特应性皮炎的综合高通量研究：通过OMICS手段探讨疾病的核心蛋白与生物标志物
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摘要
目的和意义
随着现代化和工业化进程的加快,特应性皮炎(AD)患病率增高,是皮肤病和公共卫生领域的研究热点。AD是一种慢性炎症性皮肤病,易反复发作,以湿疹性皮损分布为特征。主要病理生理表现为苔藓样硬化、瘙痒性脱皮和皮肤干燥。AD是一种系统性功能失调性疾病,可同时触发哮喘、过敏性鼻炎和食物过敏,伴有血浆IgE和嗜酸性粒细胞升高。已报道多种细胞参与了AD的发病,如嗜酸性粒细胞、T淋巴细胞、朗格汉斯细胞和角质形成细胞,这些细胞都与细胞因子、化学增活素和IgE等血液因子以及与微生物感染有关。虽然大多数AD患者血浆IgE升高,但AD疾病中存在一个亚组对空气、食物过敏原无致敏性。AD疾病可分为两个类型:内源性和外源性。外源性AD(ADe)占AD疾病的70-80%,具有Th2细胞调节的血浆IgE增高,引起IgE介导的致敏作用的特征。与ADe不同,内源性AD(ADi)占AD疾病的20-30%,没有IgE介导的致敏作用和过敏反应。
高通量筛选(HTS)方法如DNA芯片和2D-PAGE/MALDI-TOF分析已开始用于AD相关候选基因的筛选。通过HTS,可以找出大量在AD中上调或下调的蛋白质或基因,同时,相互作用组工具如PPI图能有助于核心蛋白的挑选。因为接下来的功能研究是一个耗时、花费较大的过程,所以对目标基因的筛选显得非常重要,因此,通过对HTS结果的计算机预测分析是作出正确选择的关键。PPI图中列出的候选基因为理解复杂的AD疾病提供了重要信息。一旦PPI预测的相互反应配偶体得到证实,将为疾病相关的基因调节途径提供新的见解。已知与AD疾病相关的重要因素有IgE/FcεRI、抗微生物肽、细胞因子/化学增活素、蛋白酶及其受体、脂质基因、自身抗原、细胞外基质基因、表皮分化复合物等。在这个基础上,研发出新的治疗方法,并且已经用于AD的临床治疗。尽管目前对AD的治疗有多种途径,但是AD患者在增加,能否提供有效治疗方法如快速缓解严重症状的方案仍是个严峻的挑战。因此,需要更深入的研究AD的发病机理,进一步阐明其免疫机制。
另外本研究还进行了分子水平的环境毒理学研究。环境小分子化学物进入体内,可以与体内最主要的生物大分子一蛋白质发生相互作用,除了通过共价作用形成蛋白加合物外,环境化学物可能会影响蛋白质的构象,导致蛋白质发生错误折叠,从而产生环境健康风险。这种途径是否与折叠病的环境因素有关,迄今为止,这方面的报道较少,有必要开展深入研究。本研究以脑型肌酸激酶(CK-BB)为模型,试图探讨小分子化合物对酶的结构与功能影响。在了解CK-BB去折叠与再折叠过程基础上,进一步对日常生活中人群接触的频率较高的十二烷基硫酸钠(SDS)作用及与CK-BB结合机制进行了探讨,而且阐述了两种可能与退行性疾病相关的环境化学物,丙烯酰胺与锌离子,对CK-BB的结构与功能影响的作用机制。
研究方法
1.本研究中对患者活检样本、原代细胞培养(角质形成细胞和成纤维细胞)和血清样本中的基因改变进行研究。所有的AD样本分为外源性(ADe)和内源性(ADi)两型。非特异性正常对照组(n=22),均无个人或家族史,过敏性疾病症状和体征,其样本选自整形外科手术取下的皮肤。从ADe和ADi患者(n=40)皮损处取直径3-5mm活检样本。20名ADe患者(SCORAD:50.09±15.68),20名ADi患者(SCORAD:46.38±11.77)。ADe组和ADi组总IgE水平、红细胞计数和年龄的平均值分别为:1739 U/mL,625/μL and 27.4岁和105U/mL,248/μL and 28.2岁。所有样本均取自于男性。血清按照之前报道的方法收集,等分试样后储藏在-80℃。
DNA芯片的制备按照RNA制备、标记和杂交步骤进行:分别准备正常对照、ADe、ADi样本,每个样本均进行cDNA模板合成和标记。分别采用两种芯片对不同样本进行检测:GeneChip~(?)人U133 Plus 2.0芯片和GenePlorerTwinchip人-8K芯片。基因表达比值经过图像分析,并用LOWESS回归方程标准化。应用SAM和DEG。选择q值(按照5%)有明显表达差异(＞2倍)的基因。GeneChip~(?)人U133 P1us 2.0芯片用来进行ADe和ADi皮肤组织的分析,而GenePlorer Twinchip人-8K芯片用于分析特应性角质形成细胞。自流电泳(FFE)和2-DE分别用于特应性成纤维细胞和血清处理分析。患者源性血清经多重亲和移除柱(MARC)处理后进行2-DE分析。接下来,用苯基琼脂糖树脂分段式方法进行AD血清蛋白质的分析。血清经柱子分离后TCA沉淀。
根据以上结果,可采组学工具来进行核心基因和蛋白质的预测。PPI预测使用了PPI的主要算法,包括:1)蛋白结构相互作用使用PSIMAP(蛋白质组图谱),一种使用SCOP(蛋白结构分类)数据库结构域的方法;2)蛋白质实验性相互作用PEIMAP(蛋白质组图谱),是一种普遍的使用实验性蛋白相互反应信息资源的方法,如HPRD、BIND、DIP、MINT、IntAct和BioGid。
2.重组人CK-BB按照之前报道的方法进行表达和纯化。CK-BB活性测定采用ATP和肌酸反应释放质子,在百里酚蓝指示剂存在下,其597nm光吸收变化速率对应于酶活性。内源荧光和ANS结合光谱方法检测CK-BB三级结构的影响。采用圆二色谱分光光度计,在190-250nm范围内记录远紫外圆二色谱(CD)光谱检测CK-BB二级结构的影响。等温滴定微量量热法采用3101TAMⅢ恒温箱按照之前报道的方法进行测量。从蛋白质数据库(PDB)中获得已知的CK-BB同源性结构。发现同系物牛的视黄醛肌酸激酶A链(PDB编码:1g0w)是一个良好的CK-BB结构模板(含97%相同序列)。结合模拟操作分为:1)计算相应的受体和3D配体,2)对接大小特征,3)计算网格评分,4)决定补充受体和配体对接。
结果
1.Affymetrix芯片检测出406种差异表达基因(DEG),在正常人和AD患者(ADe和ADi)之间,鉴别出130个DEG;在正常人和ADe患者之间,鉴别出327个DEG;在正常人和ADi患者之间,鉴别出146个DEG。8K cDNA芯片结果显示在原代培养的角质形成细胞AD疾病中有157个基因失调。接下来,通过FFE在AD成纤维细胞中获得93个候选基因。MARC处理后的AD血清样本检测到30个上调蛋白质和19个下调蛋白质。改进分段方法,检测到9个失调基因。Affymetrix芯片分析利用PSIMAP和PEIMAP两种不同的生物信息学计算方法得到5个和8个核心基因,分别是:PIK3R1、IGF1R、MAPK13、SFN、YWHAE和CORIN、COL14A1、CLCA4、PTPRC、IL10RA、C1S、PHLPP、SLITRK6。应用8K cDNA芯片在角质形成细胞中找出25个基因。通过FFE在AD成纤维细胞中4个核心蛋白在PSIMAP和PEIMAP算法中均检测到,即:I55423、ARR3、YWHAE和EEF1A1。AD血清样本的2-DE结果中发现26种蛋白质。AD组织中SFN和PTPRC经实时PCR进一步验证。结果发现转录水平的SFN在ADi中上调4.5倍,PTPRC在ADe和ADi中分别下调60%和46%。ELISA结果显示,MMP1和MMP10在ADi患者中均上调。ADi患者血清的ELISA研究结果表明,与正常人和ADe患者相比较,MMP1和MMP10显著上调(几乎两倍),均存在统计学差异,采用随机配对和Wilcoxon检测。
