Sonic hedgehog信号通路中Smoothened的跨膜域对其在原纤毛中定位的机制研究

发布时间：2018-05-26 04:52

本文选题：Sonic + Hedgehog　；参考：《南京医科大学》2012年硕士论文

【摘要】：Sonic Hedgehog (Shh)是哺乳动物调控组织形态发生和细胞定向分化的主要形态发生因子(morphogen)。在人类及其他高等哺乳动物中,Shh的功能与肢芽、中枢神经、骨胳、心肺、消化、生殖、及皮肤等各种组织器官系统的形成都有着至关重要的关系。由基因突变造成的Shh通路异常激活会导致很多肿瘤的产生。当Shh配基不与其受体Patched(Ptch)结合时,Ptch对G蛋白偶联受体(GPCR) Smoothened (Smo)起抑制作用。这时,Smo下游全长的Gli转录激活子(Gli activator, GliA)被蛋白酶体剪切成截短的转录抑制子形式(Glirepressor, GliR),进入细胞核,抑制Shh靶基因转录。Ptch与Shh配基结合后,它对Smo的抑制作用被解除。Smo向原纤毛迁移,调控suppressor of fused (Sufu)与Gli的蛋白复合体向原纤毛顶端转运,进而阻断转录激活子GliA被蛋白酶体剪切成Gli R。GliA进入细胞核,激活靶基因转录。在由于Shh信号通路异常激活而形成的人类肿瘤中,40%的散发性基底细胞癌和25%的早期的神经外胚层的肿瘤是由于Ptch功能缺失或是Smo的过度激活所导致的。近年来的研究已经从天然植物中发现了一大类能够抑制Smo活性的类固醇类生物碱(steroidal alkaloid)包括环多胺(cyclopamine)。目前针对Smo的环多胺药物已经作为抗皮肤基底细胞癌和小脑延髓癌的实验药物进入了临床试验阶段。 Smo是一个七次跨膜蛋白,主要包括3个结构域：N端胞外域、七次跨膜域和C端胞内域。目前,关于Smo的研究主要集中在其C端结构域,认为C端结构域是Smo的主要磷酸化区域,磷酸化后Smo构象发生改变,由无活性的单体形成2个C端相互结合的二聚体。然后该二聚体被转运进入原纤毛(primary cilia),在其中Smo活化并激活下游信号。并且Smo的C端结构域也是Smo发挥功能的主要结构域,因为Smo的活性与其保留的C端结构域长短呈正相关。但目前为止,关于Smo的七次跨膜域的功能以及七次跨膜域调节Smo在原纤毛中定位的机制尚不清楚。为了解决以上的疑问,我们首先研究了Smo七次跨膜域对其蛋白稳定性的影响。我们构建了切除Smo不同跨膜域的质粒,命名为SDTm,并在C端标记了Flag和GFP的标签。结果发现只保留了Smo第7个跨膜域的Smo(SDTm1-6)在蛋白稳定性上与全长的Smo存在差异,而其它的截断的Smo在蛋白稳定性上与全长的Smo并无差异。进一步分析我们发现SDTm1-6蛋白不稳定是由于被截的太短,使之被蛋白酶体降解造成的。既然其它的SDTm在蛋白稳定性上与全长的Smo并无差异,那么我们进而研究了Smo的七次跨膜域对下游信号传导的影响。在融合了Gli的荧光素酶报告基因的3T3细胞(Gli-Luc3T3)中,报告基因检测发现这些SDTm能与内源性的Smo协同使得下游靶基因Glil的表达量上调,说明SDTm能与内源性的Smo协同作用激活下游信号。通过进一步研究,我们用发现用病毒Ad-Cre-GFP感染Smofl/fl细胞,敲低了内源性的Smo时,下游靶基因Gli1的读值仍然有不同程度的上调,说明Smo的七次跨膜域对其下游信号传导并无显著的影响。最后,我们研究了Smo的七次跨膜域对其在原纤毛中定位的影响,发现当切除Smo的1-2号跨膜域时,Smo仍然可以在原纤毛中定位,而当切除6-7号跨膜域时,Smo不能在原纤毛中定位,说明Smo的七次跨膜域对其在原纤毛中定位起着重要的调节作用。综上所述,我们认为Smo的七次跨膜域对其蛋白稳定性和下游的信号传导并无显著的影响,而对其在原纤毛中的定位却至关重要。我们的研究阐明了Smo的七次跨膜域调节其在原纤毛中定位的部分机制,对进一步阐明Shh信号传导的机制具有深远的意义。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R3416

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