AP-2相互作用蛋白的筛选及鉴定
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摘要
转录因子AP-2是一个功能重要的家族,在哺乳动物的胚胎发育过程中,其表达受到严格的时空调控,参与多种组织和器官的细胞分化和形态发生,如神经脊、颅面部、四肢、躯干部、心脏和肾脏的发育。目前AP-2家族有五个成员被鉴定出来,分别是AP-2α、AP-2β、AP-2γ、AP-2δ和AP-2ε。这些成员都能以同源或异源二聚体的形式结合于保守的DNA序列5'-GCCNNNGGC-3'。AP-2α和AP-2β是AP-2家族中两个较早被克隆和研究较多的两个成员。其功能不仅表现为胚胎发育中的重要调控子,还通过转录调控下游靶基因参与多个生理过程的调节,包括了细胞生长,分化,癌变和凋亡等。作为一个多功能的调控因子,AP-2α和AP-2β有多个协同作用的蛋白伙伴。为了找到更多的与AP-2α和AP-2β相互作用的蛋白因子,本文采用免疫共沉淀结合质谱鉴定的方法,筛选AP-2α和AP-2β潜在的相互作用蛋白。
在小鼠的前脂肪细胞3T3L-1细胞中,筛选得到了五个新的AP-2α潜在相互作用蛋白,这其中就包括了如Acadl这种与脂肪代谢相关的蛋白。另外在人的HEK293FT细胞中筛选出两个新的AP-2β潜在相互作用蛋白,其中一个是Stomatin like protein 2(SLP-2),SLP-2据现有研究报道很可能是个肿瘤相关基因。本论文重点研究了SLP-2与AP-2β的相互作用。我们克隆了SLP-2的基因,用免疫共沉淀实验证实了外源过表达和内源的SLP-2蛋白都能与AP-2β蛋白相互作用。而且,细胞免疫荧光实验发现外源表达的Myc-AP-2β蛋白与内源的SLP-2蛋白在MCF-7和Hela细胞的胞质中有共定位。此外,过表达SLP-2能上调AP-2β的蛋白水平,但是对其转录活性水平影响不大。这些研究结果为进一部研究AP-2的功能奠定了基础。
AP-2 is a critical transcriptional family temporally and spatially regulated in mammal embryo development including neural crest,cranial face,limbs,heart and kidney.To date,five members of the AP-2 family of transcription factors,AP-2α,AP-2β,AP-2γ,AP-2δand AP-2εhave been identified and all of them can bind as homo-or hetero-dimers to the typical consensus sequence of 5'-GCCNNNGGC-3'.AP-2αand AP-2βare the early gene cloned and the relatively best characterized among the AP-2 gene family.It has been show that AP-2 is an important regulator that mediates essential events in embryo development and also participate in many biological processes such as cell growth,differentiation,carcinogenesis or apoptosis through the transcriptional contral of multiple target downstreme genes.To be a functional regulator,AP-2αand AP-2βis co-operate with a number of partners.To identify more of the interacting co-activators of AP-2αand AP-2β,we screened of AP-2αand AP-2βimmunocomplexes using Immunoprecipitation technologe combining mass spectrometry.
We screened five the functional complex of AP-2αin mice 3T3L-1 cells.These screened functional complex candidates inclued Acadl complex which was know to be related to adipose tissue metabolism.And screened two protein as a putative AP-2βinteracting protein,and some new candidates had not be reported before.SLP-2 is one of them.SLP-2 has been preported as a candidate of tumor gene.In this study,we put emphasis on the relevance between SLP-2 and AP-2β.We demonstrated both exogenously expressed and endogenous SLP-2 interacted with AP-2βin vivo by co-immunoprecipitation assays.In addition,indirect immunofluorescent analyses demonstrated that AP-2βand SLP-2 co-localized in the cytoplasm of the MCF-7 and Hela cell lines. Furthermore over-expression of SLP-2 enhanced the protein expression level of AP-2β,but without altering its transcriptional activity.This results provided a new clue for further research on the AP-2 function.
在小鼠的前脂肪细胞3T3L-1细胞中,筛选得到了五个新的AP-2α潜在相互作用蛋白,这其中就包括了如Acadl这种与脂肪代谢相关的蛋白。另外在人的HEK293FT细胞中筛选出两个新的AP-2β潜在相互作用蛋白,其中一个是Stomatin like protein 2(SLP-2),SLP-2据现有研究报道很可能是个肿瘤相关基因。本论文重点研究了SLP-2与AP-2β的相互作用。我们克隆了SLP-2的基因,用免疫共沉淀实验证实了外源过表达和内源的SLP-2蛋白都能与AP-2β蛋白相互作用。而且,细胞免疫荧光实验发现外源表达的Myc-AP-2β蛋白与内源的SLP-2蛋白在MCF-7和Hela细胞的胞质中有共定位。此外,过表达SLP-2能上调AP-2β的蛋白水平,但是对其转录活性水平影响不大。这些研究结果为进一部研究AP-2的功能奠定了基础。
AP-2 is a critical transcriptional family temporally and spatially regulated in mammal embryo development including neural crest,cranial face,limbs,heart and kidney.To date,five members of the AP-2 family of transcription factors,AP-2α,AP-2β,AP-2γ,AP-2δand AP-2εhave been identified and all of them can bind as homo-or hetero-dimers to the typical consensus sequence of 5'-GCCNNNGGC-3'.AP-2αand AP-2βare the early gene cloned and the relatively best characterized among the AP-2 gene family.It has been show that AP-2 is an important regulator that mediates essential events in embryo development and also participate in many biological processes such as cell growth,differentiation,carcinogenesis or apoptosis through the transcriptional contral of multiple target downstreme genes.To be a functional regulator,AP-2αand AP-2βis co-operate with a number of partners.To identify more of the interacting co-activators of AP-2αand AP-2β,we screened of AP-2αand AP-2βimmunocomplexes using Immunoprecipitation technologe combining mass spectrometry.
We screened five the functional complex of AP-2αin mice 3T3L-1 cells.These screened functional complex candidates inclued Acadl complex which was know to be related to adipose tissue metabolism.And screened two protein as a putative AP-2βinteracting protein,and some new candidates had not be reported before.SLP-2 is one of them.SLP-2 has been preported as a candidate of tumor gene.In this study,we put emphasis on the relevance between SLP-2 and AP-2β.We demonstrated both exogenously expressed and endogenous SLP-2 interacted with AP-2βin vivo by co-immunoprecipitation assays.In addition,indirect immunofluorescent analyses demonstrated that AP-2βand SLP-2 co-localized in the cytoplasm of the MCF-7 and Hela cell lines. Furthermore over-expression of SLP-2 enhanced the protein expression level of AP-2β,but without altering its transcriptional activity.This results provided a new clue for further research on the AP-2 function.
引文
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