rBTI的内化及诱导Hep G2细胞自噬的研究
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摘要
蛋白酶抑制剂广泛分布于自然界中,以不同形式存在于植物,动物及微生物体内,是一类可以作用于蛋白酶,并对酶有特异性抑制作用的小分子物质或蛋白质。它们按一定比例与酶结合形成可逆的蛋白质复合物,在血液凝固,补体级联反应,细胞凋亡的调控以及激素加工处理等过程中发挥重要的作用。荞麦胰蛋白酶抑制剂(Buckwheat Trypsin Inhibitor, BTI)是由69个氨基酸组成的分子量为7.9kD的耐热小分子蛋白,属于Potato I型蛋白酶抑制剂家族。本课题组的前期研究表明,重组的荞麦胰蛋白酶抑制剂(recombinant Buckwheat Trypsin Inhibitor, rBTI),对HepG2, EC9706, K562, HL60等肿瘤细胞株都有明显的抑制生长并诱导其凋亡的作用,而对正常肝细胞HL7702没有影响；rBTI可以诱导促凋亡基因表达上调,抗凋亡基因表达下降,可以使EC9706细胞周期阻滞于Go/G1期,但是具体的分子机制还不清楚。
本研究选用pExSec I表达载体,构建了含有BTI基因的表达质粒pExSecI-BTI,经原核表达和纯化,获得了无融合标签的rBTI,分析了rBTI内化进入HepG2细胞的机制,探讨了其诱导Hep G2细胞自噬的分子机制,以及与细胞表面的可能受体uPA的相互作用。主要结果如下：
1.构建了原核表达载体pExSec Ⅰ-BTI,并对其表达条件进行了优化,当诱导剂IPTG的浓度为0.5mmol/L,诱导时间为3.5h时,rBTI的表达量最高。根据rBTI执稳定性好的特点,对rBTI的纯化条件进行了优化(80℃热处理20min),经ResourceTMQ离子交换层析一步纯化即可获得纯度大于95%的rBTI。这一纯化步骤应用于rBTI的大规模制备亦十分有效。
2.将rBTI用FITC标记,综合运用多种内吞抑制剂及方法,如NH4CI, MDC,K+,蔗糖溶液等,以及RNA干扰,M期阻滞等技术和激光共聚焦显微镜研究了rBTI进入Hep G2细胞的方式。提出rBTI以细胞膜上尿激酶型纤溶酶原激活子(uPA)为受体,通过网格蛋白依赖性的内吞作用进入细胞,呈现浓度和能量依赖性,其中细胞膜电位在内吞过程中起着重要作用。
3.采用透射电子显微镜,MDC染色,流式细胞术等技术研究了rBTI诱导的HepG2细胞自噬。rBTI作用Hep G2细胞后,透射电子显微镜观察到细胞内部出现自噬体以及自噬溶酶体结构,细胞发生了自噬,细胞的自噬活性与rBTI的浓度呈正相关。通过MDC染色以及pCMV-GFP-LC3转染实验,进一步确定了rBTI可以诱导细胞产生自噬,当rBTI诱导4h后,Hep G2细胞即发生明显的自噬现象。应用激光共聚焦显微镜观察了rBTI作用前后胞内细胞器的变化。在rBTI作用12h后,Hep G2细胞内质网和溶酶体发生较大的变化,线粒体膜的通透性发生改变,但是细胞核的变化不明显。通过自噬抑制剂3-MA的联合使用以及细胞恢复实验等方法,明确了rBTI诱导的细胞死亡主要是自噬性程序性细胞死亡。
4.构建了pEGFP-N1-BTI单色荧光载体及pDsRed1-N1-EGFP-BTI双色荧光载体,观察了rBTI在Hep G2细胞内的定位,确定了rBTI主要定位于胞质中的自噬体及自噬溶酶体。据此我们提出假说：作为外源物的rBTI被细胞内的自噬相关分子选择性识别,进入自噬溶酶体被降解,过度的自噬诱发细胞失去固有的稳态,进而走向死亡。即rBTI介导的细胞死亡是一种底物特异性的死亡机制。通过RT-PCR的方法,对rBTI作用后三个自噬相关分子(mTOR, Beclinl和PI3K Ⅲ)进行了RNA水平的分析,结合3-MA抑制剂实验,初步确定rBTI诱导的细胞自噬主要受Beclin1复合物的调控。
5.通过荧光光谱法、凝胶电泳法等,进一步确定了rBTI与uPA之间存在相互作用。在体外条件下,rBTI可作为uPA的底物,被uPA水解为两个片段。采用RT-PCR的方法,对rBTI作用后,uPA以及uPA下游两个分子(MMP-2和MMP-9)进行了检测,并通过明胶酶谱和细胞划痕实验研究了rBTI对HepG2细胞迁移的影响。结果表明,rBTI可以诱导uPA, MMP-2和MMP-9的表达,并促进Hep G2细胞迁移。
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.
