本文選題：Hedgehog + Sufu��； 參考：《南京醫(yī)科大學(xué)》2015年博士論文

【摘要】：Hh蛋白家族調(diào)控著一系列發(fā)育的過(guò)程和腫瘤的產(chǎn)生,Hh通路的失調(diào)在發(fā)育紊亂和癌癥中均有作用。例如,對(duì)于指導(dǎo)從四肢到中樞神經(jīng)系統(tǒng)發(fā)育、基底細(xì)胞癌和髓母細(xì)胞瘤的產(chǎn)生,具有深遠(yuǎn)的影響。美國(guó)藥監(jiān)局批準(zhǔn)的對(duì)抗BCC轉(zhuǎn)移的Hh通路抑制劑表明了Hh在癌癥治療中的重要性;而且對(duì)于其它癌癥的研究,許多臨床試驗(yàn)仍在開(kāi)展當(dāng)中。Hh是一個(gè)形態(tài)發(fā)生因子,它沿著發(fā)育軸線形成濃度梯度而發(fā)揮作用。在神經(jīng)管發(fā)育過(guò)程中,它從脊索和底板中表達(dá),并且一直延伸到背部。Hh蛋白是Ptch的配基。Ptch是一種12次跨膜蛋白,它能反向調(diào)節(jié)7次跨膜蛋白Smo。Hh配基與Ptch結(jié)合,導(dǎo)致Smo的激活。這將導(dǎo)致Smo轉(zhuǎn)運(yùn)至原纖毛中,Hh通路也得以正常轉(zhuǎn)運(yùn)。Smo的下游是Gli家族,它們是一組鋅指轉(zhuǎn)錄因子。在哺乳動(dòng)物中具有三個(gè)同源蛋白,Gli1、Gli2和Gli3。Sufu作為Gli蛋白的分子伴侶,能夠與所有的Gli蛋白結(jié)合而形成復(fù)合物。Sufu能夠與Gli氨基端高度保守的SYGH結(jié)構(gòu)域結(jié)合,又或者通過(guò)遮擋Gli的核輸入信號(hào)(NLS),從而將Gli阻滯在細(xì)胞漿中,使得Gli蛋白被Sufu抑制。然而現(xiàn)在并不清楚Sufu對(duì)于調(diào)節(jié)Gli胞漿阻滯的具體機(jī)制。隨著Hh的刺激,Gli蛋白轉(zhuǎn)運(yùn)至細(xì)胞核中,暗示Sufu對(duì)于Gli在胞漿中的錨定功能被解除了。然而,生物化學(xué)和遺傳學(xué)研究表明Sufu調(diào)節(jié)Gli的活性是通過(guò)其它非胞漿阻滯機(jī)制。這就暗示Sufu與Gli蛋白在細(xì)胞核中仍然能夠結(jié)合。然而,Sufu調(diào)控Gli蛋白的詳細(xì)機(jī)制還不明確。由于Hh在小腦發(fā)育中起到十分重要的作用,我們利用小腦為模型進(jìn)行研究。在成年小鼠小腦中,Hh通路是處于靜息狀態(tài),但是在出生一周多之后,小腦發(fā)育處于高強(qiáng)度,Sufu的水平與Gli一樣,在外胚層顆粒神經(jīng)元前體中表達(dá)都很高。這部分細(xì)胞的發(fā)育高度依賴hh信號(hào)的輸出。在髓母細(xì)胞瘤這一來(lái)自于顆粒神經(jīng)元前體的小腦腫瘤中,hh通路被重新激活,與之前一樣的是,sufu的水平同gli1一致高度上調(diào)。除此之外,sufu在細(xì)胞核和細(xì)胞漿中均有定位。為了進(jìn)一步觀察sufu在細(xì)胞核和細(xì)胞漿中的分布,我們檢測(cè)了小鼠胚胎成纖維細(xì)胞系(mefs)。在正常的mefs中,無(wú)論shh處理與否,sufu在胞核和胞漿中均有分布。當(dāng)我們檢測(cè)gli2/3缺失的細(xì)胞時(shí),令我們驚訝的是sufu在沒(méi)有shh時(shí)幾乎處于細(xì)胞漿中。而當(dāng)有shh時(shí),sufu被誘導(dǎo)至細(xì)胞核中,在gli2/3缺失的細(xì)胞中是不存在抑制子的。這就暗示在正常mefs中,在沒(méi)有shh處理的情況下,sufu很有可能是被抑制子帶入到細(xì)胞核的。在gli2/3缺失的細(xì)胞中,shh誘導(dǎo)sufu入核是由gli1介導(dǎo)的。為了證明這一點(diǎn),我們?cè)趃li2/3缺失的細(xì)胞中穩(wěn)定表達(dá)gli1的shrna,從而構(gòu)建三種gli都沒(méi)有的細(xì)胞系。在這種細(xì)胞系中,sufu的入核不再受到shh的誘導(dǎo)。sufu能夠招募sin3a和hdac從而抑制gli的轉(zhuǎn)錄活性。我們重新考慮了這個(gè)模式并且探究sufu能否出現(xiàn)在gli結(jié)合位點(diǎn)的染色質(zhì)上。我們以gli1和ptch1的啟動(dòng)子為例,利用chip實(shí)驗(yàn),兩個(gè)位點(diǎn)在shh處理之后,gli結(jié)合元件的含量增加,暗示shh能夠誘導(dǎo)sufu招募至其靶基因啟動(dòng)子區(qū)域。有趣的是,我們同樣發(fā)現(xiàn)sap18在hh的靶基因啟動(dòng)子上,hh能夠促使其從染色質(zhì)上脫落。這些結(jié)果暗示hh能夠誘導(dǎo)gli1-sufu復(fù)合物附著在它的靶基因啟動(dòng)子區(qū)域,而且這是依賴于gli1和gli3抑制子。為了找到?jīng)Q定sufu在胞漿和胞核中分布的序列原件,我們做了缺失定位,最后確定在人類sufu的308-318殘基中具有潛在的nes信號(hào)。我們將其中兩個(gè)亮氨酸突變成丙氨酸并且在mefs中進(jìn)行檢測(cè)。相比于野生型的sufu,nes突變的sufu更多的出現(xiàn)在細(xì)胞核中。而且nes的受體crm1也不再與突變的sufu結(jié)合。本實(shí)驗(yàn)室之前報(bào)道了一個(gè)pka和gsk3β雙磷酸化位點(diǎn)能夠調(diào)節(jié)sufu的穩(wěn)定性和原纖毛轉(zhuǎn)運(yùn)。這個(gè)雙磷酸化位點(diǎn)正好處于nes的附近。在coip實(shí)驗(yàn)中,我們發(fā)現(xiàn)在342或者346位點(diǎn)不能被磷酸化而突變的sufu與crm1具有有效的結(jié)合能力,而模擬磷酸化的sufu則失去了與crm1結(jié)合的能力。最后,我們將正常Sufu-GFP與GSK-3β、PKA單獨(dú)或者二者聯(lián)合共轉(zhuǎn)染,發(fā)現(xiàn)更多的Sufu在細(xì)胞核中,但是這些激酶對(duì)非磷酸化的Sufu,即雙A突變并不能起到作用。上述結(jié)果闡明了Sufu不僅能夠參與Gli介導(dǎo)的轉(zhuǎn)錄、抑制信號(hào)通路,同時(shí)還能通過(guò)提高Gli1蛋白的細(xì)胞核轉(zhuǎn)運(yùn)和穩(wěn)定性,從而提高Hh通路活性,這對(duì)于Shh通路的最大化激活是必須的。使我們對(duì)于Sufu作為Gli蛋白分子伴侶有了新的認(rèn)識(shí)。
