本文選題：Notch3 + N端序列缺失突變　； 參考：《揚(yáng)州大學(xué)》2007年博士論文

【摘要】： Notch3信號(hào)通路在機(jī)體發(fā)育過程中調(diào)控細(xì)胞生長(zhǎng)、分化和凋亡等多種重要生物學(xué)過程。在成年人，Notch3受體特異性表達(dá)于動(dòng)脈血管平滑肌細(xì)胞(Smooth muscle cell，SMC)。Notch3是保守的Ⅰ型跨膜受體，結(jié)構(gòu)上可以分為細(xì)胞外的34個(gè)表皮生長(zhǎng)因子樣重復(fù)序列(Epidermal growth factor-like repeats，EGFR)和胞漿中的多個(gè)功能結(jié)構(gòu)域，前者與配體結(jié)合，后者介導(dǎo)細(xì)胞內(nèi)的信號(hào)傳導(dǎo)。Notch3受體合成后要經(jīng)過三次蛋白酶切割作用才能發(fā)揮其生物學(xué)活性。首先，Notch3前體蛋白在表達(dá)后被運(yùn)輸?shù)絻?nèi)質(zhì)網(wǎng)(Endoplasmic reticulum，ER)，繼而轉(zhuǎn)運(yùn)到高爾基體(Golgi)內(nèi)被弗林(Furin)蛋白酶在S1裂解位點(diǎn)切割為2個(gè)片斷，形成異二聚體后被轉(zhuǎn)運(yùn)到細(xì)胞膜表面。當(dāng)與配體結(jié)合后，Notch3蛋白被腫瘤壞死因子-α-轉(zhuǎn)化酶(TNF-αconverting enzyme，TACE)在胞膜外S2裂解位點(diǎn)切割，接著在胞膜內(nèi)被γ-促分泌酶(γ-secretase)在S3裂解位點(diǎn)切割，釋放Notch3胞內(nèi)區(qū)(Notch intracellular domain，NICD)進(jìn)入細(xì)胞核，與CLS轉(zhuǎn)錄因子結(jié)合，啟動(dòng)靶基因(如HES)的表達(dá)。 通過對(duì)Notch3與Notch蛋白家族的其它成員(Notch1，2和4)進(jìn)行序列比對(duì)分析后發(fā)現(xiàn)，Notch3蛋白N端序列包含多個(gè)連續(xù)的精氨酸、脯氨酸和亮氨酸，該特征顯著不同于Notch家族其它成員，而且在不同種屬動(dòng)物中高度保守，提示其具有重要的生物學(xué)意義。 Notch3基因的錯(cuò)義突變可以導(dǎo)致伴皮質(zhì)下梗死及白質(zhì)腦病的常染色體顯性遺傳性腦動(dòng)脈病(Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy，CADASIL)。該病臨床表現(xiàn)為偏頭痛、缺血性腦卒中和進(jìn)行性癡呆等。CADASIL病人的典型病理學(xué)變化是多發(fā)性皮質(zhì)下梗死、小動(dòng)脈血管壁增厚，動(dòng)脈平滑肌細(xì)胞之間間隙疏松，隨病程發(fā)展細(xì)胞逐漸降解，電鏡檢測(cè)發(fā)現(xiàn)在SMC周圍有顆粒狀嗜鋨樣物質(zhì)(Granular electron dense osmiophilic material，GOM)沉積。 CADASIL的相關(guān)突變發(fā)生于Notch3蛋白的EGFR內(nèi)，造成一個(gè)半胱氨酸變?yōu)槠渌被峄蛘咂渌被嶙優(yōu)榘腚装彼幔瑢?dǎo)致相應(yīng)EGFR內(nèi)半胱氨酸的數(shù)量由偶數(shù)變成奇數(shù)，進(jìn)而可能改變Notch3蛋白構(gòu)象并誘發(fā)該病，但其具體致病機(jī)制迄今不明。 基于以上背景，本研究主要有兩個(gè)目的：(1)闡明Notch3蛋白特殊的N端序列的生物學(xué)意義；(2)探討CADASIL的潛在致病機(jī)制。為此，本文構(gòu)建了Notch3蛋白N端序列的缺失突變體以及CADASIL相關(guān)的錯(cuò)義突變體，并研究了它們對(duì)Notch3蛋白的表達(dá)、加工、胞內(nèi)分布、信號(hào)轉(zhuǎn)導(dǎo)和生物學(xué)功能的影響。