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Analysis of the Sterol Sensing Domain Containing proteins during development

开发过程中含有甾醇传感结构域的蛋白质的分析

基本信息

批准号：
17570182
负责人：
NAKANO Yoshiro
金额：
$ 2.3万
依托单位：
Hyogo College of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570182/
关键词：
sterol-sensing patched hedgehog patched-related ステロールセンシングドメイン ptr ヘッジホッグ

项目摘要

Transmembrane protein Patched (Ptc) binds to Hedgehog (Hh) proteins and regulates the activity of Smoothened (Smo). Ptc contains a sterol-sensing domain (SSD) found in proteins involved in cholesterol homeostasis or trafficking. We earlier reported finding that two missense mutations in the SSD of Drosophila patched exhibit a clear dominant negative effect. Here we tested and analyzed in vivo the function of Basal Cell Nevus Syndrome missense mutations of human PTCH1 gene generating transgenic flies carrying corresponding Drosophila patched mutations. When we missexpressed all three mutations, G500V D504Y G500R, they behaved similar to wild-type patched. However none of them could rescue ptc mutant embryos. This finding suggested that not all SSD mutations act with a dominant negative and that the GxxxD motif in PTCH1 is essential for the protein function.To further elucidate SSD functions, we searched Drosophila and mouse genomes and identified a new SSD containing a gene in Drosophila (Ptr: CG11212) and four new SSD containing genes in mouse (Ptchd1 Ptchd2 Ptchd3 Ptchd4). All genes were cloned and sequenced. Their expressions were then analyzed by RT-PCR and in situ hybridization. Ptchd1 expression was observed in cerebellar primordium at E13 embryos, Ptchd2 expression in myelencephalon and spinal cord after E11, while Ptchd3 expression was restricted in spermatocyte of adult testis.Drosophila Ptr expression is very dynamic. Transcripts are detected at the germ layers except in the future mesoderm region at blastoderm stage. Later the expression becomes segmental. Anti-Ptr antibody analyses showed that Ptr protein was localized in cystoblasts and oocytes in ovary, while Ptr is detected later in centrosomes at stage 5 embryos. These expression patterns predict that one of the functions of Ptr family genes is involved in germ cell formation.

跨膜蛋白 Patched (Ptc) 与 Hedgehog (Hh) 蛋白结合并调节 Smoothened (Smo) 的活性。 Ptc 含有在参与胆固醇稳态或运输的蛋白质中发现的甾醇感应结构域 (SSD)。我们之前报道过，果蝇 SSD 中的两个错义突变表现出明显的显性负效应。在这里，我们在体内测试和分析了产生携带相应果蝇补丁突变的转基因果蝇的人类PTCH1基因的基底细胞痣综合征错义突变的功能。当我们错误表达所有三个突变（G500V D504Y G500R）时，它们的行为与野生型修补相似。然而他们都无法拯救ptc突变胚胎。这一发现表明，并非所有SSD突变都以显性失活起作用，并且PTCH1中的GxxxD基序对于蛋白质功能至关重要。为了进一步阐明SSD功能，我们搜索了果蝇和小鼠基因组，并鉴定了一个包含果蝇基因的新SSD（Ptr：CG11212）和四个包含小鼠基因的新SSD（Ptchd1 Ptchd2） Ptchd3 Ptchd4)。所有基因均被克隆并测序。然后通过 RT-PCR 和原位杂交分析它们的表达。 E13胚胎时在小脑原基中观察到Ptchd1的表达，E11后在脊髓和脊髓中观察到Ptchd2的表达，而Ptchd3的表达仅限于成年睾丸的精母细胞中。果蝇Ptr表达非常动态。在胚层中检测到转录本，但在胚盘阶段的未来中胚层区域中除外。后来这个表达式变成了分段的。抗 Ptr 抗体分析表明，Ptr 蛋白定位于卵巢的囊胚细胞和卵母细胞中，而 Ptr 随后在第 5 阶段胚胎的中心体中检测到。这些表达模式预测 Ptr 家族基因的功能之一涉及生殖细胞形成。