[Abstract]:The Hh protein family regulates a series of developmental processes and tumours, and the maladjustment of the Hh pathway plays a role in development disorders and cancers. For example, it has a far-reaching impact on the development of the limb to central nervous system, basal cell carcinoma and medulloblastoma. The Hh pathway approved by the United States Drug Administration to combat BCC metastasis The preparation shows the importance of Hh in cancer treatment; and for other cancers, many clinical trials are still developing in which.Hh is a morphogenetic factor that forms a concentration gradient along the development axis. In the process of neural tube development, it is expressed from the chord and the floor and extends to the back.Hh protein. The Ptch ligand.Ptch is a 12 transmembrane protein, which can reverse the 7 transmembrane protein Smo.Hh ligand binding with Ptch and lead to Smo activation. This will lead to the transport of Smo into the primary cilia, and the Hh pathway is also the downstream of the normal transport of.Smo, which is a group of zinc finger transcription factors. In mammals, there are three homologous proteins, Gli, Gli. 1, Gli2 and Gli3.Sufu, as a molecular chaperone of Gli protein, can bind to all Gli proteins and form complex.Sufu to bind to the highly conserved SYGH domain of the Gli amino terminal, or by blocking the nuclear input signal (NLS) of Gli, so that Gli is blocked in the cytoplasm, making Gli protein suppressed by Sufu. U is a specific mechanism for regulating Gli cytoplasmic block. With the stimulation of Hh, Gli protein is transported to the nucleus, suggesting that Sufu has been relieved of the anchoring function of Gli in the cytoplasm. However, biochemical and genetic studies have shown that the activity of Sufu to regulate Gli is through other non cytoplasmic blocking mechanisms. This implies that Sufu and Gli proteins are in the nucleus. However, the detailed mechanism of the Sufu regulation of Gli protein is still unclear. Since Hh plays a very important role in the development of the cerebellum, we use the cerebellum as a model to study. In the cerebellum of adult mice, the Hh pathway is resting state, but after more than one week of birth, the cerebellum is in high intensity, the level of Sufu and Gl The expression of I is high in the precursor of the ectodermal granular neurons. The development of this part of the cells depends on the output of the Hh signal. The Hh pathway is reactivated in the medulloblastoma, a cerebellar tumor, which comes from the precursor of granular neurons. As before, the level of Sufu is up to the same level as Gli1. In addition, Sufu is in the cell. In order to further observe the distribution of Sufu in the nucleus and cytoplasm, we detected the mouse embryonic fibroblast line (MEFs). In normal MEFs, no matter Shh treatment or not, Sufu is distributed