PP2Ac Y127位点硝基化在肾小管管周毛细血管内皮细胞转分化中的作用及干预研究

发布时间：2018-07-03 20:10

本文选题：丝/苏氨酸磷酸酶2A催化亚单位C(PP2Ac) + 酪氨酸位点硝基化　；参考：《华中科技大学》2016年博士论文

【摘要】：目的肾小管管周毛细血管内皮细胞向间充质转分化(EndMT)是新近发现的肾间质肌成纤维细胞的重要来源,其发生机制与内皮细胞紧密连接失去功能密切相关。研究表明蛋白丝/苏氨酸磷酸酶2A (PP2A)的催化亚单位C (PP2Ac)酪氨酸位点硝基化可激活PP2A并能下调紧密连接连接蛋白(Occludin)丝/苏氨酸位点磷酸化,从而参与内皮细胞紧密连接的功能调控。本研究探讨PP2Ac酪氨酸核心位点硝基化对内皮细胞紧密连接的影响,并阐明该位点是肾小管管周毛细血管EndMT的重要调控分子,参与进行性肾间质纤维化,为探索防治肾间质纤维化进行性发展的新治疗靶点提供实验依据。方法体外构建硝基化反应体系,用过氧化亚硝酸盐(Peroxynitrite)刺激重组的PP2Ac蛋白,western blotting收集对照组和刺激组中的蛋白行质谱检测,分析PP2Ac酪氨酸发生硝基化的具体位点。对上述硝基化位点进行计算机模拟分析以明确各位点空间暴露程度从而确定调控PP2A活性的核心位点。同时构建、合成以核心位点为中心的底物模拟多肽和对照多肽,体外实验中验证该多肽对EndMT的干预作用,体内实验首先应用活体成像技术检测多肽在小鼠体内的分布以及代谢状态,然后通过尾静脉联合腹腔多肽注射观察该多肽在UUO小鼠模型中对管周毛细血管EndMT的干预效应。结果：质谱检测分析PP2Ac发生硝基化的具体位点有Y284/267/265/218/130/127 。在这六个位点中,Y127在蛋白质空间结构、相邻氨基酸电荷分布上具有更稳定的空间表位优势,在PP2Ac活化过程中起核心作用。以127位点为中心构建及合成的可穿透性底物模拟多肽和对照多肽能够抑制TGF-β1诱导的EndMT。体外试验中,多肽在肝脏和肾脏中具有较高的药物积聚；较对照多肽,底物模拟多肽可明显抑制Occludin去磷酸化,减少EndMT发生,稳定外周毛细血管结构并部分减轻肾间质纤维化。结论以PP2Ac Y127为核心的底物模拟多肽可有效干预内皮细胞转分化,是肾间质纤维化治疗的潜在生物靶点。第一部分管周毛细血管EndMT是肾间质纤维化的重要组成部分目的在原发和继发性肾脏疾病以及UUO动物模型中探讨EndMT发生的证据,观察转化生长因子β1 (Transforming growth factor beta 1, TGF-β1)促进内皮细胞间充质转分化效应,探讨肾间质纤维化肌成纤维细胞内皮源性。方法选取原发性肾脏疾病(IgA肾病)以及继发性肾脏疾病(狼疮肾炎和糖尿病肾病)肾活检标本,应用血管内皮细胞特异性标志物CD31和肌成纤维细胞特异性标志物a-SMA行连续切片免疫荧光双标检测,并在阳性区域进行共聚焦三维结构重建明确CD31+a-SMA+共表达。体内实验采用C57小鼠单侧输尿管梗阻肾病模型,荧光双标结合三维重建观察CD31+a-SMA+共表达情况。体外实验采用TGF-β1 (lOng/ml)刺激人脐静脉内皮细胞72h建立EndMT细胞模型,镜下观察细胞形态变化,免疫荧光及western blot检测血管内皮钙粘素(VE-cadherin,内皮特异性标志物)和平滑肌肌动蛋白(a-SMA)表达变化。结果和对照组相比,在IgA肾病、狼疮肾炎、糖尿病肾病以及UUO动物模型肾间质纤维化区域,管周毛细血管管腔异常膨大或缩小;共聚焦显微镜下观察发现CD31和α-SMA共表达增多,差异具有统计学意义(P0.05)；对该共表达部位血管进行三维立体重建,发现部分内皮细胞可表达α-SMA。体外实验,TGF-β1刺激72h后,内皮细胞形态由铺路石状向梭型转化,细胞与细胞间距明显增大;和对照组相比,肌成纤维细胞标志物α-SMA表达显著增加,而内皮细胞标志物VE-cadherin表达下调,差异具有统计学意义(P0.05)。结论在原发和继发性肾脏病中,内皮细胞间充质转分化是肾间质纤维化肌成纤维细胞的重要组成部分。内皮细胞受到促纤维化因子刺激后,细胞表型和功能特性均可向间充质细胞转化。第二部分PP2A活化及PP2Ac硝基化对EndMT的作用研究目的探讨内皮细胞PP2A活化及PP2Ac硝基化对EndMT进程的影响方法(1)提取UUO小鼠不同梗阻时间点下肾脏组织蛋白以及不同TGF-β1刺激时间点下细胞总蛋白,继而用丝／苏氨酸蛋白磷酸酶活性试剂盒监测PP2A的活性变化;(2)采用PP2A特异性抑制剂冈田酸(Okadaic acid, OA)预处理后,观察TGF-β1刺激下α-SMA、VE-cadherin蛋白表达变化以及PP2A底物紧密连接蛋白Occludin磷酸化水平;(3) PP2Ac是PP2A的活性亚基,运用免疫组化及免疫荧光定位PP2Ac在肾脏组织的表达,并通过免疫共沉淀检测TGF-β1刺激下内皮细胞PP2Ac翻译后修饰变化及其对PP2A磷酸酶活性的影响。结果(1)和对照组相比,随着梗阻时间延长,PP2A活性逐渐增加并在UUO-14d达到峰值;体外实验,TGF-β1刺激下,PP2A的活性在15min开始升高,在60min时显著增强(P0.05) 。 (2) western blot结果显示较TGF-β1组,OA干预组α-SMA表达显著下调而VE-cadherin表达显著上调；免疫共沉淀示正常HUVE细胞紧密连接蛋白Occludin磷酸化水平维持在较高水平,TGF-β1刺激72h后磷酸化水平明显降低,OA预处理后可部分抑制Occludin磷酸化减少(P0.05)。(3)免疫荧光检测可见PP2Ac主要在肾小球及肾间质毛细血管内皮细胞表达；与对照组相比,PP2Ac蛋白表达量并无显著改变,但是PP2Ac硝基化水平明显升高;和其他翻译后修饰相比,PP2Ac硝基化可明显上调PP2A磷酸酶活性(P0.05)。结论PP2A激活可破坏内皮细胞稳定性,促进EndMT的发生,抑制PP2A活性可减弱EndMT效应。PP2Ac硝基化是内皮细胞PP2A活性增强的主要原因,提示PP2Ac硝基化在内皮细胞转分化中发挥重要的促进作用。第三部分PP2Ac Y127硝基化在EndMT中的作用研究目的前期研究表明蛋白丝/苏氨酸磷酸酶2A (PP2A)的催化亚单位C (PP2Ac)硝基化可激活PP2A并能下调紧密连接蛋白Occludin丝/苏氨酸位点磷酸化,从而参与内皮细胞紧密连接功能调控。本部分研究探讨PP2Ac酪氨酸硝基化具体位点及核心位点,并阐明该位点是肾小管管周毛细血管EndMT的关键调控位点,参与进行性管周毛细血管萎缩,为探索防治肾间质纤维化提供潜在的治疗靶点和干预策略。方法 (1)体外构建硝基化反应体系,用过氧化亚硝酸盐(Peroxynitrite)刺激重组PP2Ac蛋白,western blot收集对照组和刺激组中的蛋白行质谱检测,分析PP2Ac酪氨酸发生硝基化的具体位点。(2)对上述硝基化位点进行计算机模拟分析以明确各位点空间暴露程度从而确定调控PP2A活性的核心位点。同时构建以酪氨酸硝基化位点为中心的底物模拟多肽,偶联TAT穿膜肽,采用化学合成法获得融合多肽。(3)为了验证多肽是否能够顺利穿透进入内皮细胞,高效运载PP2Ac活性片段,我们运用FITC标记该多肽。将多肽与HUVECs共培养72小时后,荧光显微镜下观察该多肽细胞内荧光强度并采用CCK8法筛选最佳细胞药物浓度。