【作者】 王雅新；

【导师】 熊思东；

【作者基本信息】 复旦大学 ， 免疫学， 2011， 硕士

【摘要】 TRIM22 (Tripartite Motif 22)是TRIM蛋白家族成员之一。TRIM家族蛋白结构保守,与机体的多种生理功能有关,如细胞增殖、分化、发育和凋亡等。近年来研究发现多种TRIM家族蛋白在抗病毒固有免疫中发挥重要作用,其中研究最为深入的是TRIM5α。TRIM5a具有广泛的抗逆转录病毒作用,是恒河猴抵抗人类免疫缺陷病毒(HIV)感染的关键分子。值得关注的是,多种TRIM家族分子亦是干扰素(interferon,IFN)诱导基因,如TRIM5α、TRIM19、TRIM21、TRIM22、TRIM25和TRIM34等,它们在IFN介导的抗病毒固有免疫中起到重要作用,提示TRIM家族蛋白是一类新型抗病毒固有免疫分子。在正常生理条件下,TRIM22主要表达在外周血单个核细胞和淋巴组织,如胸腺和脾脏中,可能与造血细胞的分化有关。在受到IFN、脂多糖(LPS)及p53等因素刺激下,TRIM22可在多种组织细胞中显著上调表达。本课题组的前期工作发现TRIM22,而非TRIM5a,是肝细胞中被干扰素诱导表达最强的TRIM分子之一,并能有效抑制乙型肝炎病毒(HBV)的复制。急性HBV感染猩猩的肝基因组学研究亦发现,TRIM22与IFN以非细胞裂解方式清除HBV的过程相关。另有研究表明TRIM22,而非TRIM5a,参与干扰素的抗HIV作用,将TRIM22基因knockdown后,干扰素将不能有效抑制HIV的复制。这些研究结果均提示TRIM22是干扰素抗病毒过程中的关键效应分子。基于此,本课题组的前期工作对IFN-γ转录调控TRIM22的分子机制进行了研究。我们鉴定出一个在干扰素对TRIM22转录调控中起到关键作用的顺式作用元件——5’延伸的干扰素刺激反应元件(5’extended IFN-stimulating response element,5’elSRE),并发现干扰素调节因子(IFN regulatory factor, IRF)-1能与该反应元件结合,从而在IFN-y诱导TRIM22表达的过程中起到重要作用。组蛋白去乙酰化酶(Histone deacetylases. HDACs)在真核细胞基因的表观转录调控中起重要作用,HDACs通常被认为与真核细胞基因转录抑制相关。但新近研究发现HDACs对基因的转录调控比预想的要复杂得多：(1)HDACs亦可作为转录活化子来发挥功能；(2)除组蛋白外,HDACs亦可调控非组蛋白(如转录因子)的乙酰化水平；(3)HDACs亦在蛋白的泛素化修饰中起重要作用。尤为有趣的是,有研究发现HDACs抑制剂(如TSA)在IFN对某些基因的转录调控中发挥关键性的作用,但其机制仍不明确。大量研究表明,组蛋白乙酰化程度的降低与肿瘤的发生、发展密切相关。HDACs抑制剂(如TSA、NaB和VPA等)因可上调肿瘤细胞中组蛋白乙酰化水平、恢复正常的基因表达谱,从而在抗肿瘤治疗中备受关注。但人们在应用HDACs抑制剂治疗肿瘤的过程中发现,HDACs抑制剂处理可促进肝细胞中HCV和HBV的复制,这提示HDACs抑制剂可能影响某些具有重要抗病毒功能的干扰素刺激基因(IFN-stimulated genes, ISGs)的表达。因本课题组的前期研究表明TRIM22在抗HBV固有免疫应答中起重要作用,且是干扰素抗病毒的关键效应分子,我们设想HDACs抑制剂对病毒复制的促进作用可能由于其阻断了IFN-γ对TRIM22的诱导表达所导致。为此,本课题将研究HDACs抑制剂对IFN-γ诱导TRIM22表达的影响,并在此基础上进一步探讨其作用机制,以期为基于TRIM22的抗病毒策略提供新的理论依据。第一部分TSA抑制IFN-γ对TRIM22的诱导表达前期的研究结果表明,TRIM22在肝细胞中的基础表达值低,但可被IFN-γ有效诱导表达。为研究HDACs抑制剂在IFN-γ诱导TR1M22表达中的作用,我们首先检测了一种常用的HDACs抑制剂——曲古抑菌素A (Trichostain A, TSA)对TRIM22诱导表达的影响。在TSA存在或不存在的情况下,利用IFN-γ刺激HepG2细胞,再通过实时定量PCR (qPCR)和Western blot技术分别检测TRIM22 mRNA和蛋白水平的表达。结果发现,TSA可在mRNA和蛋白水平上有效抑制IFN-γ对TRIM22的上调表达,并且这种抑制作用呈剂量和时效依赖性。为阐明TSA是在哪一层面影响了IFN-γ对TRIM22基因的诱导表达,我们在IFN-γ处理的HepG2细胞中加入转录抑制剂放线菌素D (AtcD)后,再行检测TSA对TRIM22 mRNA的影响。结果发现TSA并不影响IFN-γ诱导的TRIM22 mRNA的稳定性,提示TSA在IFN-γ诱导TRIM22表达中的作用可能发生在转录水平。为此,我们将TRIM22启动子依赖的荧光素酶报告质粒(pLuc-160)转入HepG2细胞中,转染24小时后,在TSA存在或不存在的情况下,用IFN-γ刺激HepG2细胞,随后检测转染细胞裂解液中的荧光素酶活性。结果发现,TSA可有效抑制IFN-γ诱导的TRIM22启动子活性,并且这种抑制作用亦具有剂量和时效依赖性,表明TSA是在转录水平上抑制IFN-γ对TRIM22的诱导表达。为检测TSA的抑制效应是否是通过特异性抑制HDACs的活性,我们进一步检测了另一种HDACs抑制剂——NaB对IFN-γ诱导的TRIM22转录活性的影响。结果发现,TSA和NaB均可在HepG2细胞中上调组蛋白H4的乙酰化水平,表明这两种抑制剂均可有效抑制HDACs的活性；重要的是,与TSA相类似,NaB亦可以剂量依赖的方式有效抑制IFN-γ诱导的TRIM22启动子活性,表明是HDACs活性的抑制导致IFN-γ不能有效诱导TRIM22的转录。第二部分TSA抑制IFN-γ对TRIM22诱导表达的分子机制本课题组的前期工作发现IRF-1通过与5’eISRE结合在IFN-γ诱导TRIM22的过程中发挥关键作用。因上述研究结果表明TSA在转录水平上抑制了IFN-γ对TRIM22的诱导表达,我们探究TSA是否可影响在TRIM22表达调控中起关键作用的转录因子IRF-1。在TSA存在或不存在的情况下,利用IFN-γ处理HepG2细胞6小时后,抽提核蛋白,然后通过基于ELISA的转录因子分析技术检测细胞核提取物中的IRF-1蛋白与5’eISRE的结合。结果发现,TSA处理组IRF-1与5’eISRE结合作用被显著抑制。Western blot结果亦表明TSA处理可导致核内IRF-I蛋白表达量显著降低。