in the nucleus and cytoplasm. When we detect the cells missing from gli2/ 3, we are surprised that Sufu is not. Shh is almost in the cytoplasm. And when there is Shh, Sufu is induced into the nucleus, and there is no suppressor in the gli2/3 missing cells. This suggests that in normal MEFs, in the absence of Shh treatment, Sufu is likely to be brought into the nucleus by the suppressor. In gli2/3 missing cells, shh induced Sufu entry is Gli1. Mediated. In order to prove this, we stably express Gli1 shRNA in gli2/3 missing cells, thus constructing three cell lines with no Gli. In this cell line, Sufu's nucleation is no longer induced by SHH induced.Sufu to recruit sin3a and HDAC to inhibit Gli transcriptional activity. We reconsidered this pattern and explored su. Fu can appear on the chromatin of the Gli binding site. We take the promoter of Gli1 and PTCH1 as an example, using the chip experiment, after the two loci are treated with Shh, the content of the Gli binding element increases, suggesting that Shh can induce Sufu to recruit to its target gene promoter region. Interestingly, we also found sap18 in Hh target gene promoter. HH These results suggest that Hh can induce the gli1-sufu complex to attach to its target gene promoter region, and that this is dependent on the Gli1 and Gli3 suppressors. In order to find the sequence originals that determine the distribution of Sufu in the cytoplasm and nucleus, we have made the missing location and finally determined the 308-318 residue of Sufu in human beings. The base had a potential NES signal. We mutated two of the leucine into alanine and detected in MEFs. Compared to the wild type sufu, the Sufu of the NES mutation appeared more in the nucleus. And the NES receptor CRM1 was no longer associated with the mutation of sufu. A pKa and GSK3 beta diphosphorylation site was reported before this laboratory. It is possible to regulate the stability of Sufu and the transport of the primary cilium. This diphosphorylation site is just near nes. In the CoIP experiment, we found that sufu, which can not be phosphorylated at 342 or 346 sites, has an effective binding ability with CRM1, and the simulated phosphorylation of Sufu loses its ability to bind with CRM1. Finally, we will have normal Su. Fu-GFP was co transfected with GSK-3 beta, PKA alone or two, and found that more Sufu was in the nucleus, but these kinases did not play a role in the non phosphorylated Sufu, that is, double A mutation. These results illustrate that Sufu can not only participate in Gli mediated transcription, inhibit the signaling pathway, but also enhance the nuclear transport of Gli1 protein. And stability, thus enhancing the activity of the Hh pathway, which is necessary for maximizing the activation of the Shh pathway. We have a new understanding of Sufu as a molecular chaperone of the Gli protein.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R363