(4)体外实验验证上述多肽对EndMT的干预效应,进一步明确PP2Ac核心位点。结果(1)质谱结果显示,对照组仅检测出一个硝基化位点Y218,实验组PP2Ac硝基化位点增加至六个：Y284/267/265/218/130/127.计算机模拟分析显示,在这六个位点中,Y127在蛋白质空间结构、相邻氨基酸电荷分布上具有更稳定的空间表位优势,在PP2Ac活化过程中可能起核心作用。(2)以构建的TAT-127WT融合多肽为例,携带荧光标记的多肽可渗透进内皮细胞且可持续到实验终点时间72h,对内皮细胞具良好的穿透效率。CCK8法检测不同浓度下该多肽的细胞毒性,结果表明在5-10uM浓度下该多肽不仅具有良好的穿透性,对细胞的毒性作用最低。因此,在后续的细胞实验中选择10uM进行研究。(3)以各位点为中心构建合成的可穿透性底物模拟多肽抑制TGF-β1诱导的EndMT效应依次为：Y127Y265Y130284Y267。结论酪氨酸127是PP2Ac硝基化及PP2A活化的关键位点,以该位点为核心构建的底物模拟多肽可有效抑制EndMT,可能是管周毛细血管病变、间质纤维化治疗的潜在生物靶点。第四部分TAT-Y127WT对EndMT的体内外干预研究目的前期研究表明,PP2Ac Y127是PP2A全酶活性的核心位点,以PP2Ac Y127为中心构建的底物模仿多肽较其他位点具有更显著的抑制EndMT效应。本部分研究进一步探讨TAT-Y127WT及其对照多肽TAT-Y127Scr对内皮细胞转分化的体内外干预效应,为肾间质纤维化的治疗提供实验依据和策略。方法(1)合成以PP2Ac Y127为中心的底物模拟多肽(TAT-127WT)和对照多肽(TAT-127Scr),预处理HUVECs 30min,给予TGF-β1 (10ng/ml)刺激72h, western blot检测a-SMA、VE-cadherin蛋白表达变化及PP2A底物Occludin磷酸化水平。(2)运用活体动物成像技术,在多肽注射前、注射后30min、4h及24h观察其在体内的代谢情况。同时在相应时间点取出重要器官和组织进行示踪。(3)尾静脉联合腹腔注射(5 nmol/g,术前一次、术后1次／天),2周后观察该多肽对UUO模型鼠肾脏管周毛细血管内皮细胞EndMT的干预效应,免疫组化观察α-SMA、Vimentin沉积情况。结果(1) Western blot结果显示TAT-127WT组较TGF-β1组、TAT-127Scr组,a-SMA表达显著下调,而VE-cadherin和occludin磷酸化水平显著上调,差异具有统计学意义(P0.05) 。 (2) TAT-127WT在体内的代谢速度较快,4h后不足10%,24h后完全从体内代谢。肝脏和肾脏药物的富集程度较高,心、肺、脾脏程度低。检测血液中荧光强度进一步证明该融合多肽的半衰期大约为2.5h。(3)较对照组,经Y127WT干预后,肾组织微血管密度上调,具有一定EndMT抑制效应。免疫组化结果显示,干预组能够部分减少α-SMA、Vimentin在肾间质中的表达和积聚,差异具有统计学意义(P0.05)。结论通过抑制EndMT,以PP2Ac Y127为核心的底物模拟多肽对UUO小鼠肾小管管周毛细血管病变具有一定的改善作用,为肾间质纤维化治疗提供了新的思路。
[Abstract]:Objective intermesenchymal transition (EndMT) is an important source of newly discovered renal interstitial myofibroblasts. The mechanism is closely related to the close connection of endothelial cells to the loss of function. The study shows that the tyrosine loci nitro of C (PP2Ac) C (PP2Ac) is catalyzed by protein filament / threonine phosphatase (PP2A). The activation of PP2A and the downregulation of the phosphorylation of the tightly connected connexin (Occludin) silk / threonine loci are involved in the functional regulation of the tight junction of endothelial cells. This study explores the effect of nitroylation of PP2Ac tyrosine core sites on the tight junction of endothelial cells, and elucidates that this site is an important regulation of the capillary EndMT in the tubules of the renal tubules. The molecule, participating in progressive renal interstitial fibrosis, provides experimental basis for the exploration of new therapeutic targets for the prevention and treatment of renal interstitial fibrosis. Methods the nitro reaction system was constructed in vitro, the recombinant PP2Ac protein was stimulated with nitrite (Peroxynitrite), and Western blotting was used to collect the protein mass spectrometry in the control group and the stimulation group. Analysis of the specific loci of nitration of PP2Ac tyrosine. Computer simulation analysis of the nitrocellulose sites to determine the degree of space exposure to determine the core sites to regulate the activity of PP2A. At the same time, we construct, synthesize the substrate mimic and control peptides centered on the core site, and verify the polypeptide in vitro. The intervention of EndMT in vivo was first used in vivo imaging to detect the distribution