本文選題：Sonic + Hedgehog��； 參考：《南京醫(yī)科大學(xué)》2012年碩士論文

【摘要】：Sonic Hedgehog (Shh)是哺乳動(dòng)物調(diào)控組織形態(tài)發(fā)生和細(xì)胞定向分化的主要形態(tài)發(fā)生因子(morphogen)。在人類及其他高等哺乳動(dòng)物中,Shh的功能與肢芽、中樞神經(jīng)、骨胳、心肺、消化、生殖、及皮膚等各種組織器官系統(tǒng)的形成都有著至關(guān)重要的關(guān)系。由基因突變?cè)斐傻腟hh通路異常激活會(huì)導(dǎo)致很多腫瘤的產(chǎn)生。 當(dāng)Shh配基不與其受體Patched(Ptch)結(jié)合時(shí),Ptch對(duì)G蛋白偶聯(lián)受體(GPCR) Smoothened (Smo)起抑制作用。這時(shí),Smo下游全長(zhǎng)的Gli轉(zhuǎn)錄激活子(Gli activator, GliA)被蛋白酶體剪切成截短的轉(zhuǎn)錄抑制子形式(Glirepressor, GliR),進(jìn)入細(xì)胞核,抑制Shh靶基因轉(zhuǎn)錄。Ptch與Shh配基結(jié)合后,它對(duì)Smo的抑制作用被解除。Smo向原纖毛遷移,調(diào)控suppressor of fused (Sufu)與Gli的蛋白復(fù)合體向原纖毛頂端轉(zhuǎn)運(yùn),進(jìn)而阻斷轉(zhuǎn)錄激活子GliA被蛋白酶體剪切成Gli R。GliA進(jìn)入細(xì)胞核,激活靶基因轉(zhuǎn)錄。 在由于Shh信號(hào)通路異常激活而形成的人類腫瘤中,40%的散發(fā)性基底細(xì)胞癌和25%的早期的神經(jīng)外胚層的腫瘤是由于Ptch功能缺失或是Smo的過(guò)度激活所導(dǎo)致的。近年來(lái)的研究已經(jīng)從天然植物中發(fā)現(xiàn)了一大類能夠抑制Smo活性的類固醇類生物堿(steroidal alkaloid)包括環(huán)多胺(cyclopamine)。目前針對(duì)Smo的環(huán)多胺藥物已經(jīng)作為抗皮膚基底細(xì)胞癌和小腦延髓癌的實(shí)驗(yàn)藥物進(jìn)入了臨床試驗(yàn)階段。 Smo是一個(gè)七次跨膜蛋白,主要包括3個(gè)結(jié)構(gòu)域：N端胞外域、七次跨膜域和C端胞內(nèi)域。目前,關(guān)于Smo的研究主要集中在其C端結(jié)構(gòu)域,認(rèn)為C端結(jié)構(gòu)域是Smo的主要磷酸化區(qū)域,磷酸化后Smo構(gòu)象發(fā)生改變,由無(wú)活性的單體形成2個(gè)C端相互結(jié)合的二聚體。然后該二聚體被轉(zhuǎn)運(yùn)進(jìn)入原纖毛(primary cilia),在其中Smo活化并激活下游信號(hào)。并且Smo的C端結(jié)構(gòu)域也是Smo發(fā)揮功能的主要結(jié)構(gòu)域,因?yàn)镾mo的活性與其保留的C端結(jié)構(gòu)域長(zhǎng)短呈正相關(guān)。但目前為止,關(guān)于Smo的七次跨膜域的功能以及七次跨膜域調(diào)節(jié)Smo在原纖毛中定位的機(jī)制尚不清楚。 為了解決以上的疑問(wèn),我們首先研究了Smo七次跨膜域?qū)ζ涞鞍追�(wěn)定性的影響。我們構(gòu)建了切除Smo不同跨膜域的質(zhì)粒,命名為SDTm,并在C端標(biāo)記了Flag和GFP的標(biāo)簽。結(jié)果發(fā)現(xiàn)只保留了Smo第7個(gè)跨膜域的Smo(SDTm1-6)在蛋白穩(wěn)定性上與全長(zhǎng)的Smo存在差異,而其它的截?cái)嗟腟mo在蛋白穩(wěn)定性上與全長(zhǎng)的Smo并無(wú)差異。進(jìn)一步分析我們發(fā)現(xiàn)SDTm1-6蛋白不穩(wěn)定是由于被截的太短,使之被蛋白酶體降解造成的。 既然其它的SDTm在蛋白穩(wěn)定性上與全長(zhǎng)的Smo并無(wú)差異,那么我們進(jìn)而研究了Smo的七次跨膜域?qū)ο掠涡盘?hào)傳導(dǎo)的影響。在融合了Gli的熒光素酶報(bào)告基因的3T3細(xì)胞(Gli-Luc3T3)中,報(bào)告基因檢測(cè)發(fā)現(xiàn)這些SDTm能與內(nèi)源性的Smo協(xié)同使得下游靶基因Glil的表達(dá)量上調(diào),說(shuō)明SDTm能與內(nèi)源性的Smo協(xié)同作用激活下游信號(hào)。通過(guò)進(jìn)一步研究,我們用發(fā)現(xiàn)用病毒Ad-Cre-GFP感染Smofl/fl細(xì)胞,敲低了內(nèi)源性的Smo時(shí),下游靶基因Gli1的讀值仍然有不同程度的上調(diào),說(shuō)明Smo的七次跨膜域?qū)ζ湎掠涡盘?hào)傳導(dǎo)并無(wú)顯著的影響。 最后,我們研究了Smo的七次跨膜域?qū)ζ湓谠w毛中定位的影響,發(fā)現(xiàn)當(dāng)切除Smo的1-2號(hào)跨膜域時(shí),Smo仍然可以在原纖毛中定位,而當(dāng)切除6-7號(hào)跨膜域時(shí),Smo不能在原纖毛中定位,說(shuō)明Smo的七次跨膜域?qū)ζ湓谠w毛中定位起著重要的調(diào)節(jié)作用。 綜上所述,我們認(rèn)為Smo的七次跨膜域?qū)ζ涞鞍追�(wěn)定性和下游的信號(hào)傳導(dǎo)并無(wú)顯著的影響,而對(duì)其在原纖毛中的定位卻至關(guān)重要。我們的研究闡明了Smo的七次跨膜域調(diào)節(jié)其在原纖毛中定位的部分機(jī)制,對(duì)進(jìn)一步闡明Shh信號(hào)傳導(dǎo)的機(jī)制具有深遠(yuǎn)的意義。
[Abstract]:Sonic Hedgehog (Shh) is a major morphogenetic factor (morphogen) that regulates the morphogenesis and cell oriented differentiation of mammals. In human and other higher mammals, the function of Shh has a vital relationship with the formation of various tissues and organs such as limb buds, central nerves, bones, heart and lung, digestion, reproduction, and skin. The abnormal activation of Shh pathway caused by gene mutation can lead to many tumors.
When the Shh ligand is not associated with its receptor Patched (Ptch), Ptch inhibits the G protein coupling receptor (GPCR) Smoothened (Smo). At this time, the Gli transcriptional activator (Gli activator) downstream of Smo is cut into a truncated transcriptional suppressor, into the nucleus, to inhibit the transcription of the target gene. After combining with the Shh ligand, its inhibitory effect on Smo was removed from.Smo to the primary cilia, and the protein complexes of suppressor of fused (Sufu) and Gli were transported to the apex of the cilium, and then the transcriptional activator was blocked by the proteasome to be cut into Gli R.GliA into the nucleus, and the target gene was transcribed.