2.对SDS使CK-BB的折叠后的失活进行了研究。SDS能非竞争性抑制CK-BB的活性(K_i=1.22mM)。SDS与CK-BB暴露的疏水表面结合诱导其三级结构的改变。SDS配体结合热力学参数的改变如焓、吉布斯自由能和熵分别为-13±7.0MJ/mol,8.39 kJ/mol and-42.754 kJ/(K·mol)。研究明确了重要的CK-BB结构以及和SDS相互反应的折叠和抑制动力学信息。预测的结构具有0.51(?)的均方根偏差。CK-BB与SDS之间对接成功,具有显著性评分(-4.67 kcal/mol,自动对接4和-48.32 kcal/mol,DOCK6)。丙烯酰胺对脑型肌酸激酶(CK-BB)的抑制效应研究发现,CK-BB能被动态的失活并伴随疏水表面的暴露。丙烯酰胺主要与CK-BB的硫氢基(-SH)残基相互作用导致烷化。计算机模拟对接支持丙烯酰胺直接结合到CK-BB的活性位点上,主要与CYS74和GLY73残基相互作用。Zn~(2+)通过剂量依赖效应失活CK-BB(IC50=0.06 mM)。Zn~(2+)显著诱导CK-BB疏水基表面破裂导致三维结构改变。还发现Zn~(2+)能使CK-BB发生聚沉。聚沉依赖于温度以及酶和Zn~(2+)的浓度。
结论
1.为了更深入了解AD发病机制,明确在AD病情发展中有多少基因起作用,进行了大规模的微阵列研究,得到许多新的相关信息。结果主要集中在AD相关基因大规模DNA芯片研究的描述上,这将为全面了解AD疾病提供新的见解,但是结果中尚不包括详细的功能研究。在后续的实验中进行了AD皮肤组织的研究并检测到了核心基因。然后进行角质形成细胞和成纤维细胞的研究。同时还对AD患者血清中蛋白质差异表达进行了研究。已知多种因子参与了AD的发病,不同的患者中存在多种触发因素,本研究数据提示人表皮角质形成细胞和真皮成纤维细胞活化在AD疾病中起作用。然而,在原代培养的角质形成细胞和成纤维细胞中检测到的蛋白质或基因可能提示着体内也存在相应的蛋白质变化,但是mRNA水平和表达的蛋白质水平之间的差异干扰了对AD中准确作用的理解。
大规模的芯片研究结果能有助于深入了解AD发病机制的复杂的相关因素。结合表达数据分析和蛋白质相互作用组预测能找到更可靠的与AD疾病相关的基因。预测的蛋白质相互作用配体和核心蛋白能为下一步AD治疗的功能研究提供有效、可靠的靶点。AD患者数量迅速增多,急需找到特异性诊断和敏感性治疗方法。因此,OMICS手段的应用改善了对AD皮肤组织的表达形式以及组分如角质形成细胞和成纤维细胞的研究。根据本研究,得出以下结论:1)生物信息学工具结合HTS数据筛选新的核心基因或蛋白,在今后的功能学研究和临床药物开发中将发挥重要作用;2)本研究中发现多个在AD源性样本中表达显著改变的重要基因,如SFN、PTPRC、MMP1和MMP10,可能是AD疾病的生物学标记;3)本研究中预测出的大部分核心蛋白、以及实时PCR检测出的候选基因/蛋白在之前关于AD发病机制的研究中未见报道;4)结合基因组学、蛋白质组学和相互作用组分析方法等研究手段将有助于AD复杂病理机制的研究,以及准确的生物标记物或临床治疗靶点的探测。
2.本部分研究我们以脑型肌酸激酶(CK-BB)为模型,探讨了小分子工业化学物通过影响酶的结构和功能,引发蛋白质错去折叠,导致酶的失活以及聚沉的毒理途径。不管是SDS(两亲变性剂),丙烯酰胺(巯基加合物),还是Zn2+(金属离子),都可以诱导蛋白质疏水表面的暴露。尤其是Zn2+促发CK-BB发生聚沉。这种由小分子化学物诱发的聚沉现象在许多退行性病变中可能具有重要的意义,其确切的病理价值有待进一步深入研究。环境中存在大量的具有慢性神经毒作用的工业化学品。这些小分子化学物是含具有协同作用,诱导蛋白质构象与功能的变化将是一个重要的研究课题。
Background
Atopic dermatitis(AD) patient populations have been prevalent along with modernization and industrialization.AD is not only a critical skin disease,but also is the hot issue of the public health.AD is a chronic relapsing inflammatory skin disease that's characterized by the distribution of eczematous skin lesions.AD typically reveals lichenification,pruritic excoriations and dry skin with a variety of pathophysiologic manifestations.Since AD is a systemic disorder,it simultaneously triggers asthma,allergic rhinitis and food allergy,where the serum IgE and peripheral eosinophils are elevated.Several cells such as eosinophils,T lymphocytes, Langerhans cells and keratinocytes have been regarded as important for the pathogenesis of AD since these cells are directly related with several blood factors such as cytokines,chemokines and IgE,and they are also related with microbial infection.Although most AD patients have high serum IgE levels,one subgroup of AD patients lacks the sensitization to aero-allergens or food allergens.Two types of AD have been defined,and they are the extrinsic and intrinsic types.The extrinsic type of AD(ADe) has a Th2 cell mediated high serum IgE level and this condition is associated with IgE-mediated sensitization;this is seen in 70-80%of AD patients.As a counterpart to ADe,the intrinsic type(ADi) is not involved with IgE-mediated sensitization and allergies,and ADi is seen in about 20-30%of AD patients.
