Jurkat细胞中与Par3 PDZ结构域相互作用的蛋白质的蛋白质组学鉴定的研究
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摘要
细胞极性是真核生物的基本特征。在多细胞动物中,细胞极性在大多数细胞的分化和功能发挥中具有至关重要的作用。
Par-3是进化上保守的Par-3/Par-6/aPKC复合物中的一个关键组分,主要有180K、150K和100K三个亚型。进化上保守的细胞极性蛋白Par3是一个脚手架样蛋白质,它通过其PDZ1结构域与Par6和JAM相互作用,在上皮细胞极性的建立和维持中发挥重要作用。尽管Par3的作用在上皮细胞中已经被广泛地研究,但是它在其他类型细胞例如造血细胞中的作用仍然知之甚少。除了细胞极性作用之外,近来研究提示在不同的细胞株中可能有不同的与Par3相互作用的蛋白质。也有研究表明Par-3是一个多功能蛋白质,提示Par-3除了在细胞极性建立中发挥作用外也可能参与其他的生物学过程。因此Par3相互作用蛋白质的鉴定对于进一步了解Par3的功能至关重要。
目前有报道说Par3复合物在T细胞中是极化的。在血液细胞如Jurkat细胞中虽然含有Par-3蛋白质,但Par-3在这类细胞中的作用还未曾研究过,可能有不同的Par-3结合蛋白质。为了从分子水平上研究Par3的更多功能,我们用GSTpull-down与液相色谱.串联质谱结合的方法在Jurkat细胞中得到并鉴定了一些新的与Par3 PDZ结构域相互作用的蛋白质。这些被鉴定的蛋白质可能参与执行蛋白质代谢以及蛋白质运输等不同的细胞功能。Par3与这些蛋白质的相互作用提示Par-3可能参与这些生理代谢和物质运输活动。通过体外结合试验、免疫共沉淀试验和免疫荧光试验及共聚焦显微镜检测,验证了几个候选蛋白质——核运输蛋白质Importin-α4亚基、蛋白酶体激活因子PA28-β和PA28-γ通过Par3 PDZ1结构域与Par3之间的相互作用。我们的实验结果不仅有助于揭示进化上保守蛋白质Par3的新功能,更可为血液细胞中Par3新功能的研究提供重要线索。
Cell polarity is the fundamental characteristics of eukaryotic cells and of crucial importance to the differentiation and functions of most cells in the metazoans.
The partitioning-defective 3(Par-3)is a key component in the conserved Par-3/Par-6/aPKC complex.There are mainly three isoforms of Par3,180K,150K and 100K.The evolutionarily conserved cell polarity protein Par3,a scaffold-like PDZ-containing protein,plays a critical role in the establishment and maintenance of epithelial cell polarity.Although the roles of Par3 were extensively investigated in epithelial cells,its roles in other cell types such as hematopoietic cells have scarcely been explored.Recent studies have indicated that Par-3 is a multifunctional protein, and it may be involved in other biological context in addition to establishing cell polarity.Thus we believe that the identification of binding partners of Par3 is important for understanding its role.
Recently,Par3 complexes were reported being polarized in T cells.Although there are Par3 in blood cells such as Jurkat cells,the functions of Par3 in the cells remain elusive.There may be different Par3 interacting proteins in Jurkat cells.In order to obtain more information about the molecular basis of Par3 functions,we identified several potential novel binding-proteins of PDZ domains of Par3 in Jurkat cells(a T-cell line)by combining GST-pull-down approach with LC-MS/MS.The identified proteins are implicated to be involved in different cellular functions,for instance,protein metabolism,protein transportation and so on.The interaction between Par3 and these identified proteins indicates that Par3 may involve in these different biological functions.The interaction of Par3 PDZ domains with three proteins,nuclear transport protein Importin-α4,proteasome activators PA28-βand PA28-γwere further confirmed through in vitro binding assay, co-immunoprecipitation assay and immunofluorescence.Our results should not only help to uncover novel functions of this evolutionarily conserved protein Par3,but also throw new light on uncovering novel functions of the cell polarity protein Par3 in blood cells.