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王春梅,姜濤,周春燕;誘導(dǎo)心臟發(fā)生的早期信號(hào)通路[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);2004年05期

2 解文博;葉玲;;p38絲裂原活化蛋白激酶信號(hào)通路的部分研究方法[J];牙體牙髓牙周病學(xué)雜志;2009年10期

3 黎增輝;廖愛(ài)軍;;JNK信號(hào)通路[J];國(guó)際病理科學(xué)與臨床雜志;2010年03期

4 李林;;Wnt信號(hào)通路及其與骨形成的關(guān)系[J];臨床和實(shí)驗(yàn)醫(yī)學(xué)雜志;2010年20期

5 李鳳賀;辛世杰;楊棟;惠林萍;楊昱;張健;段志泉;;SHH信號(hào)通路在血小板源性生長(zhǎng)因子誘導(dǎo)的血管平滑肌細(xì)胞增殖中的作用研究[J];中國(guó)血管外科雜志(電子版);2010年04期

6 陳建勇;王聰;王娟;曹禮榮;;MAPK信號(hào)通路研究進(jìn)展[J];中國(guó)醫(yī)藥科學(xué);2011年08期

7 李寶新;李玉坤;;Wnt信號(hào)通路對(duì)骨調(diào)節(jié)研究新進(jìn)展[J];國(guó)際骨科學(xué)雜志;2008年03期

8 李佳殷;林麗珠;;JNK信號(hào)通路在惡性腫瘤中的研究進(jìn)展[J];實(shí)用中醫(yī)內(nèi)科雜志;2012年18期

9 張玉婷;劉立新;;胰島素樣生長(zhǎng)因子結(jié)合蛋白在相關(guān)信號(hào)通路中的作用研究進(jìn)展[J];中華消化病與影像雜志(電子版);2012年05期

10 席曉慧;王福文;牟艷玲;;Wnt信號(hào)通路與心血管疾病的研究進(jìn)展[J];食品與藥品;2014年01期

相關(guān)會(huì)議論文 前10條

1 程華;王文;;經(jīng)典Wnt信號(hào)通路抑制劑[A];2011全國(guó)老年癡呆與衰老相關(guān)疾病學(xué)術(shù)會(huì)議第三屆山東省神經(jīng)內(nèi)科醫(yī)師（學(xué)術(shù)）論壇論文匯編[C];2011年

2 蔣瑩;程華;張麗;艾厚喜;王文;;經(jīng)典Wnt信號(hào)通路抑制劑[A];第一屆全國(guó)麻醉藥理專業(yè)委員會(huì)第二次學(xué)術(shù)會(huì)議論文集[C];2011年