High-throughput screening(HTS) methods such as DNA microarray and 2D-PAGE/MALDI-TOF approaches are currently being used to determine the candidate genes associated with AD.With HTS,although we can obtain a great deal of genes/proteins that are overexpressed or down-regulated in AD disease,it is very difficult to choose the most important genes.Furthermore,it is hard to explain the meaning of those proteins.In this regard,interactomic tools such as performing PPI mapping may help to find the hub proteins among the detected candidate genes.Since the next step of functional studies is a time-and cost-consuming process,the number of target proteins must be limited;hence,to make the right choice,computational prediction on the basis of HTS results could be critical.The candidate genes listed in the PPI maps may provide important information to piece together the complex nature of AD disease.Regardless of the various approaches that are currently being used to treat AD,the number of AD patients has greatly increased and more effective treatments that offer quick relief of severe symptoms still remain to be developed. Thus,more insights regarding the complex gene/protein regulation are needed into the pathogenesis of AD,in addition to elucidating the immunologic aspects of this disease.
Besides AD studies,we also conducted the molecular level studies of environmental toxicology.Once the environmental chemicals absorbed into the body, they can interact with the most important biological macromolecules such as proteins. The chemicals not only can form the covalent protein adducts,but also may affect the protein conformation as well as lead to protein misfolding and thus,result in environmental health problems.In this study,we used CK-BB as a model material for trying to explore the effects of small molecules on the enzyme structure and function. Based on the understanding of CK-BB unfolding and refolding,we further studied the mechanisms of CK-BB binding with sodium dodecyl sulfate(SDS) that is abundantly presented in modern life and also,studied the effects of two environmental chemicals such as acrylamide and Zn~(2+) which may be related to degenerative diseases.
Method
1.All AD samples were divided into the two types defined as the extrinsic(ADe) and intrinsic(ADi) types.AD skin tissues were collected from non-asthmatic atopic patients.Non-atopic control subjects who had neither a personal/family history nor any signs of atopic diseases were selected for the study.Punch biopsies of 3-5 mm diameter were individually obtained from the lesional skin of both the ADe and ADi patients.We recruited 20 ADe patients,20 ADi patients and 22 normal specimen donors.The mean values for each group's total IgE level,eosinophilia count,age and SCORAD score were 1739 U/mL,625/μL,27.4 year old and 50.09 for the ADe group and 103 U/mL,248/μL,28.2 year old and 46.3 for the ADi group,respectively. The gender of all samples was male.Serum was collected according to previously reported methods and stored at-80℃after proper aliquots.
DNA microarray was performed following by the total RNA preparation, labeling and hybridization:the individually prepared normal,ADe,and ADi RNA samples were correspondingly prepared,and the further cDNA template synthesis and labeling for each of samples were conducted.The two kinds of commercial DNA chips(Affymetrix Human Genome U133 Plus 2.0 Array chip;and GenePlorer Twinchip Human-8K) were individually applied for different samples.These chips were hybridized with a mixture of fluorescently labeled cDNAs from the control and AD samples at 42℃for 16 h in the presence of hybridization solution.Image analysis to obtain the gene expression ratios was performed properly and the logged gene expression ratios were normalized by a LOWESS regression procedure.By using DEG and SAM,the data sets were analyzed.The genes showing significant expression changes(＞2-fold) were selected with a cutoff of 5%of the q value.An Affymetrix microarray was used to analyze ADe and ADi skin tissues,and the 8K cDNA microarray was used to analyze atopic keratinocytes.These tools were probed the dysregulated genes at the transcriptional levels.The free-flow electrophoresis (FFE) was used to analyze atopic fibroblasts and two-dimensional electrophoresis (2-DE) was used to analyze serums with proper treatments.Based on the above results,the interactomic tool was also adapted to predict putative hub genes or proteins.
2-DE analyses of AD patient-derived serums were conducted after treating with multiple affinity removal columns(MARC).In addition,fractionation method by using Phenyl Sepharose resin was conducted to analyze AD serum proteins.Serums were properly fractionation with a column following by TCA precipitation.
In the next step,PPI of AD related proteins were predicted by bioinformatics algorithms.The prediction of PPI uses most of the major types of PPI algorithms. They are:1) Protein Structural Interactome MAP(PSIMAP),a method that uses the structural domain of the SCOP(Structural Classification of Proteins) database;2) Protein Experimental Interactome MAP(PEIMAP),a common method that uses public resources of experimental protein interaction information such as HPRD, BIND,DIP,MINT,IntAct and BioGrid.
2.Recombinant human CK-BB was expressed and purified according to the previous reports.CK-BB activity was measured following proton generation during the reaction of ATP and creatine with the indicator thymol blue at 597 nm.The fluorescence emission spectra and the ANS labeling fluorescence were studied for monitoring the tertiary structural changes.The far-UV circular dichroism(CD) spectra were recorded in the range of 190-250 nm for measuring secondary structural changes.Isothermal titration microcalorimetric measurements were performed with a 3101 TAM III Thermostat in accordance with a previous report.We retrieved the known homologous structures of CK-BB from the Protein Data Bank(PDB) and found that bovine retinal creatine kinase chain A(PDB entry:1g0w) was a very good structural template(97%sequence identity) as a close homologue for CK-BB.The docking procedures were categorized to 1)calculate receptor and ligand 3D coordinates,2)characterize docking site,3)compute grid score,and 4)determine the docking for complementarity with receptors and ligand.
