Prosaposin （Psap） has multiple cellular functions. It is involved in the development of the reproductive system, nervous system, and prostate cancer as well as in the regulation of sphingolipid catabolism by activat...Prosaposin （Psap） has multiple cellular functions. It is involved in the development of the reproductive system, nervous system, and prostate cancer as well as in the regulation of sphingolipid catabolism by activating several lysosomal hydrolases involved in the metabolism of various sphingolipids. In this research, it was found to be a novel interaction partner for Rhox5 using yeast two-hybrid screening. The interaction between Rhox5 and the full-length prosapsoin （the transcript without exon 8） as well as the C-terminal domain of prosaposin, was further confirmed in both yeast two hybrid analysis and in vitro assay. It suggested that the C-terminal domain of prosaposin may be critical for the Rhox5-prosaposin interaction. Given the important roles played by both Rhox5 and prosaposin in maintaining the differentiation of male reproductive organs, spermatogenesis, and fertilization, the interaction between Rhox5 and prosaposin might regulate the development of male reproductive organs dynamically.展开更多
Mdfic(MyoD family inhibitor domain containing)是一个新发现的含有MyoD抑制素结构域(I-mfa domain)的转录调控因子,可能在肌细胞的分化过程中发挥重要作用.小鼠Rhox5为同源异型框基因,隶属于Rhox基因簇(reproductive homeobox on the...Mdfic(MyoD family inhibitor domain containing)是一个新发现的含有MyoD抑制素结构域(I-mfa domain)的转录调控因子,可能在肌细胞的分化过程中发挥重要作用.小鼠Rhox5为同源异型框基因,隶属于Rhox基因簇(reproductive homeobox on the X chromosome genes cluster)β亚簇.在前期证实Mdifc能结合Rhox5蛋白的基础上,进一步鉴定两者相互作用的关键结构域.生物信息学分析Mdfic的氨基酸序列,PCR方法扩增Mdfic A截短型片段(第72～247位氨基酸残基),含保守的I-mfa结构域;双向酵母双杂交和体外GST-Pull down结果表明,该截短型片段可以与Rhox5蛋白结合,且结合力度较完整的Mdfic蛋白强;将Mdfic A片段划分为两段:Mdfic B(72～191 aa,不含I-mfa结构域)和Mdfic C(191～247 aa,含I-mfa结构域).结果表明,含保守I-mfa结构域的Mdfic C截短型片段丧失了与Rhox5蛋白结合的能力,而不含I-mfa结构域的Mdfic B截短型片段可以结合Rhox5蛋白.鉴于Mdfic蛋白的非I-mfa结构域在Rhox5/Mdfic结合中发挥关键作用,Rhox5与Mdfic的结合可能进一步调控由Mdfic的I-mfa结构域参与的其他转录因子(如MyoD)的调控,三者形成一个复杂的调控网络,共同参与肌细胞发生及分化的调控.展开更多
RHOX5基因是最早发现的小鼠RHOX基因簇(reproductive homeobox on the X chromosome)成员,可特异性地在生殖系统中表达。RHOX5蛋白在胚胎发育、生殖组织的发育、精子的生成和成熟等多个环节发挥作用,但其功能的发挥途径尚不明确。在前...RHOX5基因是最早发现的小鼠RHOX基因簇(reproductive homeobox on the X chromosome)成员,可特异性地在生殖系统中表达。RHOX5蛋白在胚胎发育、生殖组织的发育、精子的生成和成熟等多个环节发挥作用,但其功能的发挥途径尚不明确。在前期筛选与RHOX5蛋白相互作用的分子中初步获得一个BRPF1的新型转录本BRPF2。进一步构建pGBKT7-BRPF2质粒,酵母双杂交实验确定其与RHOX5蛋白的相互作用,GST-pull down实验确定其在体外的直接结合;PCR扩增BRPF1基因,构建pGBKT7-BRPF1和pGADT7-BRPF1质粒,酵母双杂交实验和GST-pulldown实验证明RHOX5蛋白亦可以直接结合BRPF1蛋白。BRPF1及其新型转录本BRPF2与RHOX5蛋白间的相互作用证实暗示了BRPF2极有可能与BRPF1竞争性结合RHOX5蛋白,为三种蛋白功能的研究提供了新的思路。展开更多