而后我们检测TSA处理不同时间后(0.5、1、2、6、12、24小时)IFN-γ诱导的IRF-1蛋白的表达情况,结果显示TSA在各个时间点均可明显抑制IFN-γ对IRF-1蛋白的诱导表达。为研究TSA对IRF-1蛋白水平的影响是否是因为TSA影响了IRF-1的转录,我们通过qPCR检测了TSA对IRF-1 mRNA水平的影响。结果发现,TSA并不显著影响IFN-γ对IRF-1mRNA的诱导表达。已知STAT1在IFN-γ对IRF-1的转录调控中起关键作用,因而我们检测了TSA对STAT1磷酸化的影响,结果发现TSA处理组与未处理组在IFN-γ诱导STAT1磷酸化水平上无明显差别。这些结果表明TSA并不明显影响IRF-1的转录。随后,我们检测了TSA对IRF-1蛋白稳定性的影响,发现在TSA存在的情况下,IFN-γ诱导的IRF-1蛋白的稳定性下降,即TSA处理导致IRF-1蛋白降解加快。进一步研究发现TSA亦可导致外源性转入的IRF-1蛋白降解加快。有研究表明IRF-1蛋白半衰期短,主要通过泛素—蛋白酶体途径被降解,而亦有研究发现,HDACs抑制剂可诱导某些蛋白的泛素化。因此,我们推测TSA可能通过增强IRF-1蛋白的泛素化水平,从而导致IRF-1蛋白的降解。利用蛋白酶体抑制剂MG132处理细胞后,再检测TSA对IFN-γ诱导IRF-1蛋白的表达情况。结果显示,MG132可以抑制TSA对于1RF-1蛋白的降解。同时,我们亦检测了溶酶体抑制剂氯喹(Chloroguine)在TSA促进IRF-1蛋白降解中的作用,发现Chloroguine对这一过程并没有影响。这些结果表明,TSA可能通过泛素—蛋白酶体途径促进IRF-1蛋白的降解。为了进一步验证这一结论,我们将IRF-1过表达质粒pIRF-1-Myc与Ub-HA质粒共转染HepG2细胞,而后用偶联Myc抗体的protein A/G进行pull-down,随后再用HA抗体进行Western blot检测。结果发现,TSA可明显增强IRF-1蛋白的泛素化水平。在生理条件下,TSA的靶基因HDAC1在体内主要发挥组蛋白去乙酰化功能。但近年来研究发现,HDAC1亦可在某些蛋白的去泛素化过程中起到重要作用。通过亲和素凝胶沉淀分析发现HDAC1与IRF-1均可结合到TRIM22启动子区域的5’eISRE上,这为HDAC1发挥对IRF-1的去泛素化作用提供了空间基础。进一步的研究发现,过表达HDAC1可明显降低IRF-1蛋白的泛素化水平。综上所述,本研究证实组蛋白去乙酰化酶抑制剂TSA可通过泛素—蛋白酶体途径降解IRF-1蛋白,从而在转录水平上抑制IFN-γ对TR1M22的诱导表达。这为阐明抗乙肝病毒固有免疫分子TRIM22的调控表达和作用机制提供了新的实验依据。更多还原

【Abstract】 TRIM22 is a member of the tripartite motif (TRIM) family of proteins, which are involved in diverse cellular processes, including cell proliferation, differentiation, apoptosis, and transcriptional regulation etc. In recent years, some TRIM proteins are found to play important roles in antiviral processes. Of which, TRIM5a is the most intensively studied, which plays a crucial role in the antiviral activity against HIV-1. Notably, many TRIM proteins can be upregulated by interferons, supporting their potential role as effectors in the anti-viral cellular response. Thus, it has been speculated that the TRIM proteins might represent a new and widespread class of antiviral molecules involved in innate immunity.In physiological condition, TRIM22 is mainly expressed in lymphoid tissues, such as thymus and spleen. It has been reported that TRIM22 may be involved in hematopoietic differentiation. Although the basal expression level of TRIM22 in many other tissues is pretty low, TRIM22 expression could be strongly induced upon the stimulation of IFN, LPS or p53. Our previous data demonstrated that TRIM22, but not TRIM5a, was one of the most strongly induced TRIMs in response to IFNs treatment in human hepatocytes and could inhibit Hepatitis B virus (HBV) gene expression and replication efficiently. Intrahepatic gene expression analysis of acutely infected chimpanzees has shown that the TRIM22 is associated with the IFN-y-mediated noncytopathic inhibition of HBV replication. Additionally, it was reported that TRIM22, but not TRIM5a, was implicated in IFN-mediated anti-HIV activity. Based on these observations, we previously investigated the molecular