【作者】 王海萍；

【导师】 沈玉先； 李俊；

【作者基本信息】 安徽医科大学 ， 药理学， 2010， 博士

【摘要】 α1抗胰蛋白酶(α1-antitrypsin, AAT)主要由肝细胞合成,分泌到血液中,它是血清中的主要蛋白酶抑制剂,其作用主要是保护肺组织免受蛋白酶水解,尤其是中性粒细胞弹性蛋白酶的水解破坏。AAT缺乏症是一种常染色体隐性遗传病,发病率约1/2000~5000,它的典型临床表现是早发性肝脏疾病和肺气肿。AAT缺乏症是由于编码AAT蛋白的基因突变引起AAT分泌障碍所致,最常见的引起AAT缺乏症的突变为Z型突变,占临床病例的95%以上,该突变编码的蛋白为AAT Z突变体(α1-antitrypsin Z variant, ATZ)。在前期研究中我们发现ring-finger结构域家族泛素连接酶(Ubiquitin ligase, E3) gp78可以促进ATZ降解,本研究进一步观察另一种ring-finger结构域家族E3,Hrd1对ATZ水平的影响。目的:观察Hrd1是否可以促进ATZ的降解,并探讨其降解的机制。同时观察Hrd1介导的ATZ降解是否影响细胞的功能。方法:构建ATZ、Hrd1、Hrd1的E3活性突变体Hrd1C1A等真核表达质粒,合成siRNA-Hrd1,转染HEK 293T或HepG2细胞;逆转录聚合酶联反应(Reverse Transcription Polymerase Chain Reaction, RT-PCR)和免疫印迹(Immune Blot, IB)检测mRNA及蛋白水平的变化;流式细胞检测细胞荧光强度的变化;放线菌酮(cycloheximide, CHX)示踪、蛋白酶体抑制实验观察Hrd1对ATZ表达水平影响的作用机制;免疫细胞化学和免疫荧光观察细胞形态和蛋白定位;免疫共沉淀(Co-immunoprecipitation, Co-IP)和蛋白片段互补实验(protein fragment complementation assay, PCA)确定Hrd1和ATZ的相互作用;荧光显微镜观察和原位末端脱氧核苷酸转移酶标记法(Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL)染色检测细胞形态和凋亡情况。结果:1.Hrd1表达降低细胞内ATZ的总体水平HEK 293T细胞共转染ATZ和Hrd1的cDNA,设立ATZ共转染空载体对照,24 hrs后用Triton X-100裂解液裂解细胞并离心,IB检测裂解液上清。与对照组相比,共表达Hrd1后ATZ在上清中的总量变化不明显,但高分子量ATZ水平明显降低。由于高分子量蛋白聚集体容易形成不溶性沉淀,我们推测Hrd1可能增加ATZ的可溶性并降低沉淀中ATZ水平。为了验证这一设想,我们用IB分别检测细胞上清和沉淀中的ATZ,结果发现Hrd1能明显降低沉淀中ATZ的水平。2.下调内源性Hrd1表达能稳定细胞内ATZ水平利用siRNA技术,合成siRNA-Hrd1寡聚核苷酸,并将ATZ和siRNA-Hrd1共转染HEK 293T细胞,同时设立阴性对照。IB结果显示,与对照组相比,siRNA-Hrd1能使细胞内ATZ水平增高,尤其是SDS不溶部分的ATZ水平。3.Hrd1降低细胞内ATZ水平与E3活性有关Hrd1属于ring-finger结构域家族E3,它的E3活性依赖其指环结构的完整性。为了观察细胞内ATZ水平的降低是否与Hrd1的E3活性有关,我们分别观察野生型Hrd1和E3活性缺失体Hrd1C1A对细胞内ATZ水平的影响。HEK 293T细胞共转染ATZ与Hrd1或Hrd1C1A的cDNA,24 hrs后免疫细胞化学结果显示,野生型Hrd1表达多的细胞,ATZ聚集减少。但Hrd1C1A表达多的细胞,ATZ聚集未见减少。IB结果同样显示,Hrd1明显降低沉淀中ATZ水平,而Hrd1C1A对沉淀中ATZ水平无明显影响。上述结果提示:Hrd1降低细胞沉淀中ATZ水平是其E3活性依赖的。4.Hrd1促进ATZ的降解细胞内蛋白水平受合成和降解两方面因素影响,合成减少或/和降解增加均可以导致蛋白总体水平的降低。为了观察Hrd1引起的细胞内ATZ水平的降低是否是由于ATZ的降解增加所致,我们使用了CHX (100μg/ml)抑制蛋白质合成,然后观察加入CHX后不同时间细胞内ATZ的水平,以此来了解ATZ的降解情况。结果发现,Hrd1能明显促进SDS不溶部分ATZ的降解。同时还发现,共表达Hrd1的细胞培养上清液中ATZ的分泌减少,这进一步说明,细胞内ATZ的减少不是因为它的分泌增加,而是因为它的降解增加所致。5.Hrd1促进ATZ降解依赖其E3活性为了进一步明确Hrd1介导ATZ的降解是否与其E3活性有关,我们分别将HEK 293T细胞共转染ATZ和Hrd1或Hrd1C1A的cDNA,24 hrs后在细胞的培养上清液中加入CHX,然后检测各时间点细胞内的ATZ水平。结果发现,Hrd1C1A能增加ATZ的可溶性,但不能促进SDS不溶部分ATZ的降解。提示Hrd1介导的ATZ降解与其E3活性有关。6.Hrd1增加ATZ的泛素化细胞内的蛋白降解主要有两种途径:泛素-蛋白酶体途径(ubiquitin-proteasomepathway, UPP)和溶酶体途径。由于Hrd1是一种ring-finger结构域家族的E3,我们首先考虑Hrd1通过UPP途径促进ATZ的降解。内质网中的蛋白底物经过UPP降解时首先被泛素化修饰,然后经过逆向转运到达26S的蛋白酶体,被蛋白酶体中的蛋白水解酶降解。为了明确UPP是否参与了Hrd1介导的ATZ降解,我们观察了Hrd1对ATZ泛素化的影响。结果发现在没有蛋白酶体抑制剂MG132存在的情况下,Hrd1可明显降低多聚泛素化ATZ;在细胞的培养上清液中加入MG132,作用4 hrs后收集细胞,共转染ATZ和野生型Hrd1组出现多聚泛素化ATZ,而单独转染ATZ或者共转染ATZ与Hrd1C1A组则没有观察到明显的ATZ泛素化。该结果提示Hrd1可以促进ATZ的泛素化,这种作用也是E3活性依赖的。7.含缬酪肽蛋白(valosin-containing protein,VCP)参与Hrd1介导的ATZ降解ATZ是内质网(endoplasmic reticulum, ER)腔内的蛋白,如果经过UPP降解,需要从ER逆向转运至细胞浆,然后由26s蛋白酶体降解,即所谓的内质网相关蛋白降解(ER-associated degradation, ERAD)。在ERAD过程中,VCP在蛋白底物由ER转运至细胞浆的逆向转运中起着重要的作用。我们进一步观察了VCP是否也参与Hrd1介导的ATZ降解。我们共转染ATZ,Hrd1和VCP的突变体VCPQQ的cDNA。结果显示,与对照组相比,VCPQQ阻碍了Hrd1介导的ATZ降解,提示VCP参与了Hrd1介导的ATZ的降解。8.