Result
1.From the Affymetrix microarray results,406 genes were selected:130 genes were obtained from normal vs.AD(ADe and ADi);327 genes were obtained from normal vs.ADe;146 genes were obtained from normal vs.ADi.The 8K cDNA microarray results showed that 157 genes were significantly dysregulated in primary atopic keratinocytes compared to the control subjects.In the next step,we obtained 93 candidate proteins by using FFE in AD fibroblasts.In AD serums,30 proteins were detected as up-regulated and 19 proteins were down-regulated in AD serums after using MARC treatment.With adapting fractionation method,9 proteins were detected as dysregulated.
In Affymetrix microarray analysis,five hub genes such as the PIK3R1,IGF1R, MAPK13,SFN and YWHAE were predicted by using the PSIMAP algorithm and eight_genes such as CQRIN,COL14A1,CLCA4,PTPRC,IL10RA,C1S,PHLPP and SLITRK6 were predicted by using the PEIMAP algorithm.In 8K cDNA microarray, 25 genes were predicted for AD keratinocytes hub proteins.In FFE result, overlapping of the PEIMAP and PSIMAP algorithms detected 4 hub proteins:I55423, ARR3,YWHAE,and EEF1A1.In 2-DE results of AD serums,26 proteins were predicted for AD serum samples.
SFN and PTPRC were also further validated by real-time PCR in AD tissues. The results showed that the transcriptional level of SFN is up-regulated 4.5-fold in ADi,and PTPRC is down-regulated 60%in ADe and 46%in ADi.In the ELISA results,MMP1 and MMP10 were consistently detected as up-regulated in the serum of ADi patients.The results of ELISA studies of the serum of ADi patients showed that both MMP1 and MMP10 were significantly up-regulated(almost 2-fold) compared to normal and ADe patients.Statistical significance was confirmed by p-values.Randomized pairings and the Wilcoxon-rank sum test were applied.
2.We have also investigated the inactivation by using SDS to study CK-BB's folding behaviors.SDS strongly inhibited the CK-BB activity in a noncompetitive inhibition manner(K_i=1.22 mM).The tertiary structural change was induced by SDS binding with the exposure of hydrophobic surface.The changes of thermodynamic parameters for the SDS ligand binding such as enthalpy,Gibbs free-energy,and entropy were obtained as-13±7.0 MJ/mol,8.39 kJ/mol and-42.754 kJ/(K·mol),respectively.The predicted structure had a Root Mean Square Deviation of 0.51 A.The docking between CK-BB and SDS was successful with significant scores(-4.67 kcal/mol, AutoDock4 and-48.32 kcal/mol,DOCK6).
We studied the inhibitory effects of acrylamide on CK-BB.We found that CK-BB was kinetically inactivated by acrylamide accompanied by the disruption of the hydrophobic surface.Acrylamide mainly interacted with the thiol(-SH) residue of CK-BB and resulted in alkylation.A computational docking simulation supported that acrylamide directly bound to the active site of CK-BB where CYS74 and GLY73 residues interacted mainly.
Zn~(2+) inactivated the activity of CK-BB in a dose dependent manner(IC_(50)=0.06 mM).The spectroflurorimetry results showed that Zn~(2+) conspicuously induced the tertiary structural change of CK-BB with exposure of its hydrophobic surfaces.We also found that CK-BB aggregation was induced by Zn~(2+).This aggregation was dependent on the temperature and the enzyme and Zn~(2+) concentrations.
Conclusion
1.With respect to explaining all the events that occur in the pathogenesis of AD or for probing how many genes are involved in the development of AD,our large-scale microarray data can suggest new information.In the initial part of results,the data were mainly focused on profiling the AD associated genes in a large scale DNAchip array and this provides useful information for the overall insight into the AD disease at the level of skin tissues,but our results do not include any detailed functional study. Then,primary cultured keratinocytes and fibroblasts were studies.The profiling of differentially expressed proteins in AD serums was also conducted.In respect to the complexity of the AD pathogenesis where many factors are associated with AD and various triggers are known to exist among different patient,our data suggest that human epidermal keratinocytes and dermal fibroblasts actively participate in AD disease along with the other types of cells.However,some of the genes/proteins observed in the primary cultured keratinocytes and fibroblasts may not be representative of the in vivo changes,and the differences between the mRNA levels and the expressed protein levels limit our understanding with regard to their exact roles in AD.
Taken together,the large scale microarray provided information to further understand the complex factors associated with pathogenesis of AD.The approach of combining the expression data analysis and predicted protein interaction partners performed in large scales can bring valuable and more reliable gene targets for AD. The predicted protein interaction partners and validated hub proteins can offer valuable and reliable targets for further functional studies of AD treatment.The number of the AD patients is increasing rapidly and being able to make a specific diagnosis and administering case sensitive treatment are needed.Therefore,OMICS approaches that are based on high-throughput technologies may help researchers gain additional insight or they may reveal pieces of the puzzle into the pathogenesis of AD.
According to the results,several points are suggested in this study:i)the bioinformatic tools combined with the HTS data identified the new candidate hub genes/proteins,which can be useful in the further studies,especially for the functional studies or clinical trials of drug development;ii)several important genes such as SFN, PTPRC,MMP1 and MMP10 have been newly suggested in this study,which they are significantly up-regulated or down-regulated in AD patients-derived samples and they can be biomarkers for AD;iii)most of the predicted hub proteins and real-time PCR probing candidate genes/proteins detected in this study have not been previously reported to be involved in AD pathogenesis.This may be due to the unknown role of these factors in AD pathogenesis;iv)in addition to the fact that the combinations of several research areas such as genomics,proteomics,and interactomics can help guide research into the complex pathogenesis of AD and assist in designing precise therapeutic strategies to find biomarkers or to determine the right clinical target molecules.
2.In this section,we used CK-BB as a model enzyme to explore changes of structures and functions induced by small chemical molecules and elucidated the toxicological pathways of CK-BB unfolding and inactivation,as well as aggregation. The aggregation phenomenon has been generally recognized as an important factor of degenerative disease.Furthermore,it is worthy to study further for relationships between industrial chemicals and chronic neurotoxicity of human health.