目的:构建同源异性框基因Rhox5的真核表达质粒,转染NIH3T3细胞,建立稳定过表达Rhox5的细胞系。方法:PCR方法扩增Rhox5的全长cDNA序列,PCR产物双酶切后和人工合成的HA抗原表位标签共同克隆至pcDNA3.1(-)哺乳动物细胞表达载体中,构建pcDNA...目的:构建同源异性框基因Rhox5的真核表达质粒,转染NIH3T3细胞,建立稳定过表达Rhox5的细胞系。方法:PCR方法扩增Rhox5的全长cDNA序列,PCR产物双酶切后和人工合成的HA抗原表位标签共同克隆至pcDNA3.1(-)哺乳动物细胞表达载体中,构建pcDNA-Rhox5-HA融合表达质粒。脂质体法将经过测序成功的pcDNA-Rhox5-HA融合质粒和pcDNA3.1空载体分别转染NIH3T3细胞,潮霉素B筛选后建立阴性对照pcDNA3.1 in NIH3T3和稳定过表达Rhox5的Rhox5-HA in NIH3T3细胞系。RT-PCR和western blotting方法检测Rhox5-HA在稳定转染细胞系中的表达情况。结果:成功构建了pcDNA-Rhox5-Myc重组质粒,获得稳定过表达Rhox5的NIH3T3细胞系。RT-PCR和Western blotting结果表明,构建的稳定细胞系中成功表达Rhox5-HA融合蛋白。结论:Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达为进一步体外研究Rhox5蛋白单独的功能及其与其他分子间功能性相互作用奠定了实验基础。展开更多
基金This work was supported by the National Natural Sciences Foundation of China (No. 39770411) Doctoral Fund of Ministry of Education of China (No. 20060559006).
文摘Prosaposin （Psap） has multiple cellular functions. It is involved in the development of the reproductive system, nervous system, and prostate cancer as well as in the regulation of sphingolipid catabolism by activating several lysosomal hydrolases involved in the metabolism of various sphingolipids. In this research, it was found to be a novel interaction partner for Rhox5 using yeast two-hybrid screening. The interaction between Rhox5 and the full-length prosapsoin （the transcript without exon 8） as well as the C-terminal domain of prosaposin, was further confirmed in both yeast two hybrid analysis and in vitro assay. It suggested that the C-terminal domain of prosaposin may be critical for the Rhox5-prosaposin interaction. Given the important roles played by both Rhox5 and prosaposin in maintaining the differentiation of male reproductive organs, spermatogenesis, and fertilization, the interaction between Rhox5 and prosaposin might regulate the development of male reproductive organs dynamically.
文摘RHOX5基因是最早发现的小鼠RHOX基因簇(reproductive homeobox on the X chromosome)成员,可特异性地在生殖系统中表达。RHOX5蛋白在胚胎发育、生殖组织的发育、精子的生成和成熟等多个环节发挥作用,但其功能的发挥途径尚不明确。在前期筛选与RHOX5蛋白相互作用的分子中初步获得一个BRPF1的新型转录本BRPF2。进一步构建pGBKT7-BRPF2质粒,酵母双杂交实验确定其与RHOX5蛋白的相互作用,GST-pull down实验确定其在体外的直接结合;PCR扩增BRPF1基因,构建pGBKT7-BRPF1和pGADT7-BRPF1质粒,酵母双杂交实验和GST-pulldown实验证明RHOX5蛋白亦可以直接结合BRPF1蛋白。BRPF1及其新型转录本BRPF2与RHOX5蛋白间的相互作用证实暗示了BRPF2极有可能与BRPF1竞争性结合RHOX5蛋白,为三种蛋白功能的研究提供了新的思路。
文摘目的:构建同源异性框基因Rhox5的真核表达质粒,转染NIH3T3细胞,建立稳定过表达Rhox5的细胞系。方法:PCR方法扩增Rhox5的全长cDNA序列,PCR产物双酶切后和人工合成的HA抗原表位标签共同克隆至pcDNA3.1(-)哺乳动物细胞表达载体中,构建pcDNA-Rhox5-HA融合表达质粒。脂质体法将经过测序成功的pcDNA-Rhox5-HA融合质粒和pcDNA3.1空载体分别转染NIH3T3细胞,潮霉素B筛选后建立阴性对照pcDNA3.1 in NIH3T3和稳定过表达Rhox5的Rhox5-HA in NIH3T3细胞系。RT-PCR和western blotting方法检测Rhox5-HA在稳定转染细胞系中的表达情况。结果:成功构建了pcDNA-Rhox5-Myc重组质粒,获得稳定过表达Rhox5的NIH3T3细胞系。RT-PCR和Western blotting结果表明,构建的稳定细胞系中成功表达Rhox5-HA融合蛋白。结论:Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达为进一步体外研究Rhox5蛋白单独的功能及其与其他分子间功能性相互作用奠定了实验基础。