溶酶体通路可能参与Hrd1介导的ATZ降解为了观察溶酶体通路是否参与Hrd1介导的ATZ降解,我们共转染ATZ和Hrd1,观察溶酶体膜稳定剂氯化铵对ATZ水平的影响,发现加入氯化铵后,出现高分子量ATZ的聚集。我们同时构建了在ATZ的信号肽前方加入带有绿色荧光蛋白(green fluorescence protein, GFP)标签的ATZ表达载体,改变ATZ在细胞内质网中的定位。将表达GFP-ATZ和Hrd1的质粒共转染HEK 293T细胞,设立共转染GFP-ATZ和空载体、GFP和Hrd1及GFP和空载体的对照。24 hrs后荧光显微镜观察,与对照组相比,共转染Hrd1组GFP-ATZ绿色荧光强度明显降低;流式细胞检测同样发现,共转染Hrd1后,GFP-ATZ的荧光强度降低。而在转染GFP的对照组中,Hrd1对GFP的荧光强度没有明显影响,IB结果也显示Hrd1不能降低细胞上清和沉淀中GFP的表达水平。9.Hrd1和ATZ存在相互作用前述实验结果提示Hrd1可以通过UPP促进ATZ的降解。我们进一步观察了ATZ与Hrd1之间是否存在相互作用。共转染ATZ和Hrd1或Hrd1C1A的cDNA,免疫荧光双标结果显示ATZ与Hrd1在细胞内存在共定位。免疫共沉淀结果提示ATZ与Hrd1及Hrd1C1A均存在直接的相互作用,该结果提示ATZ与Hrd1的相互作用与其E3活性无关。为了进一步证实ATZ和Hrd1之间的相互作用,我们构建载体,将ATZ和Hrd1的N端分别连上荧光素酶的C端和N端。PCA实验观察共表达两种重组载体的细胞的荧光素酶活性。与对照组相比,实验组荧光素酶活性明显增高。该结果进一步证实Hrd1和ATZ之间存在相互作用。10.Hrd1降低ATZ的细胞毒作用ATZ的聚集可以引起肝细胞毒性,我们在体外HEK 293T和HepG2的细胞模型中同样观察到ATZ具有细胞毒作用。同时我们观察了Hrd1介导的ATZ降解对ATZ细胞毒作用的影响。免疫荧光结果显示单独表达ATZ的细胞或者共表达ATZ与Hrd1C1A的细胞变圆、突起缩短、细胞核分叶、固缩、碎裂或消失;共表达Hrd1后,细胞形态和细胞核趋于正常。统计分析ATZ阳性细胞核的完整性结果显示,Hrd1的表达有助于维持ATZ阳性细胞核的正常形态;TUNEL染色结果同样显示共表达Hrd1后,细胞凋亡减少,而Hrd1C1A没有这种作用;提示Hrd1可以降低ATZ的细胞毒作用,这种作用是E3活性依赖的。结论:以上研究结果表明,Hrd1作为E3不仅可以通过UPP促进ATZ降解,而且溶酶体通路可能参与了Hrd1介导的ATZ降解,并且Hrd1可增加ATZ的可溶性,从而降低ATZ的细胞毒作用,促进细胞存活。该研究结果可能为AAT缺乏症肝脏并发症的治疗提供潜在的药物靶点。更多还原

【Abstract】 Alpha-1-antitrypsin (AAT) is mainly synthesized in the hepatocytes and secreted into the blood. It is a major plasma serum protease inhibitor that protects tissues from enzymes, especially elastase digestion. AAT deficiency is an autosomal-recessive disorder that is characterized by the retention of malfolded AAT in the endoplasmic reticulum (ER) of hepatocytes and by a significant diminution in the serum levels of AAT, which subsequently causes liver and lung diseases. It is caused by the mutations in genes encoding AAT. The most common mutation that causes more than 95% clinical cases of severe AAT deficiency is the Z allele. The mutated gene encodes AAT Z variant (ATZ). Previous studies have demonstrated that the ubiquitin-proteasome pathway is involved in the degradation of ATZ. However, detailed mechanisms of ATZ degradation are not fully understood. We have also found gp78, an E3 with ring-finger domain, enhances ATZ degradation. In present study, we investigated whether another ring-finger domain E3, ER membrane ubiquitin ligase Hrd1 facilitated the removal of ATZ through ER-associated degradation (ERAD).Objective:To investigate whether ER membrane ubiquitin ligase Hrd1 facilitates the removal of ATZ through ER-associated degradation (ERAD) and the effect of Hrd1 on the function of cells expressing ATZ.Methods:To construct the eukaryotic expresson vectors for ATZ, wt Hrd1, E3 activity abolished mutant Hrd1C1A, etc. Small interference RNA for Hrd1 was systhesized from Genepharm. Co. LTD. The eukaryotic expresson vectors or siRNA were transfected to HEK 293T cells or HepG2 cells. Reverse transcription polymerase chain reaction (RT-PCR) and immune blot (IB) were used to detect mRNA and protein level respectively. Flow cytometry was used to monitor the GFP fluorescence