Study On The Effect Of Buckwheat Trypsin Inhibitor Helix Region On Cell Viability

Although mitochondria have their own genetic material and genetic system,more than 99% of mitochondrial proteins are encoded by nuclear genes.These mitochondrial proteins contain a leader sequence at the N-terminus of cytoplasmic synthesis,are recognized and transported by the mitochondrial outer membrane protein TOM system,and finally transport to different regions of the mitochondria to play a role.Recombinant buckwheat trypsin inhibitor(rBTI)is a type I Potato inhibitor that specifically targets mitochondria and interacts with the outer mitochondrial membrane protein Tom 20,triggering mitochondrial dysfunction and causing mitophagy to play an antitumor effect.Structural analysis showed that rBTI contained an ? helix in its three-dimensional structure,which has similar properties of the mitochondrial protein leader sequence.Whether the hydrophobic interaction of the ? helix recognized by Tom 20 is related to the hydrophobic properties of the helix,and whether the change of the hydrophobic properties of the amino acids of the helix affects the antitumor activity of rBTI is unknown.Therefore,in this paper,we will explore the recognition mechanism of rBTI targeting mitochondria,that is,the interaction between the alpha helix region and Tom20,to further clarify the influence of the amino acid composition of the helix on the antitumor effect of rBTI.The main contents are as follows:1.Tom20 intracellular region was purified by prokaryotic expression.Tom20 is an initiating receptor membrane protein imported by mitochondrial proteins,which recognize precursor sequences with different amino acid sequences.Tom20 includes the C-terminus(25-145)on the cytoplasmic side,the N-terminus on the stromal side,and the membrane space transmembrane.The intracellular region(25-145)is the region that recognizes the precursor sequence.Recombinant construction and its prokaryotic expression and purification were performed.The expression vector p GEX-4T-1 was used to obtain the recombinant Tom20 intracellularregion r Tom20,which can be expressed in large quantities,which is convenient for further research on the identification of Tom20 and the precursor sequence.2.The rBTI helical peptide was optimized and synthesized,and the interaction between the peptide and r Tom20 was studied in vitro by ultraviolet spectrophotometry and isothermal titration calorimetry.A series of rBTI helical peptides were synthesized.On the non-polar surface the low-polarity alanine A was mutated to high-hydrophobic isoleucine I,and the high-hydrophobic I and A were mutated to low-hydrophobic glutamine Q.Mutation of positively charged lysine K to hydrophobic uncharged A or hydrophilic negatively charged glutamic acid E,which increaseed or decreased the hydrophobicity of the peptide and rBTI.The interaction between r Tom20 and peptides were studied in vitro by ultraviolet spectrophotometry and isothermal titration calorimetry,and the binding constant model of the interaction between r Tom20 and peptides were calculated.The results show that the optimization of the rBTI helix region has an impact on Tom20's recognition binding and presents a dynamic balance.The larger the binding constant is,the more recognition and binding of rBTI helix polypeptide can be achieved by Tom20,and it is related to the hydrophobic nature of the rBTI helix.The hydrophobicity of the rBTI helix region is the same as that of Tom20.The high-hydrophobic helix binds to Tom20 more strongly.3.rBTI and its mutants were obtained by site directed mutagenesis,and the inhibitory effect of peptide,rBTI and their mutants on Hep G2 cell proliferation was analyzed by MTT assays.We have successfully obtained rBTI and its mutants that can be expressed in large quantities.MTT detected cell proliferation inhibition.The results of the peptide showed that compared to P0,the optimized peptide had almost no change in the cell activity of Hep G2;rBTI and its mutants showed the effect of rBTI helix optimization on Hep G2 cell proliferation.rBTI-K20 A and rBTI-K23 A have a significant proliferation inhibition effect on Hep G2,rBTI-K20 E,rBTI-K23 E,rBTI-K20/23 E also have a significant proliferation inhibition effect on Hep G2 and the inhibition effect of these three mutants was similar,but weaker than that of rBTI-K20 A and rBTI-K23 A.Compared with rBTI,non-polar amino acid modification rBTI-A21/22 I has a certain strengthening effect on the inhibition of cell proliferation,while the treatment group rBTI-I24/25 A,rBTI-A21/22I-I24/25 A,rBTI-A21/22Q-I24/25 Q,the inhibitory effect did not change much,but similar to rBTI,it showed the same cell proliferation inhibition effect.In view of the combination of hydrophobic properties and binding constants,the rBTI helical region has increased hydrophobicity and enhanced binding to Tom20,which can significantly inhibit the proliferation of Hep G2 cells.In addition,the change in the charge and hydrophobic properties of the ?-helical polar plane of rBTI is particularly important.From this,we have preliminary demonstrated that the ?-helix in rBTI can dynamically bind to Tom20.By changing the hydrophobicity of the non-polar surface of the spiral and the charge of the polar surface,it can enhance or weaken its inhibitory effect on Hep G2 cell proliferation.The above results provide the identification and optimization of rBTI's highly effective anti-tumor effect,and provide a theoretical basis for rBTI's development of a natural anti-tumor drug that targets mitochondria efficiently.