本研究选用pExSec I表达载体,构建了含有BTI基因的表达质粒pExSecI-BTI,经原核表达和纯化,获得了无融合标签的rBTI,分析了rBTI内化进入HepG2细胞的机制,探讨了其诱导Hep G2细胞自噬的分子机制,以及与细胞表面的可能受体uPA的相互作用。主要结果如下：
1.构建了原核表达载体pExSec Ⅰ-BTI,并对其表达条件进行了优化,当诱导剂IPTG的浓度为0.5mmol/L,诱导时间为3.5h时,rBTI的表达量最高。根据rBTI执稳定性好的特点,对rBTI的纯化条件进行了优化(80℃热处理20min),经ResourceTMQ离子交换层析一步纯化即可获得纯度大于95%的rBTI。这一纯化步骤应用于rBTI的大规模制备亦十分有效。
2.将rBTI用FITC标记,综合运用多种内吞抑制剂及方法,如NH4CI, MDC,K+,蔗糖溶液等,以及RNA干扰,M期阻滞等技术和激光共聚焦显微镜研究了rBTI进入Hep G2细胞的方式。提出rBTI以细胞膜上尿激酶型纤溶酶原激活子(uPA)为受体,通过网格蛋白依赖性的内吞作用进入细胞,呈现浓度和能量依赖性,其中细胞膜电位在内吞过程中起着重要作用。
3.采用透射电子显微镜,MDC染色,流式细胞术等技术研究了rBTI诱导的HepG2细胞自噬。rBTI作用Hep G2细胞后,透射电子显微镜观察到细胞内部出现自噬体以及自噬溶酶体结构,细胞发生了自噬,细胞的自噬活性与rBTI的浓度呈正相关。通过MDC染色以及pCMV-GFP-LC3转染实验,进一步确定了rBTI可以诱导细胞产生自噬,当rBTI诱导4h后,Hep G2细胞即发生明显的自噬现象。应用激光共聚焦显微镜观察了rBTI作用前后胞内细胞器的变化。在rBTI作用12h后,Hep G2细胞内质网和溶酶体发生较大的变化,线粒体膜的通透性发生改变,但是细胞核的变化不明显。通过自噬抑制剂3-MA的联合使用以及细胞恢复实验等方法,明确了rBTI诱导的细胞死亡主要是自噬性程序性细胞死亡。
4.构建了pEGFP-N1-BTI单色荧光载体及pDsRed1-N1-EGFP-BTI双色荧光载体,观察了rBTI在Hep G2细胞内的定位,确定了rBTI主要定位于胞质中的自噬体及自噬溶酶体。据此我们提出假说：作为外源物的rBTI被细胞内的自噬相关分子选择性识别,进入自噬溶酶体被降解,过度的自噬诱发细胞失去固有的稳态,进而走向死亡。即rBTI介导的细胞死亡是一种底物特异性的死亡机制。通过RT-PCR的方法,对rBTI作用后三个自噬相关分子(mTOR, Beclinl和PI3K Ⅲ)进行了RNA水平的分析,结合3-MA抑制剂实验,初步确定rBTI诱导的细胞自噬主要受Beclin1复合物的调控。
5.通过荧光光谱法、凝胶电泳法等,进一步确定了rBTI与uPA之间存在相互作用。在体外条件下,rBTI可作为uPA的底物,被uPA水解为两个片段。采用RT-PCR的方法,对rBTI作用后,uPA以及uPA下游两个分子(MMP-2和MMP-9)进行了检测,并通过明胶酶谱和细胞划痕实验研究了rBTI对HepG2细胞迁移的影响。结果表明,rBTI可以诱导uPA, MMP-2和MMP-9的表达,并促进Hep G2细胞迁移。
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.
引文
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