intensity. The mechanisms for Hrd1 mediated influences on ATZ level were explored by cycloheximide chase (CHX chase) and proteasome activity inhibition followed by IB. Cell morphologies, intracellular protein localization and quantitation were examed by immunocytochemistry and immunofluorescence. Co-immunoprecipitation (Co-IP) and protein fragment complementation assay (PCA) were employed to determine the direct interaction between Hrd1 and ATZ. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to find the apopototic cells. Results:1.Hrd1 decreases intracellular ATZ levelHEK 293T cells were co-transfected with ATZ and Hrd1 cDNA. Co-tansfection of ATZ and empty vector was used as control. Twenty four hours after transfection, cells were collected and lysed in Triton X-100 buffer containing protease inhibitors. After centrifugation, the supernatant was processed for IB AAT. ATZ in the supernatant (Triton X-100-soluble fraction) was not markedly reduced after co-transfection with Hrd1, but the high molecular weight (MW) ATZ was decreased, suggesting that Hrd1 may inhibit the formation of insoluble ATZ aggregates. To test this possibility, the transfected cells were collected and lysed with 0.1% SDS. The supernatant and the pellet were processed for IB respectively. We found that the ATZ level was significantly decreased in the pellet, though it was slightly increased in the supernatant. As a result, the total level of ATZ was decreased by Hrd1.2.Knockdown of endogenous Hrd1 with siRNA stabilized intracellular ATZ levelThe synthetic siRNA-Hrd1 was co-transfected with ATZ into HEK 293T cells. IB AAT was followed 24 hrs after transfection. The results indicated knockdown of Hrd1 with siRNA stabilized ATZ especially in the detergent-insoluble fractions.3.E3 activity is required for Hrd1-mediated reduction of intracellular ATZ levelHrd1 belongs to ring-finger domain E3 family. Its E3 activity relies on the integrity of the ring-finger domain. To assess the effect of Hrd1 E3 activity on ATZ level, the Hrd1C1A mutant that is deficient in E3 activity by the mutation of zinc ligand residue at the first cysteine site was tested. Immunocytochemistry revealed a negative relationship between intracellular ATZ and wt Hrd1 but not Hrd1C1A level. Western blotting demonstrated that the Hrd1C1A mutant did not decrease the level of ATZ in both the SDS-soluble fraction and the pellet, compared with wild type Hrd1. These results suggest that the reduction of ATZ retention by Hrd1 depends on the intact zinc ligand residues of Hrd1 and its E3 activity.4.Hrd1 facilitates ATZ degradationIntracellular protein level is determined by the balance of protein synthesis and degradation. Either blocking protein synthesis or increasing protein degradation will lead to the reduction of cellular protein levels. To determine whether the reduction of intracellular ATZ is due to the enhancement of ATZ degradation, CHX (100μg/ml) was