and metabolic state of peptides in mice. Then, the intervention effect of the polypeptide on the peripheral capillary EndMT in the UUO mouse model was observed by intraperitoneal polypeptide injection with the tail vein. The loci have Y284/267/265/218/130/127. In these six loci, Y127 has a more stable spatial epitope advantage in the spatial structure of protein, the distribution of adjacent amino acids, and plays a core role in the process of PP2Ac activation. The penetrable substrate mimic peptide and the control polypeptide can inhibit the TGF- beta 1 induced by the 127 loci as the center. In EndMT. in vitro test, peptides have high drug accumulation in the liver and kidney. Compared with the control polypeptide, the substrate mimic peptide can obviously inhibit the dephosphorylation of Occludin, reduce the occurrence of EndMT, stabilize the peripheral capillary structure and partially alleviate the renal interstitial fibrosis. Conclusion the analog peptide of substrate with PP2Ac Y127 as the core can be effectively dried. Preendothelial cell transdifferentiation is a potential biological target for the treatment of renal interstitial fibrosis. The first part of the perivascular capillary EndMT is an important part of renal interstitial fibrosis in order to explore the evidence of EndMT in primary and secondary renal diseases and UUO animal models, and to observe the transformation of growth factor beta 1 (Transforming growth factor be). TA 1, TGF- beta 1) promote the effect of mesenchymal transition in endothelial cells and explore the endotheliogenesis of myofibroblast in renal interstitial fibrosis. Methods the renal biopsy specimens of primary renal disease (IgA nephropathy) and secondary renal diseases (lupus nephritis and diabetic nephropathy) were selected, and CD31 and myofibroblast specific markers of vascular endothelial cells were used. The specific marker a-SMA was detected by continuous slice immunofluorescence, and the co confocal three-dimensional structural reconstruction was used in the positive region to clear the co expression of CD31+a-SMA+. In the body, the C57 mouse unilateral ureteral obstruction nephropathy model was used, and the co expression of CD31+a-SMA+ was observed by the fluorescence double labeling combined with three-dimensional reconstruction. The in vitro experiment was carried out with TGF- beta 1 (lOng/ml). The human umbilical vein endothelial cells (72h) were stimulated to establish a EndMT cell model. The morphological changes of the cells were observed under the microscope. The expression of vascular endothelial cadherin (VE-cadherin, endothelial specific marker) and smooth muscle actin (a-SMA) was detected by immunofluorescence and Western blot. The results were compared with those in IgA nephropathy, lupus nephritis, diabetic nephropathy and UU. O animal model renal interstitial fibrosis area, abnormal expansion or reduction of perivascular capillary tube; confocal microscope observation found that CD31 and alpha -SMA co expression increased, the difference was statistically significant (P0.05); the co expression part of the vascular three-dimensional