Mechanism Of Internal Izati On And Hep G2Autophagy Induced By RBTI

Protease inhibitors are ubiquitous in nature and are widely distributed in plants, animals and microorganisms. Their main role is to regulate the activity of proteolytic enzymes. They form reversible, stoichiometric protein-protein complexes, and play key regulatory roles in many biological processes, such as regulation of blood coagulation, the complement cascade, apoptosis, and the hormone-processing pathways. Buckwheat trypsin inhibitor (BTI) is composed of69amino acids and has a molecular weight of7.9kD, it exhibits high sequence consistency with other potato I-type inhibitors derived from different plants sources. Our previous studies have shown that rBTI exhibited high tumor suppressor activity that could induce apoptosis in certain tumor cell lines, such as EC9706, Hep G2, HeLa, K562, HL60and other solid tumor cells. Pro-apoptotic genes were up-regulated and anti-apoptotic gene expression was decreased when the tumour cells were treated with rBTI, and cell cycle arrested in G0G1phase in EC9706cells.In this study, pExSecI-BTI vector was constructed and expressed in E.coli BL21(DE3), and an rBTI for no fusion tag was obtained. FITC-BTI was used to determine the mechanism by which rBTI enters the cytosolic compartment. To investigate the autophagy induced by rBTI, transmission electron microscopy, RT-PCR, MDC staining and other technology has been used. The interaction between rBTI and uPA (urokinase-type plasminogen activator, uPA) was determined by gelatin zymography, fluorescence spectrometry and pull-down assay. The main results of this study are as follows:1. The recombinant expression plasmid pExSec Ⅰ-BTI was constructed and the vector was expressed in E.coli BL21(DE3). Prokaryotic expression conditions were optimized. When the IPTG concentration is0.5mmol/L and the induction time is3.5h, the expression of rBTI is the largest. Purification conditions were also optimized (heat treatment20min at80℃). Purified rBTI was obtained by ion exchange chromatography on ResourceTM Q.2. FITC-BTI could colocalized with labeled transferring and enter Hep G2cells in a concentration-and energy-dependent manner. Incubation of Hep G2cells with an isotonic/high K+buffer (KPBS) or an NH4C1solution abolished diffuse. FITC-BTI colocalized with a urokinase-type plasminogen activator (uPA) on the surface of Hep G2cells, implying that uPA was the target receptor for rBTI. We come to a conclusion from these results that both endocytosis and membrane potential are required for rBTI to enter into Hep G2cells.3. Transmission electron microscopy, MDC staining and flow cytometry were used to study autophagy in Hep G2cells induced by rBTI. A lot of autophagosomes and autolysosomes were observed under transmission electron microscopy. In the cytoplasm of Hep G2cells transfected with pCMV-GFP-LC3, a lot of GFP-LC3punctate structures appeared after induction by rBTI. Similar results have also been observed by MDC staining. Hep G2cell occurred autophagy when they were treated with rBTI for4hours. Further more, when cells were treated for12h, the number of endoplasmic reticulum and lysosomes were greatly reduced, membrane permeability of mitochondrial was changed, but the nucleus did not change obviously. Using the autophagy inhibitor3-MA and combining cell recovery experiments, we clear the rBTI-induced cell death is mainly autophagic programmed cell death.4. pEGFP-N1-BTI and pDsRed1-N1-EGFP-BTI were constructed to investigate the intracellular localization of rBTI and the results showed that rBTI mainly localized in the cytoplasm autophagosomes and autolysosomes. We put forward a hypothesis, as a xenobiotic, rBTI was recognized by related molecules of selective autophagy in the intracellular, and then was wrapped by autophagosomes and degraded by autolysosomes finally. Excessive autophagy was induced, the cells loss inherent steady state as a result, and then to die. Therefore rBTI mediated cell death is the death of a substrate specificity mechanism. Several autophagy related molecules (mTOR, Beclin1in and PI3K III) was analysis on RNA levels, the expression of Beclinl and PI3K III were reduced. Combining with3-MA inhibitor experiments, we initially identified rBTI induced Hep G2cells autophagy is mainly affected by Beclin1complexes.5. By fluorescence spectrometry, gel electrophoresis and other methods to further define the interaction between the rBTI with uPA. In vitro, rBTI was hydrolyzed into two fragments as a substrate of uPA. By RT-PCR method, the expression of uPA and uPA downstream molecules (MMP-2and MMP-9) were tested and studied by gelatin zymography. And cell wound healing experiments were used to investigate the effect on the Hep G2migration induced by rBTI. The results showed that expression levels of uPA and MMP-2and MMP-9were up-regulated, and rBTI coule promote cell migration.