Smurfs参与调控Sonic Hedgehog信号转导

发布时间：2017-12-31 19:15

本文关键词：Smurfs参与调控Sonic Hedgehog信号转导　出处：《南京医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：Sonic Hedgehog(Shh)信号通路是调控脊椎动物胚胎发育的最根本的信号通路之一，它在神经管、轴向骨架、肢、肺、皮肤、毛发和牙齿等的发育过程中发挥了不可或缺的作用。Shh信号通路异常是无数发育畸形的根本原因之一，同时也是人类多种肿瘤的成因或诱因。正是因其在生物发育及疾病中的重要作用，Shh信号转导机制历来是科学家们研究的重点及热点之一。在脊椎动物中，Shh信号转导始于其Shh配基与12次跨膜受体Patched1(Ptch1)的结合并内化进入细胞内，解除了其对7次跨膜受体Smoothened(Smo)的抑制作用，使Smo得以激活下游信号通路。在信号通路下游，Shh靶基因的表达依赖于转录因子Gli1、Gli2和Gli3的活性，其中Gli2和Gli3在没有Shh信号的情况下，会经蛋白酶体剪切加工生成截短的转录抑制子(GliR)，抑制靶基因的表达。当Shh信号出现时，该酶切步骤被阻断，Gli2和Gli3以转录激活子(GliA)形式入核激活靶基因表达。而Gli1则不受蛋白酶体加工处理，始终发挥转录激活作用。Smo到Gli之间的调控机制比较复杂，至今尚不清楚。在哺乳动物的正常发育过程中，细胞对不同强度的Shh信号接收能力至关重要。研究表明，在Shh配基被分泌并转运到接收细胞附近时，跨膜受体Ptch内化入细胞，Smo富集到细胞膜上这一分子转运过程在接收不同强度信号的精密调控时发挥了重要作用。在膜受体从细胞膜转运至胞内体的过程中，它可能有两种去向，一是被转运至溶酶体并降解，二是又循环转运回到细胞膜上。其中，泛素化修饰，尤其是多位点的单泛素化修饰是促进膜受体内化转运的关键信号之一。并且，许多HECT家族E3泛素连接酶能参与调控膜受体泛素化并内吞转运的作用已有报道，但在Shh信号转导过程中，它们是否参与又如何调控膜受体的泛素化至今尚无确切的认识。为此，，我们首先建立了一套泛素连接酶的筛选系统，并对HECT家族的E3泛素连接酶进行了筛选。通过荧光素酶报告基因系统和细胞免疫荧光图象系统的两轮检测，我们筛选到了三个HECT E3泛素连接酶—Smurf1、Smurf2和Wwp1---不仅能调节Ptch1、Gli3蛋白的原纤毛定位，也可以增加通路下游转录因子Gli1的活性。其中，Smurf1和Smurf2特异性促进Ptch1蛋白的泛素化修饰，其降解受到溶酶体途径和蛋白酶体途径的共同调控。由此，为研究Shh信号转导的调控机制提供了新的重要线索。
[Abstract]:Sonic Hedgehog (Shh) signal pathway is one of the most fundamental regulation of vertebrate embryonic development in neural tube, axial skeleton, limb, lung, skin, hair and teeth etc. the development process played a role in the.Shh signaling pathway is one of the indispensable abnormal root cause numerous malformations, but also a variety of human the tumor causes or inducements. It is because of its important role in biological development and diseases, Shh signal transduction mechanism has always been one of the focus of scientists.
In vertebrates, Shh signal transduction from its Shh ligand and 12 transmembrane receptor Patched1 (Ptch1) binding and internalization into cells, relieve the 7 transmembrane receptor Smoothened (Smo) inhibited the Smo to activate downstream signaling pathways in the signaling pathway downstream target gene expression of Shh depending on the transcription factor Gli1, Gli2 and Gli3 activity, Gli2 and Gli3 in the absence of Shh signal, through the transcription of proteasome inhibitor produced a truncated shear (GliR), inhibit the expression of target genes. When the Shh signal occurs, the digestion step is blocked, Gli2 and Gli3 transcription activator (GliA) in the form of nuclear activation of target gene expression. But Gli1 is not affected by the proteasome processing, always play transcriptional activation to.Smo regulation mechanism between Gli complex is still not clear.
In the normal development of the mammalian cells, is critical to Shh signal receiving ability of different intensity. The results show that Shh ligand is secreted and transported to the receiving cells nearby, transmembrane receptor Ptch internalization into cells, Smo concentration in the cell membrane of the molecular transport processes play an important role in the regulation of different receiving precision when the signal strength. In the process of membrane receptors from the cell membrane to the cell body, it may have two to one, were transported to the lysosomes and degradation, two is returned to the cell membrane transport cycle. Among them, ubiquitination, especially monoubiquitin multilocus is one of the key signal induced membrane receptor internalization transport. Also, many of the HECT family of E3 ubiquitin ligase can participate in the regulation of membrane receptor ubiquitination and endocytosis transport effects have been reported, but in the Shh signal transduction process, whether they participate in again Know what regulation of membrane receptor ubiquitination has no exact. Therefore, we first established a screening system of ubiquitin ligase, and the HECT family of E3 ubiquitin ligase were screened by luciferase reporter assays and immunofluorescence image system of the two round of testing, we screened three HECT ubiquitin E3 Smurf1 Smurf2 Wwp1--- ligase, and can not only regulate the Ptch1, primary ciliary localization of Gli3 protein, can also increase the downstream transcription factor Gli1 activity. Among them, Smurf1 and Smurf2 specifically promote the ubiquitination of Ptch1 protein and its degradation regulated lysosomal pathway and proteasome pathway. Thus, provide important clues the regulatory mechanism of Shh signal transduction.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R363

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