本文關(guān)鍵詞：Smurfs參與調(diào)控Sonic Hedgehog信號(hào)轉(zhuǎn)導(dǎo)　出處：《南京醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： SonicHedgehog信號(hào)通路 泛素化修飾 HECTE3泛素連接酶 蛋白降解

【摘要】：Sonic Hedgehog(Shh)信號(hào)通路是調(diào)控脊椎動(dòng)物胚胎發(fā)育的最根本的信號(hào)通路之一，它在神經(jīng)管、軸向骨架、肢、肺、皮膚、毛發(fā)和牙齒等的發(fā)育過(guò)程中發(fā)揮了不可或缺的作用。Shh信號(hào)通路異常是無(wú)數(shù)發(fā)育畸形的根本原因之一，同時(shí)也是人類多種腫瘤的成因或誘因。正是因其在生物發(fā)育及疾病中的重要作用，Shh信號(hào)轉(zhuǎn)導(dǎo)機(jī)制歷來(lái)是科學(xué)家們研究的重點(diǎn)及熱點(diǎn)之一。 在脊椎動(dòng)物中，Shh信號(hào)轉(zhuǎn)導(dǎo)始于其Shh配基與12次跨膜受體Patched1(Ptch1)的結(jié)合并內(nèi)化進(jìn)入細(xì)胞內(nèi)，解除了其對(duì)7次跨膜受體Smoothened(Smo)的抑制作用，使Smo得以激活下游信號(hào)通路。在信號(hào)通路下游，Shh靶基因的表達(dá)依賴于轉(zhuǎn)錄因子Gli1、Gli2和Gli3的活性，其中Gli2和Gli3在沒(méi)有Shh信號(hào)的情況下，會(huì)經(jīng)蛋白酶體剪切加工生成截短的轉(zhuǎn)錄抑制子(GliR)，抑制靶基因的表達(dá)。當(dāng)Shh信號(hào)出現(xiàn)時(shí)，該酶切步驟被阻斷，Gli2和Gli3以轉(zhuǎn)錄激活子(GliA)形式入核激活靶基因表達(dá)。而Gli1則不受蛋白酶體加工處理，始終發(fā)揮轉(zhuǎn)錄激活作用。Smo到Gli之間的調(diào)控機(jī)制比較復(fù)雜，至今尚不清楚。 在哺乳動(dòng)物的正常發(fā)育過(guò)程中，細(xì)胞對(duì)不同強(qiáng)度的Shh信號(hào)接收能力至關(guān)重要。研究表明，在Shh配基被分泌并轉(zhuǎn)運(yùn)到接收細(xì)胞附近時(shí)，跨膜受體Ptch內(nèi)化入細(xì)胞，Smo富集到細(xì)胞膜上這一分子轉(zhuǎn)運(yùn)過(guò)程在接收不同強(qiáng)度信號(hào)的精密調(diào)控時(shí)發(fā)揮了重要作用。在膜受體從細(xì)胞膜轉(zhuǎn)運(yùn)至胞內(nèi)體的過(guò)程中，它可能有兩種去向，一是被轉(zhuǎn)運(yùn)至溶酶體并降解，二是又循環(huán)轉(zhuǎn)運(yùn)回到細(xì)胞膜上。其中，泛素化修飾，尤其是多位點(diǎn)的單泛素化修飾是促進(jìn)膜受體內(nèi)化轉(zhuǎn)運(yùn)的關(guān)鍵信號(hào)之一。并且，許多HECT家族E3泛素連接酶能參與調(diào)控膜受體泛素化并內(nèi)吞轉(zhuǎn)運(yùn)的作用已有報(bào)道，但在Shh信號(hào)轉(zhuǎn)導(dǎo)過(guò)程中，它們是否參與又如何調(diào)控膜受體的泛素化至今尚無(wú)確切的認(rèn)識(shí)。為此，，我們首先建立了一套泛素連接酶的篩選系統(tǒng)，并對(duì)HECT家族的E3泛素連接酶進(jìn)行了篩選。通過(guò)熒光素酶報(bào)告基因系統(tǒng)和細(xì)胞免疫熒光圖象系統(tǒng)的兩輪檢測(cè)，我們篩選到了三個(gè)HECT E3泛素連接酶—Smurf1、Smurf2和Wwp1---不僅能調(diào)節(jié)Ptch1、Gli3蛋白的原纖毛定位，也可以增加通路下游轉(zhuǎn)錄因子Gli1的活性。其中，Smurf1和Smurf2特異性促進(jìn)Ptch1蛋白的泛素化修飾，其降解受到溶酶體途徑和蛋白酶體途徑的共同調(diào)控。由此，為研究Shh信號(hào)轉(zhuǎn)導(dǎo)的調(diào)控機(jī)制提供了新的重要線索。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R363

【共引文獻(xiàn)】

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