Ubiquitin activating enzyme activates ubiquitin by a three

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 cc-5013 (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). Ginsenoside Re-lysine mixture had increased anticancer effects upon heat processing by regulating Bcl-2 and Bax (Yamabe et al., 2013). Ginsenoside Re (20mg/kg) inhibited NF-κB activation by inhibiting LPS binding to TLR4 on macrophages in TNBS-treated mice (Lee, Hyam, Jang, Han, & Kim, 2012). The chemistry, metabolism, and pharmacokinetics of ginsenoside Re were shown in Peng et al. (2012). Ginsenoside Re only decreased the chymotrypsin-like activity of 26S proteasome by 10% (Chang et al., 2008). Fifty micrometres ginsenoside Re inhibited 40% of the E1 activity. Therefore, ginsenosides Re and Rg1 are in a novel class of ubiquitin E1 inhibitors within functional foods application or herbal medicine. Ginsenoside Rb1 can protect cells from UV radiation-induced apoptosis by inducing DNA repair (Cai, Jin, Luo, Lin, & Gao, 2009). Treating HepG2 cells with ginsenoside Rd significantly inhibited metastasis, most likely by blocking MMP activation and MAPK signaling pathways involved in cancer cell migration (Yoon, Choi, Cha, & Lee, 2012). Ginsenoside Rd, Re, Rb1, and Rg1 (100μg/mL) decreased MDR1 protein levels in MCF-7/ADR cells. Ginsenoside Rd most potently inhibited MDR1 protein expression without cytotoxicity (Pokharel, Kim, Han, Oh, & Kang, 2010). In our previous study, ginsenosides Rb1 and Rd inhibited 30% and 52.9% of the chymotrypsin-like activity but did not increase caspase-like and tryptic-like activities in the 26S proteasome (Chang et al., 2008). In this study, we demonstrated that ginsenosides Rb1, Rb2, Rc, and Rd increased E1 activity (Fig. 3). Fifty micrometres ginsenosides Rb1 and Rd increased E1 activity by 2.2- and 1.74-fold.