reconstruction, the expression of partial endothelial cells can express alpha -SMA. in vitro experiment, TGF- beta 1 prickly After stimulated 72h, the morphology of endothelial cells was transformed from paving stone to spindle type, and the distance between cells and cells increased obviously. Compared with the control group, the expression of myofibroblast marker alpha -SMA increased significantly, while the expression of VE-cadherin was down down, the difference was statistically significant (P0.05). Conclusion endothelial cells were in primary and secondary renal diseases. Mesenchymal stromal differentiation is an important component of myofibroblast in renal interstitial fibrosis. Endothelial cells are stimulated by fibrotic factors. Cell phenotypes and functional properties can be transformed into mesenchymal cells. Second the activation of PP2A and the effect of PP2Ac nitro on EndMT, the purpose of this study is to explore the activation of endothelial cells PP2A and PP2Ac nitration. The influence method of EndMT process (1) extracts the total protein of the renal tissue protein in UUO mice and the time point of different TGF- beta 1 at different time points. Then the activity changes of PP2A are monitored with silk / threonine protein phosphatase activity kit. (2) PP2A specificity inhibitor, Okadaic acid, OA, is used to observe the TGF The expression of alpha -SMA, VE-cadherin protein expression and PP2A substrate close connexin Occludin phosphorylation level under the stimulation of beta 1; (3) PP2Ac is the active subunit of PP2A, using immunohistochemistry and immunofluorescence to locate the expression of PP2Ac in the renal tissue, and to detect the changes of PP2Ac post-translational modification of endothelial cells stimulated by TGF- beta 1 by immunofluorescence. The effect of PP2A phosphatase activity. (1) compared with the control group, the activity of PP2A increased gradually and reached the peak at UUO-14d with the prolongation of the obstruction time. In vitro, under the stimulation of TGF- beta 1, the activity of PP2A increased in 15min and increased significantly in 60min (P0.05). (2) Western blot results showed the TGF- beta 1 group, the OA intervention group expressed a significant expression. The expression of VE-cadherin was significantly up-regulated, and immunoprecipitation showed that the phosphorylation level of the close connexin Occludin in normal HUVE cells was maintained at a high level. The phosphorylation level of TGF- beta 1 stimulated 72h significantly decreased, and Occludin phosphorylation decreased partially after OA pretreatment (P0.05). (3) immunofluorescence detection showed that PP2Ac was mainly in the glomerulus Compared with the control group, the expression of PP2Ac protein was not significantly changed, but the level of PP2Ac nitrochemical increased significantly. Compared with other post-translational modifications, PP2Ac nitro could obviously increase the activity of PP2A phosphatase (P0.05). Conclusion PP2A activation could destroy the stability of endothelial cells and promote the occurrence of EndMT. Inhibition of PP2A activity can weaken