| Objective:Cell membrane penetrating peptide is a kind of polypeptide which can penetrate cell biofilm barrier by direct penetrating membrane or endocytosis,about 5～55bp in length.It has become an important research object in drug delivery system in recent years because of its ability to enter the cell to exert its efficacy through covalent bond binding or complexation of various types of drugs and particles.HMGB4 protein,as a newly discovered member of the high mobility group protein HMGB family,has a special HMG-box DNA binding domain and can bind DNA molecules nonspecificly.Its amino acid sequence is rich in basic amino acid residues such as arginine and lysine,positively charged and structurally composed of box A and box B in series,and lacking acidic tails compared to other members of the HMGB family.Studies have shown that HMGB proteins,as nuclear proteins,play a role in DNA binding and transcriptional regulation in physiological state,while they can trigger a series of inflammatory signal transduction by actively secreting or passively leaking out to the outside of inflammation or necrosis,binding to pattern recognition receptors such as target cell surface TLR.We are interested in the intracellular transport of HMGB protein family members to explore whether HMGB proteins have the ability to cross cell membranes and provide experimental evidence for further HMBG applications According to HMGB4,as a new member of the high mobility family protein family,many functions are not clear and there is more research potential.This study used bioinformatics technology to carry out a series of analysis of HMGB4,screened out candidate CPPs and further bioinformatics analysis and evaluation,and then verify its cell membrane penetration ability by culture cells.Methods(1)The HMGB4 protein fragments with the ability to penetrate membrane were predicted by data analysis by biological information.In this study,the CPP was predicted more comprehensively and accurately by the amino acid molecular composition,isoelectric point,hydrophilicity,solvent accessibility,surface charge,amino acid secondary structure analysis,composition features relevant to flexibility and disorder propensity of amino acid residues,and the evaluation of various CPPs prediction software.One segment(81～90)containing ten amino acid residues at the end of a HMGB4-Box A-C with a full length of 181 bp(abbreviated as box A-C)is predicted by statistical analysis can have the ability to penetrate the film,which will be further verified with the experimental method.(2)In combination with cell experiments,box A-C-FITC/NCO-FITC short peptides withfluorescent labels were incubated with box A-C/NCO and so on in vitro,and the intracellular fluorescence distribution was observed by fluorescence microscope.The changes of intracellular fluorescence in different conditions were detected by setting the concentration gradient of box A-C,the time gradient of incubation with cells,the temperature conditions of incubation,the effect of DMSO and the comparison with known penetrating peptides,which in turn reflected the efficiency of membrane penetration.Results(1)The results of data analysis HMGB4-box A the C end of the protein,a segment of 10 amino the polypeptide is likely to be a new CPPs,its amino acid sequence:KRRKRRKRDP,rich in alkaline arginine and lysine,a cationic polypeptide,peptide surface large-area distribution of positive charge,conducive to electrostatic adsorption with negatively charged cell membrane,through the 20 amino acids to promote disorder or order tendency and flexibility or rigidity analysis,HMGB4-box A-C peptide contains arginine,lysine,aspartic acid and proline are better disorder and flexibility.acids,especially arginine and lysine,while it has been reported CPPs the statistical results in the database are more structurally biased towards disorder,which may be more conducive to their membrane penetration,and CPPs more pliable traits also enhance its ability to flexibly penetrate the lipid-coating bimolecular layer.Studies have shown that the secondary structure of CPPs amino acids is mostly in the form coil irregular curl structure and α-spiral structure,while the data show that the secondary structure of HMGB4-box A-C peptide amino acids exists in the form of coil class.prediction of the water solubility of the peptide may suggest that its penetrating mechanism is likely to be direct diffusion.(2)The results of cell experiments on HMGB4-box A-C peptides showed that different concentrations of box A-C could penetrate the cell membrane into the cell,and the intracellular fluorescence increased with the increase of concentration in a certain concentration range(2.5μM～10.0μM),and the intracellular fluorescence increased with incubation time in a certain time range(0.5 h～2.0h).5% DMSO can obviously improve the box A-C penetration efficiency;and the penetration efficiency increased significantly with the decrease of temperature at 37℃,16℃ and 4℃,indicating that the penetration mechanism may be energy-independent direct diffusion,and the increased membrane permeability at low temperature may promote the penetration efficiency.Conclusion: A new kind of membrane-penetrating peptide has been successfully discovered by bioinformatics and cel lexperiments,and it calls:HMGB4-Box A-C. |
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