Par-3是进化上保守的Par-3/Par-6/aPKC复合物中的一个关键组分,主要有180K、150K和100K三个亚型。进化上保守的细胞极性蛋白Par3是一个脚手架样蛋白质,它通过其PDZ1结构域与Par6和JAM相互作用,在上皮细胞极性的建立和维持中发挥重要作用。尽管Par3的作用在上皮细胞中已经被广泛地研究,但是它在其他类型细胞例如造血细胞中的作用仍然知之甚少。除了细胞极性作用之外,近来研究提示在不同的细胞株中可能有不同的与Par3相互作用的蛋白质。也有研究表明Par-3是一个多功能蛋白质,提示Par-3除了在细胞极性建立中发挥作用外也可能参与其他的生物学过程。因此Par3相互作用蛋白质的鉴定对于进一步了解Par3的功能至关重要。
目前有报道说Par3复合物在T细胞中是极化的。在血液细胞如Jurkat细胞中虽然含有Par-3蛋白质,但Par-3在这类细胞中的作用还未曾研究过,可能有不同的Par-3结合蛋白质。为了从分子水平上研究Par3的更多功能,我们用GSTpull-down与液相色谱.串联质谱结合的方法在Jurkat细胞中得到并鉴定了一些新的与Par3 PDZ结构域相互作用的蛋白质。这些被鉴定的蛋白质可能参与执行蛋白质代谢以及蛋白质运输等不同的细胞功能。Par3与这些蛋白质的相互作用提示Par-3可能参与这些生理代谢和物质运输活动。通过体外结合试验、免疫共沉淀试验和免疫荧光试验及共聚焦显微镜检测,验证了几个候选蛋白质——核运输蛋白质Importin-α4亚基、蛋白酶体激活因子PA28-β和PA28-γ通过Par3 PDZ1结构域与Par3之间的相互作用。我们的实验结果不仅有助于揭示进化上保守蛋白质Par3的新功能,更可为血液细胞中Par3新功能的研究提供重要线索。
Cell polarity is the fundamental characteristics of eukaryotic cells and of crucial importance to the differentiation and functions of most cells in the metazoans.
The partitioning-defective 3(Par-3)is a key component in the conserved Par-3/Par-6/aPKC complex.There are mainly three isoforms of Par3,180K,150K and 100K.The evolutionarily conserved cell polarity protein Par3,a scaffold-like PDZ-containing protein,plays a critical role in the establishment and maintenance of epithelial cell polarity.Although the roles of Par3 were extensively investigated in epithelial cells,its roles in other cell types such as hematopoietic cells have scarcely been explored.Recent studies have indicated that Par-3 is a multifunctional protein, and it may be involved in other biological context in addition to establishing cell polarity.Thus we believe that the identification of binding partners of Par3 is important for understanding its role.
Recently,Par3 complexes were reported being polarized in T cells.Although there are Par3 in blood cells such as Jurkat cells,the functions of Par3 in the cells remain elusive.There may be different Par3 interacting proteins in Jurkat cells.In order to obtain more information about the molecular basis of Par3 functions,we identified several potential novel binding-proteins of PDZ domains of Par3 in Jurkat cells(a T-cell line)by combining GST-pull-down approach with LC-MS/MS.The identified proteins are implicated to be involved in different cellular functions,for instance,protein metabolism,protein transportation and so on.The interaction between Par3 and these identified proteins indicates that Par3 may involve in these different biological functions.The interaction of Par3 PDZ domains with three proteins,nuclear transport protein Importin-α4,proteasome activators PA28-βand PA28-γwere further confirmed through in vitro binding assay, co-immunoprecipitation assay and immunofluorescence.Our results should not only help to uncover novel functions of this evolutionarily conserved protein Par3,but also throw new light on uncovering novel functions of the cell polarity protein Par3 in blood cells.
引文
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