the EndMT effect of.PP2Ac nitration is the main reason for the enhancement of PP2A activity in endothelial cells, suggesting that PP2Ac nitration plays an important role in the transformation of endothelial cells. Third the role of PP2Ac Y127 nitration in EndMT The nitrification of C (PP2Ac) can activate PP2A and reduce the phosphorylation of the close connexin Occludin / threonine site, thus participating in the regulation of the tight junction function of endothelial cells. This part studies the position and core loci of PP2Ac tyrosine nitroylation, and clarifies that this site is the key regulation of the EndMT in the capillary tube of the renal tubule. Loci, participating in progressive perivascular capillary atrophy, provides potential therapeutic targets and intervention strategies for the prevention and treatment of renal interstitial fibrosis. Methods (1) the nitro reaction system was constructed in vitro, the recombinant PP2Ac protein was stimulated with nitrite (Peroxynitrite), and Western blot was used to collect protein mass spectrometry in the control group and the stimulation group. Analysis of the specific loci of nitro formation of PP2Ac tyrosine. (2) computer simulation analysis of the nitro loci was carried out to determine the degree of space exposure to determine the core loci that regulate the activity of PP2A. At the same time, the substrate simulated polypeptides with tyrosine nitro site as the center, coupled with TAT membrane peptide, were obtained by chemical synthesis. (3) (3) in order to verify whether the polypeptide can penetrate into the endothelial cells successfully and efficiently carry the active fragment of the polypeptide, we use FITC to mark the polypeptide. After co culture of the polypeptide and HUVECs for 72 hours, the fluorescence intensity of the polypeptide cell is observed under the fluorescence microscope and the best cell drug concentration is screened by CCK8 method. (4) in vitro experiment The interference effect of the polypeptide to EndMT was verified and the core site of PP2Ac was further clarified. Results (1) the results of mass spectrometry showed that only a nitrochemical site Y218 was detected in the control group, and the nitrochemical site of PP2Ac in the experimental group was increased to six: Y284/267/265/218/130/127. computer simulation analysis showed that in the six loci, Y127 was in the protein space junction. Structure, the adjacent amino acid charge distribution has a more stable spatial epitope advantage and may play a core role in the process of PP2Ac activation. (2) taking the constructed TAT-127WT fusion peptide as an example, the polypeptide carrying fluorescent labeled peptides can permeate into the endothelial cells and continue to the end of the experiment at 72h, which has good penetration efficiency.CCK8 assay for endothelial cells. The cytotoxicity of the polypeptide was measured at different concentrations. The results showed that the polypeptide not only had good penetration but the lowest toxicity to the cells at the concentration of 5-10uM. Therefore, 10uM was selected in the subsequent cell experiments. (3) a