Target Prediction And Bioactivity Research Of Rice Bran Active Peptide KF-8

Active peptides are natural substances that come from various sources and have a wide range of physiological functions.They have the potential to be anti-inflammatory,antioxidant,and immunomodulatory,and can alleviate and treat various diseases.Nowadays,more and more food-derived natural products are used to extract biologically active peptides and applied in the research and development of nutraceuticals and additives.In our previous study,we identified an octapeptide peptide with potential bioactivity from rice:KF-8.This paper utilized advanced computationalbased analytical methods and experimental tests to explore the bioactivity of KF-8.The study focused on identifying its possible bioactivity and targets,with a particular emphasis on its potential antioxidant activity.The research and results of this paper are summarized as follows:(1)Targets prediction and analysis for KF-8 based on computational models.Target prediction tools based on different principles were used to predict the potential targets of KF-8.Target screening and ranking were conducted based on both of recommendation thresholds and knowledge.Finally,12 potential targets related to antioxidant were selected.After analyzing existing literature studies and using knowledge-based discrimination,we selected SIRT1 and CXCR4 for further study.(2)Mechanism exploration for KF-8 based on molecular docking.By comparing the similarities and differences in the interactions of KF-8 with SIRT1 and CXCR4 by small molecule docking software and peptide docking software,we found that the different docking strategies both suggest that KF-8 can effectively bind to SIRT1 and CXCR4 at the corresponding agonist and inhibitor sites.The different docking software suggested that the main residues of KF-8 interacting with SIRT1 included Asn226,Glu230,Ile227,Thr219,Ile223,Gln222,and the main interaction types included hydrogen bonding,π-π stacking,ionic bonding,and hydrophobic interactions.The main residues that KF-8 interacts with CXCR4 included asp187,Glu288,Arg188,Asp97,Asp 193.The main interaction types included hydrogen bonding,H-pi,ionic bonding,hydrophobic interaction forces,etc.Meanwhile,our findings suggested that software specifically designed for peptide-protein docking provided a more reasonable evaluation of peptide flexibility compared to small molecule docking software.(3)In vitro experimental validation.Three cell lines,PMT339,GST,and EGFP,were constructed by plasmid transfection of HUVEC cells,and KF-8 carrying a fluorescent tag was identified to be stably expressed in HUVEC cells.Then the cell viability of different groups of cells was measured by CCK8 method,and it was found that the cells transfected with KF-8 had a significant resistance to oxidative damage.In addition,the results of laser confocal experiments showed that KF-8F co-localized with SIRT1 and CXCR4,respectively,and the co-localization coefficients reached:0.5879 and 0.5684,which tentatively demonstrated that KF-8 interacted with both proteins.(4)Screening of bioactive peptides similar with KF-8.In order to further validate the antioxidant activity of KF-8 and broaden its potential applications,we screened and compared food peptides from the bioactive library BP and antioxidant library AP that may have similar bioactivity to KF-8.The eligible peptides were screened and filtered by setting stepwise thresholds based on both sequence similarity and structural similarity strategies.Finally,87 food peptides were obtained based on sequence similarity,88 food peptides were filtered based on structural similarity,and 17 peptides were selected to have similar activities to KF-8 after cross-tabulation analysis.In summary,we used various advanced computational methods to explore the biological activity of food peptides,and successfully found the possible antioxidant targets of KF-8,and preliminarily evaluated the antioxidant effect of KF-8 by interacting with SIRT1 and CXCR4 through in vitro experiments.In addition,the results of expanding the activity of KF-8 have laid the foundation for exploring the bioactivity of KF-8-like peptides in the future.The research findings of this project contribute to a comprehensive understanding of the antioxidant activity of KF-8 and provide new solutions for studying the bioactivity of food-derived peptides.