used to inhibit protein synthesis. CHX was added to the cells 24 hrs after transfection and the cells were collected at different time intervals. The results show that Hrd1 mainly accelerated ATZ clearance in the SDS-insoluble fraction, not in the supernatant. On the contrary, Hrd1 increased the amount of ATZ in the supernatant. These data indicate that Hrd1 increases the solubility of ATZ and facilitates ATZ degradation.5.E3 activity is required for Hrd1-mediated ATZ degradationTo further determine whether Hrd1 E3 activity is involved in ATZ degradation, Hrd1C1A and ATZ were co-transfected into HEK 293T cells. CHX chase was conducted to detect protein degradation. The results indicate Hrd1C1A mutant stabilized ATZ in the cells, especially in the supernatant compared to wt Hrd1. This result suggests that one cysteine residue mutation in the ring-finger domain does not affect the function of Hrd1 on ATZ solubility, but attenuates its activity on ATZ degradation.6.Hrd1 increases ATZ ubiquitinationThere are two major protein degradation pathways for intracellular protein, namely the ubiquitin-proteasome pathway (UPP) and the lysosomal pathway. ATZ was shown to be degraded by both of them. Since Hrd1 is a ring-finger domain E3 and degrades ATZ dependently on its intact ring-finger domain, we reason that Hrd1 targets ATZ for degradation at least in part through UPP. To test this hypothesis, we first investigated whether ubiquitination was required for Hrd1-mediated ATZ degradation. The plasmid encoding ubiquitin was co-transfected into 293T cells to enhance ATZ ubiquitination. We found that Hrd1 decreased soluble polyubiquitinated ATZ. After inhibition of proteasome activity by MG132, the high MW ATZ was present in the cells co-expressing Hrd1, but not Hrd1C1A. These results indicate that the presence of the high MW ATZ depends on Hrd1 E3 activity and proteasome inhibition, and that Hrd1 mediates degradation of polyubiquitinated ATZ.7 . valosin-containing protein (VCP) is required for Hrd1-mediated ATZ degradationATZ is an endoplasmic reticulum (ER) lumen protein and its degradation for proteasome requires retrotranslocation from the ER into the cytosol. Therefore, VCP, together with its cofactors, must play an essential role in the retrotranslocation of ATZ for UPP. To test this possibility, VCPQQ, a mutant form of p97/VCP that does not have ATPase activity and does not function in ERAD was used. The result showed that VCPQQ stabilized ATZ, especially in the SDS-soluble fraction, suggesting that soluble ATZ degradation was blocked by VCPQQ. This result indicates that VCP is required for Hrd1-mediated soluble ATZ degradation and also provides additional evidence that Hrd1 increases the solubility of ATZ.8. Lysosomal pathway maybe involved in Hrd1-mediated ATZ degradationTo clarify whether lysosomal pathway is involved in Hrd1-mediated ATZ degradation. Ammonium chloride was used as lysosome