In human tumors due to abnormal activation of the Shh signaling pathway, 40% of sporadic basal cell carcinoma and 25% of the early neuroectoderm tumors are caused by Ptch dysfunction or over activation of Smo. Recent studies have discovered a large class of steroid species that can inhibit Smo activity from natural plants. Steroidal alkaloid includes cyclic polyamines (cyclopamine). At present, cyclic polyamines against Smo have been used as experimental drugs for anti skin basal cell carcinoma and cerebellopontine carcinoma.
Smo is a seven time transmembrane protein, mainly including 3 domains: the N end cell outer domain, seven transmembrane domain and C endpoint. At present, the research on Smo is mainly concentrated in its C terminal domain. The C terminal domain is the main phosphorylation area of Smo, the Smo conformation changes after phosphorylation, and 2 C ends are formed by the inactive monomer two. The polymer is then transported to the original cilia (primary cilia), in which the Smo activates and activates the downstream signal. And the C end domain of the Smo is also the main domain of the Smo function, because the activity of the Smo is positively related to the length of the retained C end domain. But at present, the function of the seven transmembrane domains of Smo and seven times The mechanism by which transmembrane domains regulate the localization of Smo in the original cilia is unclear.
In order to solve the above questions, we first studied the effect of the seven transmembrane domains of Smo on its protein stability. We constructed the plasmids of different transmembrane domains of Smo, named SDTm, and labeled Flag and GFP at the C end. The results showed that only the Smo (SDTm1-6) in the seventh transmembrane domains of Smo retained the protein stability with the full length of Smo. In the difference, the other truncated Smo did not differ from the full length of Smo in protein stability. Further analysis we found that the instability of SDTm1-6 protein was due to the short cut of the intercepted, which was caused by proteasome degradation.
Since other SDTm is not different from the full length of Smo in protein stability, then we have studied the effect of the seven transmembrane domains on the downstream signal transduction in the Smo. In the 3T3 cells (Gli-Luc3T3) that fused the luciferase reporter gene of Gli (Gli-Luc3T3), the reporter gene detection found that these SDTm can cooperate with endogenous Smo to make the target genes downstream. The expression of Glil is up-regulated, indicating that SDTm can activate downstream signals in synergy with endogenous Smo. Through further study, we found that the reading value of the downstream target gene Gli1 is still in varying degrees when the virus Ad-Cre-GFP is infected with Smofl/fl cells and the endogenous Smo is knocked down, indicating that the seven transmembrane domain of Smo is transmitted to its downstream signal. There is no significant impact on guidance.
Finally, we studied the effect of the seven transmembrane domains of Smo on its location in the original cilium. It was found that when the 1-2 transmembrane domain was removed, Smo could still be located in the original cilium, while Smo could not be located in the original cilium when the 6-7 transmembrane domain was excised. It indicated that the seven transmembrane domains of Smo played an important role in its localization in the original cilium. Use.
To sum up, we think that the seven transmembrane domains of Smo have no significant effect on its protein stability and downstream signal transduction, but it is crucial to its location in the primary cilium. Our study elucidated the mechanism of the seven transmembrane domains to regulate its localization in the primary cilia, and to further clarify the mechanism of the Shh signal transduction. There is a profound meaning.
【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】：2012
【分類號(hào)】：R3416

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1 濮曉紅;Sonic hedgehog信號(hào)通路中Smoothened的跨膜域?qū)ζ湓谠w毛中定位的機(jī)制研究[D];南京醫(yī)科大學(xué);2012年

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本文編號(hào)：1936007