以上研究為全面認(rèn)識(shí)Notch3蛋白的生物學(xué)特性和闡明CADASIL的致病機(jī)制奠定了基礎(chǔ)。 1 Notch3基因N端序列的缺失突變對(duì)其相關(guān)生物學(xué)特性的影響 1．1 Notch3蛋白N端序列缺失突變體Δ14-Notch3、Δ39-Notch3和Sec-Δ39-Notch3的構(gòu)建 根據(jù)Notch3受體N端序列的特征，我們構(gòu)建了三個(gè)N端序列缺失的突變體。Δ14-Notch3缺少含有多個(gè)精氨酸的前14個(gè)氨基酸，并用第二個(gè)甲硫氨酸作為翻譯起始位點(diǎn)。與野生型的Notch3相比，，該克隆的構(gòu)建是為了闡明前14個(gè)氨基酸的潛在生物學(xué)功能。Δ39-Notch3缺少前39個(gè)氨基酸，刪除了完整的潛在信號(hào)肽序列(經(jīng)生物信息學(xué)軟件分析)以及靠近于信號(hào)肽切割位點(diǎn)的多個(gè)連續(xù)亮氨酸。與野生型的Notch3相比，該克隆用于研究前39個(gè)氨基酸對(duì)該蛋白的靶向性分泌以及信號(hào)轉(zhuǎn)導(dǎo)的影響。最后，我們用IgKappa的分泌信號(hào)肽代替前39個(gè)氨基酸構(gòu)建了Sec-Δ39-Notch3，其目的是將Notch3自身的N端信號(hào)肽序列與已知的高效分泌信號(hào)肽的功能進(jìn)行比較。酶切及測(cè)序結(jié)果表明以上突變體均構(gòu)建成功。 1．2 Notch3蛋白N端序列對(duì)其表達(dá)、加工和胞漿分布的影響 間接免疫熒光染色(Indirect immunofluorescent assay，IFA)、Western blot以及胞漿抽提等分析結(jié)果顯示，與WT-Notch3相比，Δ14-Notch3不影響蛋白表達(dá)水平和加工效率，但顯著增高了蛋白的胞漿分布；Δ39-Notch3不僅顯著降低蛋白表達(dá)水平和加工效率，而且極顯著地提高了蛋白胞漿分布；Sec-Δ39-Notch3不影響蛋白表達(dá)水平，但可提高加工效率，抑制蛋白胞漿分布。 1．3 Notch3受體N端序列對(duì)其信號(hào)轉(zhuǎn)導(dǎo)、促平滑肌細(xì)胞生長(zhǎng)和抗凋亡活性的影響 我們用與表達(dá)配體Jagged1或Delta1的L細(xì)胞共培養(yǎng)來活化Notch3受體，用受Notch3靶基因啟動(dòng)子調(diào)控的報(bào)告系統(tǒng)(HES-luciferase)分析Notch3受體的信號(hào)轉(zhuǎn)導(dǎo)活性。結(jié)果表明，Δ14-Notch3和Sec-Δ39-Notch3轉(zhuǎn)染組顯示出與WT-Notch3相似的活性，但Δ39-Notch3的信號(hào)轉(zhuǎn)導(dǎo)能力顯著降低。 鑒于正常Notch3信號(hào)具有促進(jìn)血管平滑肌細(xì)胞生長(zhǎng)和抗凋亡的作用，我們研究了Notch3受體N端序列突變對(duì)這些生物學(xué)活性的影響。結(jié)果表明，WT-Notch3、Δ14-Notch3和Sec-Δ39-Notch3可顯著促進(jìn)細(xì)胞生長(zhǎng)，但Δ39-Notch3對(duì)細(xì)胞生長(zhǎng)促進(jìn)效果顯著降低。 通過碘化丙啶(Propidium iodide，PI)染色進(jìn)行細(xì)胞凋亡分析，我們比較了轉(zhuǎn)染野生型以及突變型Notch3的血管平滑肌細(xì)胞對(duì)Fas配體(Fas ligand，F(xiàn)as-L)的敏感性。結(jié)果表明，WT-Notch3、Δ14-Notch3和Sec-Δ39-Notch3能有效抵抗由Fas-L所誘導(dǎo)的細(xì)胞凋亡，而Δ39-Notch3則喪失了相應(yīng)的保護(hù)效果。 1．4胞漿Notch3與蛋白酶體PSMA1亞基相互作用并抑制蛋白酶體活性 前面研究發(fā)現(xiàn)，富含精氨酸的前14個(gè)氨基酸序列對(duì)降低Notch3蛋白的胞漿分布具有重要作用。