mechanisms of TRIM22 induction by IFN-y. We identified a special cis-element named 5’extended IFN-stimulating response element (5’eISRE) that was crucial for IFN-y-induced TRIM22 expression. Furthermore, we demonstrated that interferon regulatory factor (IRF)-1 played a key role in the expression of TRIM22 induced by IFN-y via binding to this cis-element.Histone deacetylases (HDACs) play an important role in the epigenetic transcriptional regulation of many genes. Histone deacetylation enzymes have often been associated with the suppression of eukaryotic gene transcription. However, recent studies show that the effect of HDACs on gene transcriptional regulation is much more complicated than previously expected. First, although HDACs often act as suppressors for gene expression, they can also be crucial for the transcriptional activation of some genes. Second, HDACs can also regulate the acelytion of non-histones, such as transcription factors. Third, HDACs can also play an import role in the ubiquitination of some proteins. Interestingly, some studies demonstrated that HDACs inhibitors, such as TSA, played a crucial role in the transcriptional regulation of some genes by IFNs, although the underlying mechanisms remained unclear.Accumulating data show that the low acelyted level of histons correlates closely with pathogenesis of malignant tumours. HDACs inhibitors, such as TSA, NaB and VPA, are therefore received much attention in anti-tumor therapy for their role in regulation of protein acelytion. However, during the course of HDACs inhibitors-mediated anti-tumor therapy, there are some reports that HDACs inhibitors treatment can lead to the higher replication of HCV or HBV, indicating HDACs inhibitors may interrupt the expression of some ISGs. As we previously demonstrated that TRIM22 played an important role in the innate immunity against HBV and it was a key effector in IFN-mediated antiviral activity, we hypothesize that the enhancement of viral replication by HDACs inhibitors may be due to their inhibitory effect on IFN-y induction of TRIM22. To address this question, we have studied on the effect of HDACs inhibitors on IFN-y induction of TRIM22 and its molecular mechanisms. This study may help to further decipher the molecular mechanisms of TRIM22 induction by IFN-y, and might also be useful for designing TRIM22-based antiviral strategy.Part I TSA blocked the IFN-y-induced TRIM22 expressionOur previous data showed that the expression level of TRIM22 in quicient hepatocytes was pretty low, but could be strongly induced in response to IFN-y stimulation. To examine the role of HDACs inhibitors in the IFN-y-induced TRIM22 expression, we treated HepG2 cells with TSA plus IFN-y. After 24 hours, the protein expression of TRIM22 was determined. Results showed that TSA could