synthetic penetrable substrate analogue polypeptide was constructed to inhibit the EndMT induced by TGF- beta 1. The effect is: Y127Y265Y130284Y267. conclusion tyrosine 127 is the key site of PP2Ac nitration and PP2A activation. The substrate mimic peptide constructed with this site can effectively inhibit EndMT. It may be a potential biological target for the treatment of interstitial fibrosis and interstitial fibrosis. The fourth part of TAT-Y127WT is a pre research on the body and body of EndMT in the body. The previous study showed that PP2Ac Y127 was the core site of the whole enzyme activity of PP2A, and that the substrate constructed by PP2Ac Y127 as the center was more significant than the other loci in inhibiting the EndMT effect. This part of the study further explored the effect of TAT-Y127WT and its control polypeptide TAT-Y127Scr on the transdifferentiation of endothelial cells in vitro and in vivo. The treatment of interstitial fibrosis provides experimental basis and strategy. Methods (1) synthesize PP2Ac Y127 centered substrate analog peptide (TAT-127WT) and control polypeptide (TAT-127Scr), pretreat HUVECs 30min, give TGF- beta 1 (10ng/ml) to stimulate 72h, Western blot detection a-SMA, protein expression and substrate phosphorylation level. (2) In vivo animal imaging technique was used to observe the metabolism in the body before injection of polypeptide, 30min, 4H and 24h after injection. At the same time, the important organs and tissues were taken out to trace. (3) the tail vein was injected with intraperitoneal injection (5 nmol/g, one time before, 1 days after operation), and after 2 weeks, the polypeptide was observed on the kidney tube Zhou Maoxi of UUO model rat Intervention effect of vascular endothelial cell EndMT, immunohistochemical observation of alpha -SMA, Vimentin deposition. Results (1) Western blot results showed that TAT-127WT group was more than TGF- beta 1 group, TAT-127Scr group, a-SMA expression was significantly down, and VE-cadherin and occludin phosphorylation level was significantly up, difference was statistically significant (2) in vivo The metabolic rate was faster, less than 10% after 4h, and after 24h, the concentration of liver and kidney was high, heart, lung and spleen were low. The fluorescence intensity in the blood showed that the half-life of the fusion polypeptide was about 2.5h. (3) compared with the control group. After the Y127WT dry prognosis, the density of the renal tissue was up regulated, with a certain EndMT suppression. The results of immuno histochemistry showed that the intervention group could partially reduce the expression and accumulation of alpha -SMA, Vimentin in the renal interstitium, and the difference was statistically significant (P0.05). Conclusion by inhibiting EndMT, the analog peptide of the substrate with PP2Ac Y127 as the core has a certain improvement in the renal tubule Zhou Mao fine vascular lesions of UUO mice, and it is the renal interstitium. The treatment of fibrosis provides a new way of thinking.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692

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