In Situ PLA技术原位分析MTA1与NuRD复合体其他组分的相互作用

目的原位检测肿瘤转移相关蛋白MTA1与NuRD复合体其他组分-Mi2、HDAC2及MBD3的相互作用并分析MTA1/NuRD复合体在HCT116细胞中的分布情况。方法首先利用co-IP技术体外验证MTA1与NuRD复合体其他组分Mi2、HDAC2及MBD3在HCT116细胞裂解液中的相互作用。然后利用In Situ PLA技术,体内原位检测MTA1与三者的相互作用,并根据镜下阳性荧光信号数目,比较MTA1与Mi2、HDAC2及MBD3之间的作用强弱;根据相互作用位点的亚细胞分布情况,分析间期及M期MTA1/NuRD复合体的核质分布情况。结果首次从体内原位水平证实MTA1与NuRD复合体其他组分-Mi2、HDAC2及MBD3的相互作用;其作用强度HDAC2最高,Mi2次之,MBD3最弱;首次发现MTA1/NuRD复合体存在很明显的胞质分布(约占总量的1/4);另外,我们还首次发现在M期MTA1/NuRD复合体不再位于核区,而是位于染色体外周。结论In Situ PLA技术是一项有效的原位检测蛋白相互作用的新技术;MTA1/NuRD复合体可能参与MTA1胞质中功能的发挥;并且其胞质分布可能参与M期进程调控。

ZMYND8与NuRD复合物核心组分RBBP4相互作用界面的鉴定

核小体重塑与去乙酰化酶(Nucleosome remodeling and histone deacetylase,NuRD)复合物由多个亚基组成,在转录调节和DNA损伤修复的过程中发挥着重要的功能。人源ZMYND8(Zinc finger MYND-type containing 8)能与NuRD形成复合物参与转录调节和DNA损伤修复的过程中。利用昆虫杆状病毒表达系统,经过镍柱和分子筛柱层析纯化获得电泳纯的NuRD核心亚基RBBP4蛋白质。采用等温量热滴定技术鉴定ZMYND8与RBBP4的相互作用界面,结果显示RBBP4能够结合位于ZMYND8氮端的43到54位富含碱性氨基酸残基的区域。RBBP4上的氨基酸残基点突变实验初步确定二者的相互作用界面。结合以往的研究发现,ZMYND8可以通过其氮端的碱性区域和碳端的MYND结构域环绕结合在NuRD复合物的表面。

JARID2 coordinates with the NuRD complex to facilitate breast tumorigenesis through response to adipocyte-derived leptin

Background Proteins containing the Jumonji C(JmjC)domain participated in tumorigenesis and cancer progression.However,the mechanisms underlying this effect are still poorly understood.Our objective was to investigate the role of Jumonji and the AT-rich interaction domain-containing 2(JARID2)—a JmjC family protein—in breast cancer,as well as its latent association with obesity.Methods Immunohistochemistry,The Cancer Genome Atlas,Gene Expression Omnibus,and other databases were used to analyze the expression of JARID2 in breast cancer cells.Growth curve,5-ethynyl-2-deoxyuridine(EdU),colony formation,and cell invasion experiments were used to detect whether JARID2 affected breast cancer cell proliferation and invasion.Spheroidization-based experiments and xenotumor transplantation in NOD/SCID mice were used to examine the association between JARID2 and breast cancer stemness.RNA-sequencing,Kyoto Encyclopedia of Genes and Genomes,and Gene Set Enrichment Analysis were used to identify the cell processes in which JARID2 participates.Immunoaffinity purification and silver staining mass spectrometry were conducted to search for proteins that might interact with JARID2.The results were further verified using co-immunoprecipitation and glutathione S-transferase(GST)pull-down experiments.Using chromatin immunoprecipitation(ChIP)sequencing,we sought the target genes that JARID2 and metastasis-associated protein 1(MTA1)jointly regulated;the results were validated by ChIP-PCR,quantitative ChIP(qChIP)and ChIP-reChIP assays.A coculture experiment was used to explore the interactions between breast cancer cells and adipocytes.Results In this study,we found that JARID2 was highly expressed in multiple types of cancer including breast cancer.JARID2 promoted glycolysis,lipid metabolism,proliferation,invasion,and stemness of breast cancer cells.Furthermore,JARID2 physically interacted with the nucleosome remodeling and deacetylase(NuRD)complex,transcriptionally repressing a series of tumor suppressor genes such as BRCA2 DNA repair associated(BRCA2),RB transcriptional corepressor 1(RB1),and inositol polyphosphate-4-phosphatase type II B(INPP4B).Additionally,JARID2 expression was regulated by the obesity-associated adipokine leptin via Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)pathway in the breast cancer microenvironment.Analysis of various online databases also indicated that JARID2/MTA1 was associated with a poor prognosis of breast cancer.Conclusion Our data indicated that JARID2 promoted breast tumorigenesis and development,confirming JARID2 as a target for cancer treatment.

Corepressor metastasis-associated protein 3 modulates epithelial-to-mesenchymal transition and metastasis

Worldwide,metastasis is the leading cause of more than 90%of cancer-related deaths.Currently,no specific therapies effectively impede metastasis.Metastatic processes are controlled by complex regulatory networks and transcriptional hierarchy.Corepressor metastasis-associated protein 3(MTA3)has been confirmed as a novel component of nucleosome remodeling and histone deacetylation(NuRD).Increasing evidence supports the theory that,in the recruitment of transcription factors,coregulators function as master regulators rather than passive passengers.As a master regulator,MTA3 governs the target selection for Nu RD and functions as a transcriptional repressor.MTA3dysregulation is associated with tumor progression,invasion,and metastasis in various cancers.MTA3 is also a key regulator of E-cadherin expression and epithelial-to-mesenchymal transition.Elucidating the functions of MTA3 might help to find additional therapeutic approaches for targeting components of NuRD.