inhibitor. The result indicated that ammonium chloride increased the high molecular weight ATZ level. For more intuitive observation of Hrd1 on the level of non-ER localized intracellular ATZ, we constructed GFP-tagged ATZ expression vector and put GFP before the signal peptide of ATZ to change the intracellular localization of ATZ. GFP-ATZ and Hrd1 were expressed in HEK 293T cells. The GFP fluorenscence intensity was detected by both fluorenscencent microscope and FCS 24 hrs after transfection. Hrd1 significantly reduced the GFP fluorescence intensity of GFP-ATZ expressing cells compared to the control. By contrast, in the control cells expressing GFP, Hrd1 had no effect on the level of GFP either in supernatant or in pellet, suggesting the effect of Hrd1 on ATZ is specific.9.Hrd1 interacts with ATZThe previous data indicates that Hrd1 targets ATZ degradation through UPP. We next explore whether Hrd1 interacts with ATZ. In the cells co-transfected with FLAG-tagged Hrd1 and ATZ, double immunostaining with anti-AAT and anti-FLAG antibodies revealed that Hrd1 and ATZ were strongly co-localized. Immunoprecipitation assay was also conducted to confirm the interaction of Hrd1 and ATZ. The results indicated that ATZ was co-immunmoprecipitated with both wt Hrd1 and Hrd1C1A, suggesting this interaction did not depend on the intact ring-finger domain. We got further proof for this interaction from PCA. We fused C (G1) and N terminal (G2) of luciferase to N terminal of Hrd1 and ATZ respectively and the two constructs were co-transfected to HEK 293T cells. The relative hGluc activity significantly increased in the cells co-transfected with Hrd1-G1 and ATZ-G2 compared with Hrd1-G1 or ATZ-G2 alone.10.Hrd1 relieves the toxicity of ATZIntracellular accumulation of ATZ is toxic to hepatocytes. We wondered whether the removal of the accumulated ATZ by Hrd1 promoted cell survival. Double immunofluorescent labeling was conducted to show the over-expressed proteins. In both HEK 293T and HepG2 cells, ATZ-expressing resulted in abnormal morphologies. The cells became round, detached, decreased in size, and shortened in cell processes. The nuclei were especially reduced in ATZ-positive cells. However, the amount of ATZ was decreased and the proportion of ATZ-positive cells with normal morphologies increased after wt Hrd1 co-transfection. Moreover, Hrd1C1A mutant did not improve the abnormal morphologies of the ATZ-positive cells. In situ cell death detection assay indicated wt Hrd1 but not Hrd1C1A reduced cell apoptosis. These datas indicate Hrd1 relieves ATZ toxicity and it requires its E3 activity.Conclusions:Our findings demonstrated that Hrd1 acts as an E3 that mainly decreases the retention of detergent-insoluble ATZ in ER by ERAD and increases its solubility. Accordingly, Hrd1 relieves ATZ toxicity and promotes cell survival, which implies a potential target in the treatment of AAT deficiency liver complications.更多还原