為研究該蛋白胞漿分布的潛在作用，我們通過酵母雙雜交(Yeast two-hybrid，YTH)和免疫共沉淀(Coimmunoprecipitation，Co-IP)證實(shí)了胞漿表達(dá)的Notch3蛋白能與蛋白酶體的PSMA1亞基特異性結(jié)合；通過蛋白酶體活性分析發(fā)現(xiàn)了Notch3可以抑制蛋白酶體功能。提高Notch3蛋白的胞漿表達(dá)水平可以顯著增強(qiáng)與PSMA1亞基的相互作用并更加顯著地抑制蛋白酶體活性。Notch3和PSMA1相互作用代表了一種新型的、由膜蛋白胞漿轉(zhuǎn)位所介導(dǎo)的蛋白酶體活性調(diào)節(jié)方式。 綜上所述，該研究主要發(fā)現(xiàn)為：(1) Notch3蛋白N端1—39位氨基酸殘基(包含多個(gè)連續(xù)的精氨酸、脯氨酸和亮氨酸)是其表達(dá)、加工、細(xì)胞內(nèi)分布以及生物活性所必需的；(2) N端1—14位氨基酸殘基(包含七個(gè)連續(xù)的精氨酸)可抑制Notch3蛋白的胞漿分布，但并不影響其表達(dá)、加工以及生物活性；(3)胞漿內(nèi)Notch3蛋白能與蛋白酶體的PSMA1亞基特異性結(jié)合，并顯著抑制蛋白酶體的活性。以上研究表明，Notch3蛋白N端保守序列具有多重功能，除了促進(jìn)蛋白靶向到分泌通路以外，還可以減少蛋白的胞漿分布，從而避免對(duì)蛋白酶體的抑制作用。 2 CADASIL相關(guān)的錯(cuò)義突變對(duì)Notch3蛋白相關(guān)生物學(xué)特性的影響 2．1 CADASIL相關(guān)突變體的構(gòu)建 為了探討CADASIL的潛在致病機(jī)制，我們構(gòu)建了兩個(gè)臨床存在的CADASIL相關(guān)的Notch3突變體C49Y(Notch3第49位氨基酸由C突變?yōu)閅)和R90C(Notch3第90位氨基酸由R突變?yōu)镃)，它們分別代表所在EGFR失去或獲得一個(gè)半胱氨酸的突變，導(dǎo)致EGFR內(nèi)半胱氨酸的數(shù)量由偶數(shù)變成奇數(shù)。我們研究了以上突變體對(duì)蛋白的表達(dá)、加工、細(xì)胞內(nèi)分布以及促細(xì)胞生長(zhǎng)和抗凋亡活性的影響。為了高度特異且敏感地反映CADASIL相關(guān)突變對(duì)信號(hào)轉(zhuǎn)導(dǎo)能力的影響，我們應(yīng)用了新型的GV融合蛋白(GAL4結(jié)合區(qū)域—VP16活化區(qū)域)報(bào)告系統(tǒng)。在該體系中，WT-Notch3、C49Y和R90C的胞內(nèi)區(qū)分別被GV融合蛋白所取代。當(dāng)Notch3信號(hào)活化后，Notch3-GV經(jīng)歷S3位點(diǎn)切割，釋放GV融合肽段進(jìn)入細(xì)胞核，與報(bào)告載體(pFR-luciferase)所含有的GAL4序列特異性結(jié)合并活化報(bào)告基因的表達(dá)。酶切及測(cè)序結(jié)果表明以上突變體均構(gòu)建成功。 2．2 R90C和C49Y對(duì)Notch3蛋白表達(dá)水平、加工效率和細(xì)胞漿內(nèi)分布的影響 通過IFA比較了WT-Notch3、C49Y和R90C的表達(dá)，結(jié)果發(fā)現(xiàn)三者熒光強(qiáng)度相當(dāng)；Western blot分析也進(jìn)一步證實(shí)突變蛋白不影響表達(dá)水平。繼而，我們通過計(jì)算蛋白加工后形成的胞內(nèi)區(qū)和未加工全長(zhǎng)蛋白的比值評(píng)價(jià)了蛋白的加工效率，發(fā)現(xiàn)突變蛋白與正常蛋白水平相當(dāng)。最后，我們抽提了WT-Notch3、C49Y和R90C轉(zhuǎn)染細(xì)胞的胞漿蛋白，發(fā)現(xiàn)突變蛋白也不影響蛋白的胞漿分布。 2．3 R90C和C49Y對(duì)Notch3蛋白信號(hào)轉(zhuǎn)導(dǎo)的影響 我們構(gòu)建了高特異性的Notch-GV新型報(bào)告系統(tǒng)，并在CADASIL的靶細(xì)胞(血管平滑肌細(xì)胞)上評(píng)價(jià)了CADASIL相關(guān)突變對(duì)Notch3信號(hào)轉(zhuǎn)導(dǎo)能力的影響。