随着现代化和工业化进程的加快,特应性皮炎(AD)患病率增高,是皮肤病和公共卫生领域的研究热点。AD是一种慢性炎症性皮肤病,易反复发作,以湿疹性皮损分布为特征。主要病理生理表现为苔藓样硬化、瘙痒性脱皮和皮肤干燥。AD是一种系统性功能失调性疾病,可同时触发哮喘、过敏性鼻炎和食物过敏,伴有血浆IgE和嗜酸性粒细胞升高。已报道多种细胞参与了AD的发病,如嗜酸性粒细胞、T淋巴细胞、朗格汉斯细胞和角质形成细胞,这些细胞都与细胞因子、化学增活素和IgE等血液因子以及与微生物感染有关。虽然大多数AD患者血浆IgE升高,但AD疾病中存在一个亚组对空气、食物过敏原无致敏性。AD疾病可分为两个类型:内源性和外源性。外源性AD(ADe)占AD疾病的70-80%,具有Th2细胞调节的血浆IgE增高,引起IgE介导的致敏作用的特征。与ADe不同,内源性AD(ADi)占AD疾病的20-30%,没有IgE介导的致敏作用和过敏反应。
高通量筛选(HTS)方法如DNA芯片和2D-PAGE/MALDI-TOF分析已开始用于AD相关候选基因的筛选。通过HTS,可以找出大量在AD中上调或下调的蛋白质或基因,同时,相互作用组工具如PPI图能有助于核心蛋白的挑选。因为接下来的功能研究是一个耗时、花费较大的过程,所以对目标基因的筛选显得非常重要,因此,通过对HTS结果的计算机预测分析是作出正确选择的关键。PPI图中列出的候选基因为理解复杂的AD疾病提供了重要信息。一旦PPI预测的相互反应配偶体得到证实,将为疾病相关的基因调节途径提供新的见解。已知与AD疾病相关的重要因素有IgE/FcεRI、抗微生物肽、细胞因子/化学增活素、蛋白酶及其受体、脂质基因、自身抗原、细胞外基质基因、表皮分化复合物等。在这个基础上,研发出新的治疗方法,并且已经用于AD的临床治疗。尽管目前对AD的治疗有多种途径,但是AD患者在增加,能否提供有效治疗方法如快速缓解严重症状的方案仍是个严峻的挑战。因此,需要更深入的研究AD的发病机理,进一步阐明其免疫机制。
另外本研究还进行了分子水平的环境毒理学研究。环境小分子化学物进入体内,可以与体内最主要的生物大分子一蛋白质发生相互作用,除了通过共价作用形成蛋白加合物外,环境化学物可能会影响蛋白质的构象,导致蛋白质发生错误折叠,从而产生环境健康风险。这种途径是否与折叠病的环境因素有关,迄今为止,这方面的报道较少,有必要开展深入研究。本研究以脑型肌酸激酶(CK-BB)为模型,试图探讨小分子化合物对酶的结构与功能影响。在了解CK-BB去折叠与再折叠过程基础上,进一步对日常生活中人群接触的频率较高的十二烷基硫酸钠(SDS)作用及与CK-BB结合机制进行了探讨,而且阐述了两种可能与退行性疾病相关的环境化学物,丙烯酰胺与锌离子,对CK-BB的结构与功能影响的作用机制。
研究方法
1.本研究中对患者活检样本、原代细胞培养(角质形成细胞和成纤维细胞)和血清样本中的基因改变进行研究。所有的AD样本分为外源性(ADe)和内源性(ADi)两型。非特异性正常对照组(n=22),均无个人或家族史,过敏性疾病症状和体征,其样本选自整形外科手术取下的皮肤。从ADe和ADi患者(n=40)皮损处取直径3-5mm活检样本。20名ADe患者(SCORAD:50.09±15.68),20名ADi患者(SCORAD:46.38±11.77)。ADe组和ADi组总IgE水平、红细胞计数和年龄的平均值分别为:1739 U/mL,625/μL and 27.4岁和105U/mL,248/μL and 28.2岁。所有样本均取自于男性。血清按照之前报道的方法收集,等分试样后储藏在-80℃。
DNA芯片的制备按照RNA制备、标记和杂交步骤进行:分别准备正常对照、ADe、ADi样本,每个样本均进行cDNA模板合成和标记。分别采用两种芯片对不同样本进行检测:GeneChip~(?)人U133 Plus 2.0芯片和GenePlorerTwinchip人-8K芯片。基因表达比值经过图像分析,并用LOWESS回归方程标准化。应用SAM和DEG。选择q值(按照5%)有明显表达差异(＞2倍)的基因。GeneChip~(?)人U133 P1us 2.0芯片用来进行ADe和ADi皮肤组织的分析,而GenePlorer Twinchip人-8K芯片用于分析特应性角质形成细胞。自流电泳(FFE)和2-DE分别用于特应性成纤维细胞和血清处理分析。患者源性血清经多重亲和移除柱(MARC)处理后进行2-DE分析。接下来,用苯基琼脂糖树脂分段式方法进行AD血清蛋白质的分析。血清经柱子分离后TCA沉淀。
根据以上结果,可采组学工具来进行核心基因和蛋白质的预测。PPI预测使用了PPI的主要算法,包括:1)蛋白结构相互作用使用PSIMAP(蛋白质组图谱),一种使用SCOP(蛋白结构分类)数据库结构域的方法;2)蛋白质实验性相互作用PEIMAP(蛋白质组图谱),是一种普遍的使用实验性蛋白相互反应信息资源的方法,如HPRD、BIND、DIP、MINT、IntAct和BioGid。
2.重组人CK-BB按照之前报道的方法进行表达和纯化。CK-BB活性测定采用ATP和肌酸反应释放质子,在百里酚蓝指示剂存在下,其597nm光吸收变化速率对应于酶活性。内源荧光和ANS结合光谱方法检测CK-BB三级结构的影响。采用圆二色谱分光光度计,在190-250nm范围内记录远紫外圆二色谱(CD)光谱检测CK-BB二级结构的影响。等温滴定微量量热法采用3101TAMⅢ恒温箱按照之前报道的方法进行测量。从蛋白质数据库(PDB)中获得已知的CK-BB同源性结构。发现同系物牛的视黄醛肌酸激酶A链(PDB编码:1g0w)是一个良好的CK-BB结构模板(含97%相同序列)。结合模拟操作分为:1)计算相应的受体和3D配体,2)对接大小特征,3)计算网格评分,4)决定补充受体和配体对接。
结果
1.Affymetrix芯片检测出406种差异表达基因(DEG),在正常人和AD患者(ADe和ADi)之间,鉴别出130个DEG;在正常人和ADe患者之间,鉴别出327个DEG;在正常人和ADi患者之间,鉴别出146个DEG。8K cDNA芯片结果显示在原代培养的角质形成细胞AD疾病中有157个基因失调。接下来,通过FFE在AD成纤维细胞中获得93个候选基因。MARC处理后的AD血清样本检测到30个上调蛋白质和19个下调蛋白质。改进分段方法,检测到9个失调基因。Affymetrix芯片分析利用PSIMAP和PEIMAP两种不同的生物信息学计算方法得到5个和8个核心基因,分别是:PIK3R1、IGF1R、MAPK13、SFN、YWHAE和CORIN、COL14A1、CLCA4、PTPRC、IL10RA、C1S、PHLPP、SLITRK6。应用8K cDNA芯片在角质形成细胞中找出25个基因。通过FFE在AD成纤维细胞中4个核心蛋白在PSIMAP和PEIMAP算法中均检测到,即:I55423、ARR3、YWHAE和EEF1A1。AD血清样本的2-DE结果中发现26种蛋白质。AD组织中SFN和PTPRC经实时PCR进一步验证。结果发现转录水平的SFN在ADi中上调4.5倍,PTPRC在ADe和ADi中分别下调60%和46%。ELISA结果显示,MMP1和MMP10在ADi患者中均上调。ADi患者血清的ELISA研究结果表明,与正常人和ADe患者相比较,MMP1和MMP10显著上调(几乎两倍),均存在统计学差异,采用随机配对和Wilcoxon检测。