3 蔣瑩;程華;張麗;艾厚喜;王文;;經(jīng)典Wnt信號(hào)通路抑制劑[A];中國(guó)成人醫(yī)藥教育論壇（4）[C];2011年

4 蔣瑩;程華;張麗;艾厚喜;王文;;經(jīng)典Wnt信號(hào)通路抑制劑[A];中國(guó)藥理學(xué)會(huì)補(bǔ)益藥藥理專業(yè)委員會(huì)成立大會(huì)暨人參及補(bǔ)益藥學(xué)術(shù)研討會(huì)會(huì)議論文集[C];2011年

5 李艷菲;艾厚喜;孫芳玲;張麗;蔣瑩;王文;;Wnt信號(hào)通路研究新進(jìn)展[A];2013年中國(guó)藥學(xué)大會(huì)暨第十三屆中國(guó)藥師周論文集[C];2013年

6 李艷菲;艾厚喜;孫芳玲;張麗;蔣瑩;王文;;Wnt信號(hào)通路研究新進(jìn)展[A];2013年全國(guó)老年性癡呆與相關(guān)疾病學(xué)術(shù)會(huì)議論文匯編[C];2013年

7 李艷菲;艾厚喜;孫芳玲;張麗;蔣瑩;王文;;Wnt信號(hào)通路研究新進(jìn)展[A];第三屆中國(guó)藥理學(xué)會(huì)補(bǔ)益藥藥理專業(yè)委員會(huì)學(xué)術(shù)研討會(huì)論文集[C];2013年

8 季宇彬;胡哲清;鄒翔;;MAPK信號(hào)通路及其研究進(jìn)展[A];轉(zhuǎn)化醫(yī)學(xué)研討會(huì)論文集[C];2010年

9 程華;孫芳玲;艾厚喜;張麗;蔣瑩;王文;;Wnt信號(hào)通路與血管發(fā)生[A];全國(guó)第三次麻醉藥理學(xué)術(shù)會(huì)議論文集[C];2012年

10 李林;;利用小分子探針研究經(jīng)典Wnt信號(hào)通路的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制[A];中國(guó)生物化學(xué)與分子生物學(xué)會(huì)第十一次會(huì)員代表大會(huì)暨2014年全國(guó)學(xué)術(shù)會(huì)議論文集——專題報(bào)告七[C];2014年

相關(guān)重要報(bào)紙文章 前6條

1 劉海英;美發(fā)現(xiàn)兩種細(xì)胞信號(hào)通路互動(dòng)機(jī)制[N];科技日?qǐng)?bào);2009年

2 記者 劉海英;英發(fā)現(xiàn)細(xì)胞信號(hào)通路新“剎車”蛋白[N];科技日?qǐng)?bào);2012年

3 衣曉峰　陳英云 記者 李麗云;結(jié)腸癌發(fā)生中新的信號(hào)通路找到[N];科技日?qǐng)?bào);2009年

4 記者 許琦敏;減肥信號(hào)受控于腦中“通訊員”[N];文匯報(bào);2008年

5 記者 劉海英;一種蛋白決定人的抗壓能力[N];科技日?qǐng)?bào);2011年

6 通訊員 喬蕤琳 記者 衣曉峰 聶松義;癌癥中小分子和微小核苷酸有關(guān)聯(lián)[N];健康報(bào);2012年

相關(guān)博士學(xué)位論文 前10條

1 王森;乙型肝炎病毒表面抗原抑制TLR2和TLR4信號(hào)通路的機(jī)制研究[D];復(fù)旦大學(xué);2014年

2 尚果果;NRF2抗氧化應(yīng)激信號(hào)通路的活化在糖尿病腎病中的保護(hù)作用及其機(jī)制研究[D];復(fù)旦大學(xué);2014年