efficiently inhibit the TRIM22 induction by IFN-y at both mRNA and protein levels in a dose-and time-dependent manner. To determine at which level TSA exerted its effect on IFN-y induction of TRIM22, we examined TRIM22 mRNA stability in DMSO-or TSA-treated cells using an actinomycin D inhibition assay. Results showed the half-life of TRIM22 mRNA induced by IFN-y appeared to be similar between DMSO-and TSA-treated cells for the indicated time period, indicating TSA didn’t affect TRIM22 mRNA stability. The data agreed with the notion that the inhibitory effect of TSA on TRIM22 induction by IFN-y was likely at the transcriptional level. Then, we tansfected TRIM22 promoter dependent luciferase reporter plasmid, pLuc-160, into HepG2 cells, and treated these cells with different concentrations of TSA in the presence of IFN-y.24 h later, luciferase activity in the cell Iysate was examined. Results showed that TSA could effectively inhibit the IFN-y-induced promoter activity of TRIM22 in a dose-and time-dependent manner, indicating TSA inhibited the IFN-y-induced TRIM22 expression at the transcriptional level. To further verify the role of TSA on IFN-y induction of TRIM22 is due to its specific inhibitory effect on HDACs, we examined the effect of another HDACs inhibitor NaB on IFN-y-induced TRIM22 transcriptional activity. It was found that both TSA and NaB could enhance the acelytion of histone H4 significantly, indicating both of them could inhibit the HDACs activity in HepG2 cells dramatically. Further study showed that NaB could also inhibit the IFN-y-induced promoter activity of TRIM22, indicating it was the blockage of HDACs activity that led to the inability of IFN-y to induce TRIM22 transcription.Part II TSA blocked the IFN-y-induced TRIM22 expression via enhancing the degradation of IRF-1 through ubiquitin-proteasome pathwayOur previous work found that IRF-1 played an important role in IFNs-induced TRIM22 expression via binding with 5’eISRE. As the above-mentioned data indicated that TSA inhibited IFN-y-induced TRIM22 expression at the transcriptional level, we therefore investigated whether TSA could have effect on the IFN-y-induced expression of IRF-1, which is the key transcription factor for TRIM22 induction. The ELISA-based transcription factor assay showed that the binding of nuclear IRF-1 from TSA-treated cells with 5’eISRE was decreased significantly. Western blot results also showed that upon TSA treatment, the expression level of nuclear IRF-1 was reduced dramatically. Next, we examined the IFN-y-induced IRF-1 protein expression at different time points (0.5,1,2,6,12,24 h) after TSA treatment. Results showed that TSA could inhibit IFN-y-induced IRF-1 protein expression at each time point, further confirming the effect of TSA on IRF-1 protein expression. To determine