2.对SDS使CK-BB的折叠后的失活进行了研究。SDS能非竞争性抑制CK-BB的活性(K_i=1.22mM)。SDS与CK-BB暴露的疏水表面结合诱导其三级结构的改变。SDS配体结合热力学参数的改变如焓、吉布斯自由能和熵分别为-13±7.0MJ/mol,8.39 kJ/mol and-42.754 kJ/(K·mol)。研究明确了重要的CK-BB结构以及和SDS相互反应的折叠和抑制动力学信息。预测的结构具有0.51(?)的均方根偏差。CK-BB与SDS之间对接成功,具有显著性评分(-4.67 kcal/mol,自动对接4和-48.32 kcal/mol,DOCK6)。丙烯酰胺对脑型肌酸激酶(CK-BB)的抑制效应研究发现,CK-BB能被动态的失活并伴随疏水表面的暴露。丙烯酰胺主要与CK-BB的硫氢基(-SH)残基相互作用导致烷化。计算机模拟对接支持丙烯酰胺直接结合到CK-BB的活性位点上,主要与CYS74和GLY73残基相互作用。Zn~(2+)通过剂量依赖效应失活CK-BB(IC50=0.06 mM)。Zn~(2+)显著诱导CK-BB疏水基表面破裂导致三维结构改变。还发现Zn~(2+)能使CK-BB发生聚沉。聚沉依赖于温度以及酶和Zn~(2+)的浓度。
结论
1.为了更深入了解AD发病机制,明确在AD病情发展中有多少基因起作用,进行了大规模的微阵列研究,得到许多新的相关信息。结果主要集中在AD相关基因大规模DNA芯片研究的描述上,这将为全面了解AD疾病提供新的见解,但是结果中尚不包括详细的功能研究。在后续的实验中进行了AD皮肤组织的研究并检测到了核心基因。然后进行角质形成细胞和成纤维细胞的研究。同时还对AD患者血清中蛋白质差异表达进行了研究。已知多种因子参与了AD的发病,不同的患者中存在多种触发因素,本研究数据提示人表皮角质形成细胞和真皮成纤维细胞活化在AD疾病中起作用。然而,在原代培养的角质形成细胞和成纤维细胞中检测到的蛋白质或基因可能提示着体内也存在相应的蛋白质变化,但是mRNA水平和表达的蛋白质水平之间的差异干扰了对AD中准确作用的理解。
大规模的芯片研究结果能有助于深入了解AD发病机制的复杂的相关因素。结合表达数据分析和蛋白质相互作用组预测能找到更可靠的与AD疾病相关的基因。预测的蛋白质相互作用配体和核心蛋白能为下一步AD治疗的功能研究提供有效、可靠的靶点。AD患者数量迅速增多,急需找到特异性诊断和敏感性治疗方法。因此,OMICS手段的应用改善了对AD皮肤组织的表达形式以及组分如角质形成细胞和成纤维细胞的研究。根据本研究,得出以下结论:1)生物信息学工具结合HTS数据筛选新的核心基因或蛋白,在今后的功能学研究和临床药物开发中将发挥重要作用;2)本研究中发现多个在AD源性样本中表达显著改变的重要基因,如SFN、PTPRC、MMP1和MMP10,可能是AD疾病的生物学标记;3)本研究中预测出的大部分核心蛋白、以及实时PCR检测出的候选基因/蛋白在之前关于AD发病机制的研究中未见报道;4)结合基因组学、蛋白质组学和相互作用组分析方法等研究手段将有助于AD复杂病理机制的研究,以及准确的生物标记物或临床治疗靶点的探测。
2.本部分研究我们以脑型肌酸激酶(CK-BB)为模型,探讨了小分子工业化学物通过影响酶的结构和功能,引发蛋白质错去折叠,导致酶的失活以及聚沉的毒理途径。不管是SDS(两亲变性剂),丙烯酰胺(巯基加合物),还是Zn2+(金属离子),都可以诱导蛋白质疏水表面的暴露。尤其是Zn2+促发CK-BB发生聚沉。这种由小分子化学物诱发的聚沉现象在许多退行性病变中可能具有重要的意义,其确切的病理价值有待进一步深入研究。环境中存在大量的具有慢性神经毒作用的工业化学品。这些小分子化学物是含具有协同作用,诱导蛋白质构象与功能的变化将是一个重要的研究课题。
Background
Atopic dermatitis(AD) patient populations have been prevalent along with modernization and industrialization.AD is not only a critical skin disease,but also is the hot issue of the public health.AD is a chronic relapsing inflammatory skin disease that's characterized by the distribution of eczematous skin lesions.AD typically reveals lichenification,pruritic excoriations and dry skin with a variety of pathophysiologic manifestations.Since AD is a systemic disorder,it simultaneously triggers asthma,allergic rhinitis and food allergy,where the serum IgE and peripheral eosinophils are elevated.Several cells such as eosinophils,T lymphocytes, Langerhans cells and keratinocytes have been regarded as important for the pathogenesis of AD since these cells are directly related with several blood factors such as cytokines,chemokines and IgE,and they are also related with microbial infection.Although most AD patients have high serum IgE levels,one subgroup of AD patients lacks the sensitization to aero-allergens or food allergens.Two types of AD have been defined,and they are the extrinsic and intrinsic types.The extrinsic type of AD(ADe) has a Th2 cell mediated high serum IgE level and this condition is associated with IgE-mediated sensitization;this is seen in 70-80%of AD patients.As a counterpart to ADe,the intrinsic type(ADi) is not involved with IgE-mediated sensitization and allergies,and ADi is seen in about 20-30%of AD patients.
