迟发性脊椎骨骨骺发育不良蛋白Sedlin在哺乳动物细胞中的作用机制

Spondyloepiphyseal dysplasia tarda (SEDT, MIM 313400) is a genetically heterogeneous disorder of X-linked recessive (XR). The main clinical features of it are skeletal disorders including osteoarthritis, short stature dwarf due to platyspondyly and dysplasia of large joints...SEDT is caused by the mutation of SEDL gene (MIM 300202), the product of which is called as Sedlin composed of 140-amino acid residues. The SEDL gene is 20 kb in length containing the six exons, and is mapped in Xp22. The highly homology between its orthlogs is found from yeast to human. Sedlin's ortholog in yeast is Trs20p, a subunit of TRAPP (transport protein particle) complex. The TRAPP has two functional forms, TRAPPⅠand TRAPPⅡ. Both of the TRAPPⅠand TRAPPⅡhave the Trs20p subunit and its deletion has lethal effect on yeast cells that can be resecued by Sedlin with microinjection. So Sedlin may play important roles in the vesicle trafficking from the endoplasmic reticulum to the Golgi complex. Sedlin has been reported to bind to MBP-1, a transcription repressor for the c-myc promoter,it suggests that Sedlin may also contribute to regulation of cell survival and proliferation...We have confirmed that Sedlin can be associated with CLIC1 and PAM14 in mammalian cells. PAM14,a binding partner for the transcription factor MRG15,has been reported to regulate Rb-induced E2F-responsive promoter repression, thus facilitating cell cycle progression. And now we reveal that Sedlin and Rb can interact each other in mammalian cells.Localization of Sedlin in the nucleolus suggests that Sedlin may have a broad biological role in addition to its potential role in regulation of gene transcription by binding to PAM14...To further examine the potential function of Sedlin in mammalian cells, we will try to perform multiple methods including GST-pull down, co-immunoprecipitation,immune electron microscopy, RNA interference,mass spectrometry. We will focus on the fine mechanism among Sedlin, PAM14 and Rb and try to find the additional candidate proteins and their ulterstructure in the bingding complex. We hope that our study can provide a noval molecular mechanism for the occurrence of SEDT.

迟发性脊椎骨骨骺发育不良病(SEDT)是一种X连锁隐性遗传病，多发病于儿童期，发病率高，危害大。SEDT的发生与Sedlin蛋白的基因突变有关，并伴随Sedlin的性质或数量发生改变，但SEDT发病的分子机制尚不明确。已知包含Sedlin亚基的TRAPP复合物参与物质运输，并可能与生长发育相关。我们先前的结果表明Sedlin与氯离子通道蛋白1以及PAM14蛋白结合，而且能和Rb蛋白直接相互作用。提示Sedlin可能参与胞内物质运输及基因的转录调节等多种功能。本研究拟在此基础上，利用GST-pull down、免疫沉淀、免疫电镜、RNA干扰、质谱法等多种实验手段确定Sedlin和PAM14以及Rb之间结合的精细机制、Sedlin在与PAM14或Rb形成的复合物中的其他结合蛋白和结合部位的超微结构，进而探讨Sedlin对Rb蛋白功能的影响。从而为SEDT的发病机制提供崭新的分子水平的解释。

迟发性脊椎骨骨骺发育不良病(SEDT)是一种X连锁隐性遗传病。SEDT的发生与Sedlin蛋白的基因突变有关，并伴随Sedlin的性质或数量发生改变，但其发病的分子机制尚不明确。Rb1蛋白是调节哺乳动物细胞周期进入和进行的关键蛋白。一般认为Rb的主要作用是通过与转录因子E2F家族成员的结合来控制细胞通过G1/S期，即活性的次磷酸化形式的Rb蛋白与E2F结合，阻止E2F靶基因的活化，而超磷酸化形式的Rb蛋白不再与E2F结合，释放E2F以活化其靶基因，以便合成为S期所必需的分子。鉴于我们之前已证实在哺乳动物细胞中，Sedlin和PAM14是相互作用的，而且酵母酵母双杂交结果显示Sedlin与Rb1相互结合。有报道认为，MRG15可与Rb蛋白和PAM14结合，并通过Rb蛋白介导对转录的活化作用。本研究拟在此基础上，利用GST-pull down、免疫沉淀、免疫电镜、RNA干扰、质谱法等多种实验手段确定Sedlin和PAM14以及Rb之间结合的精细机制、Sedlin在与PAM14或Rb形成的复合物中的其他结合蛋白和结合部位的超微结构，进而探讨Sedlin对Rb蛋白功能的影响。从而为SEDT的发病机制提供崭新的分子水平的解释。.本研究已通过蛋白质相互作用的经典方法即GST Pulldown、免疫荧光、免疫沉淀等方法分别证实了在细胞核内Sedlin和Rb1、MRG15、E2F1分别存在直接相互作用，而且揭示了它们与Sedlin结合的关键结构域；通过RNA干扰技术，证明了Sedlin对肿瘤细胞的促生长、增殖作用。另外，利用本项目，我们也进一步揭示了Sedlin与CLIC家族成员的相互作用机制，Sedlin与COX6B1相互作用的机制以及Sedlin与DEPP的相互作用。.我们的一系列研究结果表明，Sedlin在细胞内具有广泛的生物学功能，为进一步理解、揭示SEDT的发病机制奠定了重要的基础。

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