagonist of the ubiquitin-activating enzyme E1.
    Materials and methods
    Results Protein degradation via UPP involves two steps. One step is the conjugation of multiple ubiquitin to a substrate. The other step is the degradation of tagged protein by the downstream 26S proteasome complex. Protein ubiquitination is the E1–E2–E3 enzymatic cascade (Hershko & Ciechanover, 1998). E1-activating enzyme activates ubiquitin via an adenylated intermediate and catalyzes its transfer to an E2 enzyme. There are eight human E1s that activate ubiquitin or an ubiquitin-like protein and transfer it to the E2-conjugating enzyme (Chen et al., 2011). In this study, we attempted to determine which ginsenoside can inhibit both the first and final steps of UPP. We therefore constructed a red fluorescent protein referred to as mRFP-Ub to study the role of active ginseng components in the E1-Ub activation of UPP.
    Discussion The ubiquitin–proteasome pathway clearly represents an important area of research in cancer biology (Spataro, Norbury, & Harris, 1998) because protein ubiquitination and degradation play important roles in the regulation of eukaryotic cell functions, such as the cell cycle and apoptosis. Proteasome inhibitors are considered to be a relatively nonspecific way to target ubiquitin-dependent protein degradation. There has been speculation that inhibiting the degradation of a large number of proteins by proteasome inhibitors assures that multiple pro-apoptotic, anti-survival activities can proceed, and inhibiting E3 ligase using specific inhibitors has a limited effect because of compensation by other enzymes (Mattern et al., 2012). Bortezomib was the first proteasome inhibitor to make it through clinical trials, and it is currently approved to treat patients with multiple myeloma. The proteasome is the nonspecific final step of targeted ubiquitin-dependent protein degradation, and E1 is the first step of ubiquitination. MLN4924 is currently in a Phase II clinical trial of E1 inhibitor for hematologic cancers (Chen et al., 2011). Blocking the first step of E1 in ubiquitination using ginsenosides is a new direction for preventing diseases such as cancer.