High-throughput screening(HTS) methods such as DNA microarray and 2D-PAGE/MALDI-TOF approaches are currently being used to determine the candidate genes associated with AD.With HTS,although we can obtain a great deal of genes/proteins that are overexpressed or down-regulated in AD disease,it is very difficult to choose the most important genes.Furthermore,it is hard to explain the meaning of those proteins.In this regard,interactomic tools such as performing PPI mapping may help to find the hub proteins among the detected candidate genes.Since the next step of functional studies is a time-and cost-consuming process,the number of target proteins must be limited;hence,to make the right choice,computational prediction on the basis of HTS results could be critical.The candidate genes listed in the PPI maps may provide important information to piece together the complex nature of AD disease.Regardless of the various approaches that are currently being used to treat AD,the number of AD patients has greatly increased and more effective treatments that offer quick relief of severe symptoms still remain to be developed. Thus,more insights regarding the complex gene/protein regulation are needed into the pathogenesis of AD,in addition to elucidating the immunologic aspects of this disease.
Besides AD studies,we also conducted the molecular level studies of environmental toxicology.Once the environmental chemicals absorbed into the body, they can interact with the most important biological macromolecules such as proteins. The chemicals not only can form the covalent protein adducts,but also may affect the protein conformation as well as lead to protein misfolding and thus,result in environmental health problems.In this study,we used CK-BB as a model material for trying to explore the effects of small molecules on the enzyme structure and function. Based on the understanding of CK-BB unfolding and refolding,we further studied the mechanisms of CK-BB binding with sodium dodecyl sulfate(SDS) that is abundantly presented in modern life and also,studied the effects of two environmental chemicals such as acrylamide and Zn~(2+) which may be related to degenerative diseases.
Method
1.All AD samples were divided into the two types defined as the extrinsic(ADe) and intrinsic(ADi) types.AD skin tissues were collected from non-asthmatic atopic patients.Non-atopic control subjects who had neither a personal/family history nor any signs of atopic diseases were selected for the study.Punch biopsies of 3-5 mm diameter were individually obtained from the lesional skin of both the ADe and ADi patients.We recruited 20 ADe patients,20 ADi patients and 22 normal specimen donors.The mean values for each group's total IgE level,eosinophilia count,age and SCORAD score were 1739 U/mL,625/μL,27.4 year old and 50.09 for the ADe group and 103 U/mL,248/μL,28.2 year old and 46.3 for the ADi group,respectively. The gender of all samples was male.Serum was collected according to previously reported methods and stored at-80℃after proper aliquots.
DNA microarray was performed following by the total RNA preparation, labeling and hybridization:the individually prepared normal,ADe,and ADi RNA samples were correspondingly prepared,and the further cDNA template synthesis and labeling for each of samples were conducted.The two kinds of commercial DNA chips(Affymetrix Human Genome U133 Plus 2.0 Array chip;and GenePlorer Twinchip Human-8K) were individually applied for different samples.These chips were hybridized with a mixture of fluorescently labeled cDNAs from the control and AD samples at 42℃for 16 h in the presence of hybridization solution.Image analysis to obtain the gene expression ratios was performed properly and the logged gene expression ratios were normalized by a LOWESS regression procedure.By using DEG and SAM,the data sets were analyzed.The genes showing significant expression changes(＞2-fold) were selected with a cutoff of 5%of the q value.An Affymetrix microarray was used to analyze ADe and ADi skin tissues,and the 8K cDNA microarray was used to analyze atopic keratinocytes.These tools were probed the dysregulated genes at the transcriptional levels.The free-flow electrophoresis (FFE) was used to analyze atopic fibroblasts and two-dimensional electrophoresis (2-DE) was used to analyze serums with proper treatments.Based on the above results,the interactomic tool was also adapted to predict putative hub genes or proteins.
2-DE analyses of AD patient-derived serums were conducted after treating with multiple affinity removal columns(MARC).In addition,fractionation method by using Phenyl Sepharose resin was conducted to analyze AD serum proteins.Serums were properly fractionation with a column following by TCA precipitation.
In the next step,PPI of AD related proteins were predicted by bioinformatics algorithms.The prediction of PPI uses most of the major types of PPI algorithms. They are:1) Protein Structural Interactome MAP(PSIMAP),a method that uses the structural domain of the SCOP(Structural Classification of Proteins) database;2) Protein Experimental Interactome MAP(PEIMAP),a common method that uses public resources of experimental protein interaction information such as HPRD, BIND,DIP,MINT,IntAct and BioGrid.
2.Recombinant human CK-BB was expressed and purified according to the previous reports.CK-BB activity was measured following proton generation during the reaction of ATP and creatine with the indicator thymol blue at 597 nm.The fluorescence emission spectra and the ANS labeling fluorescence were studied for monitoring the tertiary structural changes.The far-UV circular dichroism(CD) spectra were recorded in the range of 190-250 nm for measuring secondary structural changes.Isothermal titration microcalorimetric measurements were performed with a 3101 TAM III Thermostat in accordance with a previous report.We retrieved the known homologous structures of CK-BB from the Protein Data Bank(PDB) and found that bovine retinal creatine kinase chain A(PDB entry:1g0w) was a very good structural template(97%sequence identity) as a close homologue for CK-BB.The docking procedures were categorized to 1)calculate receptor and ligand 3D coordinates,2)characterize docking site,3)compute grid score,and 4)determine the docking for complementarity with receptors and ligand.
