Active sites of peptides Asp-Asp-Asp-Tyr and Asp-Tyr-Asp-Asp protect against cellular oxidative stress
Introduction
Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, organic peroxides, and the hydroxyl radical, can cross cell membranes, leading to oxidative stress (Akhtar, Ahamed, Alhadlaq, & Alshamsan, 2017). Additionally, the antioxidant defense mechanisms of organisms to reduce ROS levels in vivo include both enzymatic and non-enzymatic systems. Specifically, enzymatic systems involve superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), whereas non-enzymatic systems mainly consists of exogenous antioxidants (Uribe, López-Giraldo, & Decker, 2018). Further, the mechanisms of free radical scavenging are primarily based on the transfer of hydrogen atoms or their supply to stop free radical reactions (Foti, Johnson, Vinqvist, Wright, Lrc, & Ingold, 2002).
It is also well known that antioxidant peptides can reduce free radical levels, and the antioxidant activities of such peptides have been determined primarily based on their amino acid compositions and peptide structures (Elias, Kellerby, & Decker, 2008). Specifically, amino acid structure (such as side chains and sequences) affects the antioxidant activities of peptides (Matsui et al., 2018), and compared with synthetic antioxidants, antioxidant peptides have fewer side effects and are readily digested and absorbed owing to their low molecular weights (500–1800 Da) and amino acid contents (2–20); they also have favorable nutritional and processing properties (Wattanasiritham, Theerakulkait, Wickramasekara, Maier, & Stevens, 2016). It has also been reported that some antioxidant peptides can be absorbed by the intestine-like Caco-2 cells; thus, it is probable that they affect the human body (Liu, Wu, & Wang, 2018). It has also been demonstrated that peptides derived from black soybean and chickpea exhibit free radical scavenging activities (Kou et al., 2013, Ren, 2010). In a previous study, we isolated the peptides Asp-Asp-Asp-Tyr (DDDY) and Asp-Tyr-Asp-Asp (DYDD) from DPP (Dendrobium aphyllum after Lactobacillus amylolyticus solid-state optimized fermentation peptides) (Liu, Ma, Yin, & Wu, 2018) and demonstrated that DPP shows chemical antioxidant activity. Therefore, we believe that DDDY and DYDD from DPP may have free radical-scavenging potential, indicating that they can be used as potential antioxidants. However, the mechanism of their antioxidant activity as well as their structure–related antioxidant activity are unclear.
Theoretical calculation-based analytical methods, such as the density functional theory (DFT), are tools that can be employed to explore the structure–activity relationships of chemical compounds (Chen, Xiao, Zheng, & Liang, 2015). Further, the quantum chemical parameters of antioxidants include the highest occupied and lowest unoccupied molecular orbital (HOMO and LUMO) energy, bond length, and molecular shape parameters (Ananda et al., 2012, Johns and Platts, 2014), and the active sites of antioxidant peptides can be identified by analyzing their HOMO energy and bond lengths (Cheng, Luo, Zeng, Li, Xiao, Bu, et al., 2015).
Given that DYDD and DDDY have a similar amino acid composition, it would be interesting to explore how their peptide sequences affect their antioxidant capacities. Therefore, in this study, we examined the mechanisms of the antioxidant activities of these peptides as follows: a) fluorescent-labeled peptides were used to determine dynamic changes; b) cytoprotective effects were evaluated by assessing oxidant-related signaling pathways; c) the structure–antioxidant activity relationships of the peptides were evaluated via quantum chemistry theoretical calculations and active site prediction; and d) the predicted active sites of the peptides were methylated, and ROS levels were determined for verification. The findings of this study clarify the underlying relationship between the structures of amino acid and their free radical scavenging activities. They may also facilitate the development of new antioxidant peptides with similar structures.
Section snippets
Materials
The peptides DYDD, DDDY, FITC-6-aminocaproic acid (acp)-DDDY, FITC-acp-DYDD, methylated-DDDY, and methylated-DYDD (Supplementary Figs. 1–4) were synthesized by Shanghai Science Peptide Biological Technology Co., Ltd. (Shanghai, China). The antioxidant enzymes assay kits were obtained from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China), and the ROS assay kits were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). TRIzol reagent was obtained from Life
Cell viability
Before determining the activities of the peptides DYDD and DDDY (each with purity greater than 98%), we first of all confirmed their non-toxic effects on HepG2 cells by conducting cell viability tests. Thus, it was observed that after all the treatments, the viability of the HepG2 cells remained above 90%. Reportedly, a cell viability above 80% is indicative of a non-cytotoxic effect of peptide treatment on HepG2 cells (J. Wu, Huo, Huang, Zhao, Luo, & Sun, 2017). In line with this previous
Conclusion
DDDY and DYDD showed the ability to directly scavenge free radicals to the end of protecting AAPH-treated HepG2 cells intracellularly and extracellularly, instead of the activation of the Nrf2/Keap1 signaling pathway. Based on quantum chemistry theoretical calculations, O58-H59 on DYDD and N10-H12 on DDDY were identified as antioxidant active sites (Fig. 4). Our findings provide insights into the structure–activity relationships of antioxidant peptides with different amino acid arrangements.
Funding sources
This study was funded by the Fundamental and Applied Basic Research Fund for Science and Technology Planning Project of Guangzhou (Project No. 202002030383), the Basic Research and Applied Basic Research Funding Project of Guangdong Province (Grant No. 2019A1515011283), Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology (Grant No. 2021B1212040013).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References (40)
- et al.
Mechanism of ROS scavenging and antioxidant signalling by redox metallic and fullerene nanomaterials: Potential implications in ROS associated degenerative disorders
Biochimica et Biophysica Acta
(2017) - et al.
Molecular structure, quantum mechanical calculation and radical scavenging activities of (E)-4,6-dibromo-2-[(3,5-dimethylphenylimino)methyl]-3-methoxyphenol and (E)-4,6-dibromo-2-[(2,6-dimethylphenylimino)methyl]-3-methoxyphenol compounds
Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy
(2014) - et al.
Structures and properties of antioxidative peptides derived from royal jelly protein
Food Chemistry
(2009) - et al.
Antioxidative, anti-inflammatory and hepatoprotective effects of resveratrol on oxidative stress-induced liver damage in tilapia (Oreochromis niloticus)
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
(2019) - et al.
Structure–activity relationships of new 4-hydroxy bis-coumarins as radical scavengers and chain-breaking antioxidants
Biochimie
(2010) - et al.
Purification and identification of antioxidant peptides from chickpea (Cicer arietinum L.) albumin hydrolysates
LWT - Food Science and Technology
(2013) - et al.
Characteristic analysis of peptide fraction extracted from dendrobium aphyllum after in vitro gastrointestinal digestion and fermentation by human fecal microbiota
International Journal of Peptide Research and Therapeutics
(2018) - et al.
Designing antioxidant peptides based on the antioxidant properties of the amino acid side-chains
Food Chemistry
(2018) - et al.
Computer-modeling-based QSARs for analyzing experimental data on biotransformation and toxicity
Toxicol In Vitro
(2001) - et al.
Isolation and identification of antioxidant peptides from enzymatically hydrolyzed rice bran protein
Food Chemistry
(2016)