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  • Protein degradation via UPP involves two steps One step is

    2020-05-21

    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 LY2409881 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. Ubiquitin-activating enzyme activates ubiquitin by a three-step process with ATP as a cofactor (Chen et al., 2011, Haas and Rose, 1982, Haas et al., 1982). We demonstrated that ATP is required for mRFP-Ub–E1 formation under non-reducing conditions (Fig. 2A). A time course of radioactive ATP production in ATP-PPi exchange assays shows a dramatic increase in 20min (Chen et al., 2011). As in a previous report, the cycle of mRFP-Ub–E1 formation is approximately 5–20min (Fig. 2B). We showed that mRFP-Ub–E1 formation experienced a robust increase and in turn increased the E1 concentration. The effects of mRFP-Ub–E1 formation as induced by E1 from 150nM to 300nM were increased by approximately 7.8-fold. However, the effects of mRFP-Ub–E1 formation induced by mRFP-Ub from 83.6nM to 150nM were increased by approximately 2.1-fold. Therefore, the E1 enzyme concentration is more significant to the formation of mRFP-Ub–E1. Natural antioxidants present in fruits and vegetables, such as vitamins and phenolic compounds, are considered to be responsible for these chemopreventive effects (Amarowicz and Pegg, 2013, Deng et al., 2013, Lin et al., 2013, Roupasa and Keogh, 2012, Senanayake, 2013). Ginsenoside Rg1 has been shown to activate Nrf2, to have antioxidant activities, and to have potential synergistic effects in combination with other ginsenosides, which could be important for cancer chemopreventive activities (Saw et al., 2012). Ginsenoside Rg1 may activate ERα via MEK/ERK in a ligand-independent manner in human breast cancer MCF-7 cells (Lau, Chen, Chan, Guo, & Wong, 2009). Ginsenoside Rg1 served as a functional ligand for the glucocorticoid receptor and in estrogen-like activities (Lau et al., 2008, Lee et al., 2003). Ginsenoside Rg1 can also be used as a novel therapeutic modality for inducing angiogenesis (Leung, Pon, Wong, & Wong, 2006). As our data have shown, ginsenoside Rg1 inhibited 32% the chymotrypsin-like activity but increased caspase-like and tryptic-like activities of the 26S proteasome (Chang et al., 2008). In vitro and in vivo protein degradation is reduced significantly only when either the trypsin-like or caspase-like sites are inhibited, together with the chymotrypsin-like sites (Kisselev, Callard, & Goldberg, 2006). However, 50μM ginsenoside Rg1 decreased E1 activity by 89.2% (Fig. 3).