whether the effect of TSA on IRF-1 protein expression is due to its effect on IRF-1 transcription, we examined the effect of TSA on IRF-1 mRNA expression. Results showed that TSA did not affect the IFN-y-induced IRF-1 mRNA expression. It was well known that IRF-1 transcription was under control of STAT1, we therefore investigated the effect of TSA on IFN-y-induced STAT1 phosphorylation. It was found there was no difference between DMSO-or TSA-treated cells in the context of IFN-y-induced STAT1 phosphorylation. These data indicated that TSA did not affect IRF-1 transcription. We then tested the stability of IRF-1 protein in the presence of TSA, and found that TSA treatment decreased the stability of IFN-y-induced IRF-1 protein significantly. We also tested the effect of TSA on the stability of ectopic IRF-1 protein and found that TSA could also accelerate the degradation of ectopic IRF-1 protein. Taken together, these data indicated that TSA could decrease the protein level of IRF-1 via enhancing the degradation of IRF-1 protein.Studies have reported that, half-life of IRF-1 protein is very short, and its degradation occurs mainly through the ubiquitin-proteasome pathway. We therefore tested the effect of the proteasome inhibitor MG132 on TSA-mediated degradation of IRF-1 protein. We found that MG132 could inhibit TSA-mediated degradation of IRF-1 protein. Meanwhile, we also tested the effect of lysosome inhibitor Chloroquine on TSA-mediated degradation of IRF-1 protein. Results showed that Chloroquine failed to block the effect of TSA. These data indicated that TSA might enhance the degradation of IRF-1 via the ubiquitin-proteasome pathway. To further verify this phenomenon, we transfected pIRF-1-Myc and Ub-HA plasmids into HepG2 cells, then conducted Co-IP with anti-Myc for pull-down and with Ub or HA antibodies for detection. Results showed that TSA could increase the ubiquitination level of IRF-1 protein significantly.HDAC1 is the target gene of TSA. Under physiological conditions, the main function of HDAC1 is to reduce the acetylation of histone. However, recent study showed that HDAC1 may function as a deubiquintin ligase. We found that both IRF-1 and HDAC1 could bind to the 5’eISRE of TRIM22 promoter as determined by avidin gel precipitation test, which might act as a platform to facilitate the deubiquitination of IRF-1. Further study showed that overexpression HDAC1 could decrease the level of IRF-1 ubiquitination significantly.In summary, this study demonstrated that the histone deacetylase inhibitor TSA could block the transcription of IFN-y-induced TRIM22 expression through enhancing the degradation of IRF-1 protein via ubiquitin-proteasome pathway. It provides new data for explanation of the regulatory and functionary mechanism of the anti-HBV innate molecule-TRIM22.更多还原