3 孫鼎琪;Akt信號(hào)通路在膀胱腫瘤的進(jìn)展及化療敏感性的實(shí)驗(yàn)研究[D];山東大學(xué);2015年

4 白秀峰;新基因Gsdma3對(duì)小鼠毛囊周期及Wnt/β-catenin信號(hào)通路作用的研究[D];第三軍醫(yī)大學(xué);2015年

5 張俊波;PI3K/Akt信號(hào)通路調(diào)控胞內(nèi)布魯氏菌16M存活的分子機(jī)制研究[D];石河子大學(xué);2014年

6 成旭東;黃芪活性物質(zhì)篩選及基于PKC-ERK信號(hào)通路抑制肺癌的作用機(jī)理研究[D];南京中醫(yī)藥大學(xué);2015年

7 路康;STAT3信號(hào)通路介導(dǎo)CLL和MCL發(fā)生組蛋白去乙�；敢种苿┠退幍臋C(jī)制研究[D];山東大學(xué);2015年

8 趙睿;Wnt信號(hào)通路在臂叢神經(jīng)根性撕脫傷誘發(fā)的神經(jīng)病理性痛中的作用機(jī)制研究[D];第四軍醫(yī)大學(xué);2015年

9 劉海燕;Sonic hedgehog信號(hào)通路對(duì)正常角質(zhì)形成細(xì)胞增殖及凋亡的影響及其分子機(jī)制[D];第四軍醫(yī)大學(xué);2015年

10 祁義軍;胃腸道間質(zhì)瘤中Shh信號(hào)通路作用的實(shí)驗(yàn)研究[D];安徽醫(yī)科大學(xué);2015年

相關(guān)碩士學(xué)位論文 前10條

1 韓紹芳;小鼠主要嗅覺(jué)表皮組織內(nèi)AC3缺失對(duì)相關(guān)因子及信號(hào)通路的影響[D];河北大學(xué);2015年

2 李玉娟;Hedgehog信號(hào)通路在大鼠急性胰腺炎中的作用及其對(duì)TNF-α、IL-10的影響[D];河北醫(yī)科大學(xué);2015年

3 趙玲玲;FLCN與PAT1相互作用調(diào)控mTORC1信號(hào)通路的機(jī)理研究[D];西北農(nóng)林科技大學(xué);2015年

4 努爾古麗·蘇里坦;mir-260和mir-240調(diào)控錢蟲(chóng)壽命的研究[D];華中師范大學(xué);2015年

5 王瑜;晚期糖基化終末產(chǎn)物和骨髓間充質(zhì)干細(xì)胞對(duì)血管內(nèi)皮細(xì)胞VEGF信號(hào)通路的影響及其機(jī)制探討[D];河北醫(yī)科大學(xué);2015年

6 楊瑩瑩;microRNA-203通過(guò)APC介導(dǎo)活化Wnt/β-catenin信號(hào)通路在佐劑性關(guān)節(jié)炎大鼠成纖維樣滑膜細(xì)胞中的調(diào)控作用及機(jī)制研究[D];安徽醫(yī)科大學(xué);2015年

7 徐貞貞;PM2.5對(duì)人支氣管上皮細(xì)胞JAK/STAT信號(hào)通路的影響[D];山西醫(yī)科大學(xué);2015年

8 徐國(guó)棟;芪蛭皺肺顆粒調(diào)控肺泡Ⅱ型上皮細(xì)胞NF-κB信號(hào)通路的研究[D];甘肅中醫(yī)藥大學(xué)(原名：甘肅中醫(yī)學(xué)院);2015年

9 陸兆雙;多聚左旋精氨酸促進(jìn)LPS誘導(dǎo)的NCI-H292細(xì)胞釋放炎癥因子IL-6、IL-8的信號(hào)通路研究[D];安徽醫(yī)科大學(xué);2015年

10 修麗梅;P38MAPK信號(hào)通路在肢體缺血預(yù)處理腦保護(hù)中的作用[D];山西醫(yī)科大學(xué);2015年

，

本文編號(hào)：1962635