A Preliminary Study On The Mechanism Of Buckwheat Trypsin Inhibitor Inhibited Cell Proliferation By Targeting Mitochondria

Mitochondria,which have their own genetic material and genetic system,are a semi autonomous organelle.There are about 1000 proteins in mitochondria,but mitochondria can synthesize only 1% of the protein by themselves;more than 99% of of which are encoded in the nucleus and synthesized in cytosol as precursor protein.The precursors are then imported into mitochondria.The import machincry of mitochondrial proteins which are located in different areas.Most of the precursor proteins contain the location separation signal in the N terminal,known as the leader peptide,which can guide its introduction to mitochondria.rBTI was obtained by gene recombination,prokaryotic expression and purification,which is a Potato I inhibitor with a molecular weight of 7.9 kD.It has been previously shown that rBTI can interact with the mitochondrial membrane protein Tom 20,causing mitochondrial damage to induce mitochondrial autophagy and then inhibit cell proliferation.These results suggest that rBTI can target mitochondria.The three-dimensional structure of rBTI contains an alpha helix,which is very similar to the mitochondrial leader peptide.However,it is not clear whether the rBTI alpha helix has a function similar to the mitochondrial leader peptide and its role in inhibiting the proliferation of tumor cells.In order to further investigate the molecular mechanisms of rBTI inhibiting the proliferation of tumor cells,the following contents are carried out in this article.1.MTT assay,ATP assay,qRT-PCR,Western Blot,Immunofluorescence and Determination of enzyme activity were used to analyze the effects of rBTI on the transcriptional level of glycolysis key enzymes,particularly HK II protein level,activity and spatial location in Hep G2 cells.The results showed that rBTI could reduce the HK II transcription and protein expression in dose and time dependent manner,and reduce the HK II in the space location of mitochondria,destroy the combination of HK II and VDAC,cause mitochondrial dysfunction,lead to mitochondrial autophagy,and inhibit the proliferation of HepG2 cells.2.Three mutants of rBTI-K20 A,rBTI-K23 A and rBTI-K20A/K23 A have obtained by site-specific mutagenesis the positive charge of the hydrophilic surface lysine(K)into alanine(A).The inhibitory activity and the secondary structure of the mutant were analyzed by the activity of trypsin,the determination of inhibition constant and circular dispectrometry.When the positive charge of the rBTI alpha helix structure was mutated,rBTI-K20 A,rBTI-K23 A and rBTI-K20A/K23 A had strong trypsin inhibitory activity,and could form stable enzyme inhibitor complexes.The secondary structure remained stable,and was no significant change after the mutation.3.MTT assay,electron microscope and laser scanning confocal microscope were used to analyze the inhibition effect of mutant on Hep G2,HCT116 and FHC,as well as the influence on cell morphology,mitochondrial morphology,and energy metabolism on ATP level and HK II expression.The results showed that rBTI-K20 A and rBTI-K23 A had no toxicity to normal cell FHC,but had obvious proliferation inhibition to Hep G2 and HCT116.Compared with rBTI,rBTI-K20 A and rBTI-K23 A treatment group,the cells were round,decreased adherent ability;enhanced mitochondria autophagy,mitochondrial morphology changed from reticulation to punctate,distributed around the nucleus;in the energy metabolism,rBTI-K20 A and rBTI-K23 A treatment group,the decrease of ATP level was more obvious,total HK II expression was significantly decreased.There,we have preliminarily demonstrated that the alpha helix in rBTI has the similar function of the mitochondrial leader peptide.After the hydrophilic surface charge is mutated,the rBTI can be blocked in the mitochondrial membrane,resulting in greater mitochondrial damage,thereby inhibiting the proliferation of tumor cells.rBTI may target the mitochondrial matrix,rBTI-K20 A and rBTI-K23 A may target the outer mitochondrial membrane,rBTI-K20A/K23 A may be blocked outside the mitochondria due to absence of a positive charge in the alpha helix and lose the interaction withTom 22.The above results provide a theoretical basis for the development of rBTI as a natural and targeted mitochondria anti-tumor drug.