Result
1.From the Affymetrix microarray results,406 genes were selected:130 genes were obtained from normal vs.AD(ADe and ADi);327 genes were obtained from normal vs.ADe;146 genes were obtained from normal vs.ADi.The 8K cDNA microarray results showed that 157 genes were significantly dysregulated in primary atopic keratinocytes compared to the control subjects.In the next step,we obtained 93 candidate proteins by using FFE in AD fibroblasts.In AD serums,30 proteins were detected as up-regulated and 19 proteins were down-regulated in AD serums after using MARC treatment.With adapting fractionation method,9 proteins were detected as dysregulated.
In Affymetrix microarray analysis,five hub genes such as the PIK3R1,IGF1R, MAPK13,SFN and YWHAE were predicted by using the PSIMAP algorithm and eight_genes such as CQRIN,COL14A1,CLCA4,PTPRC,IL10RA,C1S,PHLPP and SLITRK6 were predicted by using the PEIMAP algorithm.In 8K cDNA microarray, 25 genes were predicted for AD keratinocytes hub proteins.In FFE result, overlapping of the PEIMAP and PSIMAP algorithms detected 4 hub proteins:I55423, ARR3,YWHAE,and EEF1A1.In 2-DE results of AD serums,26 proteins were predicted for AD serum samples.
SFN and PTPRC were also further validated by real-time PCR in AD tissues. The results showed that the transcriptional level of SFN is up-regulated 4.5-fold in ADi,and PTPRC is down-regulated 60%in ADe and 46%in ADi.In the ELISA results,MMP1 and MMP10 were consistently detected as up-regulated in the serum of ADi patients.The results of ELISA studies of the serum of ADi patients showed that both MMP1 and MMP10 were significantly up-regulated(almost 2-fold) compared to normal and ADe patients.Statistical significance was confirmed by p-values.Randomized pairings and the Wilcoxon-rank sum test were applied.
2.We have also investigated the inactivation by using SDS to study CK-BB's folding behaviors.SDS strongly inhibited the CK-BB activity in a noncompetitive inhibition manner(K_i=1.22 mM).The tertiary structural change was induced by SDS binding with the exposure of hydrophobic surface.The changes of thermodynamic parameters for the SDS ligand binding such as enthalpy,Gibbs free-energy,and entropy were obtained as-13±7.0 MJ/mol,8.39 kJ/mol and-42.754 kJ/(K·mol),respectively.The predicted structure had a Root Mean Square Deviation of 0.51 A.The docking between CK-BB and SDS was successful with significant scores(-4.67 kcal/mol, AutoDock4 and-48.32 kcal/mol,DOCK6).
We studied the inhibitory effects of acrylamide on CK-BB.We found that CK-BB was kinetically inactivated by acrylamide accompanied by the disruption of the hydrophobic surface.Acrylamide mainly interacted with the thiol(-SH) residue of CK-BB and resulted in alkylation.A computational docking simulation supported that acrylamide directly bound to the active site of CK-BB where CYS74 and GLY73 residues interacted mainly.
Zn~(2+) inactivated the activity of CK-BB in a dose dependent manner(IC_(50)=0.06 mM).The spectroflurorimetry results showed that Zn~(2+) conspicuously induced the tertiary structural change of CK-BB with exposure of its hydrophobic surfaces.We also found that CK-BB aggregation was induced by Zn~(2+).This aggregation was dependent on the temperature and the enzyme and Zn~(2+) concentrations.
Conclusion
1.With respect to explaining all the events that occur in the pathogenesis of AD or for probing how many genes are involved in the development of AD,our large-scale microarray data can suggest new information.In the initial part of results,the data were mainly focused on profiling the AD associated genes in a large scale DNAchip array and this provides useful information for the overall insight into the AD disease at the level of skin tissues,but our results do not include any detailed functional study. Then,primary cultured keratinocytes and fibroblasts were studies.The profiling of differentially expressed proteins in AD serums was also conducted.In respect to the complexity of the AD pathogenesis where many factors are associated with AD and various triggers are known to exist among different patient,our data suggest that human epidermal keratinocytes and dermal fibroblasts actively participate in AD disease along with the other types of cells.However,some of the genes/proteins observed in the primary cultured keratinocytes and fibroblasts may not be representative of the in vivo changes,and the differences between the mRNA levels and the expressed protein levels limit our understanding with regard to their exact roles in AD.
Taken together,the large scale microarray provided information to further understand the complex factors associated with pathogenesis of AD.The approach of combining the expression data analysis and predicted protein interaction partners performed in large scales can bring valuable and more reliable gene targets for AD. The predicted protein interaction partners and validated hub proteins can offer valuable and reliable targets for further functional studies of AD treatment.The number of the AD patients is increasing rapidly and being able to make a specific diagnosis and administering case sensitive treatment are needed.Therefore,OMICS approaches that are based on high-throughput technologies may help researchers gain additional insight or they may reveal pieces of the puzzle into the pathogenesis of AD.
According to the results,several points are suggested in this study:i)the bioinformatic tools combined with the HTS data identified the new candidate hub genes/proteins,which can be useful in the further studies,especially for the functional studies or clinical trials of drug development;ii)several important genes such as SFN, PTPRC,MMP1 and MMP10 have been newly suggested in this study,which they are significantly up-regulated or down-regulated in AD patients-derived samples and they can be biomarkers for AD;iii)most of the predicted hub proteins and real-time PCR probing candidate genes/proteins detected in this study have not been previously reported to be involved in AD pathogenesis.This may be due to the unknown role of these factors in AD pathogenesis;iv)in addition to the fact that the combinations of several research areas such as genomics,proteomics,and interactomics can help guide research into the complex pathogenesis of AD and assist in designing precise therapeutic strategies to find biomarkers or to determine the right clinical target molecules.
2.In this section,we used CK-BB as a model enzyme to explore changes of structures and functions induced by small chemical molecules and elucidated the toxicological pathways of CK-BB unfolding and inactivation,as well as aggregation. The aggregation phenomenon has been generally recognized as an important factor of degenerative disease.Furthermore,it is worthy to study further for relationships between industrial chemicals and chronic neurotoxicity of human health.
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