The UPP broadly involves proteolysis in biochemical processe
The UPP broadly involves proteolysis in biochemical processes and is a potential target for cancer therapy. The UPP degrades unfolding or damaged proteins by an ATP-dependent mechanism (Ciechanover, Elias, Heller, Ferber, & Hershko, 1980). It also plays an important role in semagacestat synthesis regulation, DNA damage repair, and has implications for tumourigenesis (Tu et al., 2012). Ubiquitination is catalyzed by the sequential action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin protein ligase (E3) (Hershko & Ciechanover, 1998). In humans, there is a single essential E1 and over 30 distinct E2s. E3s, of which there are estimated to be between 500 and 1000, are largely responsible for conferring specificity to ubiquitination (Ciechanover and Schwartz, 2004, Fang and Weissman, 2004). E1 is the common first step in ubiquitination, whether the proteasome represents the final destination of degradation for many ubiquitinated proteins.
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 amino acids 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