肺腺癌细胞中PRDM5抑癌作用的分子机制初探

目的:解析肺腺癌中PR结构域锌指蛋白5(PRDM5)发挥抑癌功能的分子机制,寻找与PRDM5相互作用的蛋白因子。方法:免疫共沉淀实验(Co-IP)检测肺腺癌细胞系A549和H1299中PRDM5与核小体重塑和去乙酰化酶复合物(NuRD复合物)各组分之间内源性结合。构建含有GST标签的原核表达载体pGEX-4T-1-HDAC1、pGEX-4T-1-MTA1、pGEX-4T-1-MTA2以及带有His标签的原核表达载体pET-28a(+)-PRDM5。将上述重组质粒转化入大肠埃希菌(E.coli)BL21中诱导融合蛋白成功表达并纯化相应的蛋白。GST-pull down体外实验检测PRDM5与NuRD复合物直接结合的组分。结果:在肺腺癌细胞系A549和H1299中证实PRDM5与NuRD复合物多个主要组分CHD3、CHD4、MTA1/2、HDAC1/2、RbAp46/48、MBD2/3间存在内源性结合。体外GST-pull down实验证实PRDM5与NuRD复合物中的组蛋白去乙酰化酶1(HDAC1)、转移相关蛋白1(MTA1)和转移相关蛋白2(MTA2)组分存在直接结合。结论:肺腺癌细胞中PRDM5与NuRD复合物存在相互作用。

MTA1与肿瘤侵袭转移的研究进展

肿瘤转移相关基因 1(MTA1)是NuRD的亚单位之一,它通过促进组蛋白脱乙酰基,影响基因的转录和复制。研究发现,它与乳腺癌、食道癌、胃肠癌、胰腺癌等多种人类上皮源性肿瘤侵袭转移的能力紧密相关,它的高表达与肿瘤的侵袭深度与病理分级呈正相关,可作为评价预后的一个指标。

核小体重塑及组蛋白去乙酰化酶复合物的负染电镜结构分析

目的·利用负染电镜技术分析人源核小体重塑及组蛋白去乙酰化酶复合物(nucleosome remodeling and deacetylase complex,NuRD复合物)结构,获得人源NuRD复合物的轮廓信息。方法·将C端带有3×Flag标签的MBD3(methyl-CpG binding domain protein 3)和N端带有10×His标签的GATAD2A(GATA zinc finger domain containing 2A)克隆至pMLink表达载体中,采用聚乙烯亚胺瞬时转染过表达的方式在人源Expi293F悬浮细胞里表达NuRD复合物中的蛋白质组分;依次通过Ni-NTA亲和层析、Flag(DYKDDDDK)标签亲和层析和Superose 6 Increase 5/150凝胶过滤层析分离纯化NuRD复合物;利用蛋白质印迹法(Western blotting)和液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)对复合物进行组分鉴定;利用负染电镜技术结合单颗粒重构技术研究NuRD复合物的空间结构;通过UCSF Chimera软件将蛋白质数据库(Protein Data Bank,PDB)中已有亚复合物的原子结构模型(7AO9,5FXY)与生成的结构模型进行自动匹配及比对,预测多个蛋白组分在负染结构模型中的定位。结果·利用两步亲和层析,成功富集了带有纯化标签的MBD3、GATAD2A蛋白及其他内源蛋白组分,通过进一步的凝胶过滤层析分离得到了均一性良好的复合物;通过Western blotting和LC-MS/MS鉴定,确认纯化得到的复合物为组分完整的人源NuRD复合物。利用负染电镜技术及单颗粒重构技术初步解析了NuRD复合物的空间结构,其整体轮廓特征明显,呈现为不对称的长条形;通过进一步的三维优化处理,最终获得了人源NuRD复合物分辨率约为17A(1A=0.1 nm)的初步三维结构模型;已有的亚复合物原子结构模型(PDB:7AO9,5FXY)与NuRD复合物的初步三维结构模型自动匹配后,初步确定了MTA1/2/3(metastasis-associated protein 1/2/3)、HDAC1/2(histone deacetylase 1/2)、RBBP4/7(retinoblastoma-binding protein 4/7)及MBD2/3(methyl-CpG-binding domain protein 2/3)蛋白亚基在NuRD复合物负染结构模型中的定位。结论·利用单颗粒重构技术搭建了人源NuRD复合物的低分辨率负染结构模型。

肿瘤转移相关蛋白家族在雌激素受体α信号转导中的作用

雌激素受体α(estrogen responseα,ERα)通过调控多种靶基因的转录来发挥其重要的生物学功能,这一过程依赖于共调节因子的调控。肿瘤转移相关蛋白(metastasis-associated protein,MTA)是近年来受到广泛重视的ERα共抑制因子,其家族成员分别是不同的核小体重构和组蛋白脱乙酰基酶(nucleosome remodeling and histone deacetylase,NuRD)复合体的亚基,在ERα的信号通路中发挥着不同的作用,与乳腺癌、肝癌、卵巢癌等的发生、发展有密切联系。MTA1既可抑制又可刺激ERα的转录;MTA2能够对ERα的转录活性产生抑制作用;而MTA3的表达受ERα的调节。主要就MTA1、2、3在ERα信号通路中的作用以及它们对肿瘤发生转移过程的影响作一综述。