結(jié)果表明，C49Y-GV和R90C-GV的信號(hào)轉(zhuǎn)導(dǎo)活性均顯著低于WT-Notch3-GVP。而且，當(dāng)WT-Notch3-GVP分別與WT-Notch3、R90C和C49Y共轉(zhuǎn)染入血管平滑肌細(xì)胞后，R90C和C49Y能顯著抑制Notch3-GV的信號(hào)轉(zhuǎn)導(dǎo)活性。該發(fā)現(xiàn)為CADASIL致病機(jī)制的闡明提供了線索。 2．4 R90C和C49Y對(duì)Notch3蛋白促平滑肌細(xì)胞生長(zhǎng)和抗凋亡活性的影響 利用LDH活性檢測(cè)進(jìn)行細(xì)胞總數(shù)分析顯示，WT-Notch3可顯著促進(jìn)血管平滑肌細(xì)胞A7r5的生長(zhǎng)，而R90C和C49Y對(duì)細(xì)胞生長(zhǎng)的促進(jìn)作用減弱。通過PI染色確定轉(zhuǎn)染W(wǎng)T-Notch3、R90C和C49Y的血管平滑肌細(xì)胞A7r5對(duì)Fas-L的敏感性。結(jié)果表明，WT-Notch3能有效保護(hù)細(xì)胞抵抗由Fas-L所誘導(dǎo)的細(xì)胞凋亡，但R90C和C49Y不能產(chǎn)生明顯的保護(hù)效應(yīng)。以上結(jié)果表明，CADASIL相關(guān)突變體不僅減弱Notch3信號(hào)轉(zhuǎn)導(dǎo)能力，而且直接降低了對(duì)動(dòng)脈血管平滑肌細(xì)胞的保護(hù)作用。 綜上所述，該研究主要發(fā)現(xiàn)為：(1)CADASIL突變體(R90C和C49Y)不影響Notch3蛋白的表達(dá)、加工和胞漿分布；(2)R90C和C49Y降低Notch3蛋白信號(hào)轉(zhuǎn)導(dǎo)活性，而且當(dāng)與野生型Notch3同時(shí)存在時(shí)，突變蛋白可抑制野生蛋白的信號(hào)轉(zhuǎn)導(dǎo)活性，發(fā)揮顯性抑制作用；(3)R90C和C49Y促血管平滑肌細(xì)胞生長(zhǎng)的活性下降，同時(shí)抗凋亡的活性喪失。以上研究為CADASIL致病機(jī)制的探索奠定了基礎(chǔ)。
[Abstract]:Notch3 signaling pathways regulate cell growth, differentiation and apoptosis during the development of the body. In adults, the Notch3 receptor is specifically expressed in arterial vascular smooth muscle cells (Smooth muscle cell, SMC).Notch3 as a conservative type I transmembrane receptor, which can be divided into 34 epidermal growth factors like extracellular matrix. Epidermal growth factor-like repeats (EGFR) and multiple functional domains in the cytoplasm, the former is combined with the ligand, and the latter mediates the synthesis of signal transduction.Notch3 receptors in cells after three protease cleavage. First, Notch3 precursor protein is transported to the endoplasmic reticulum (E) after expression. Ndoplasmic reticulum, ER), then transshipped to the Golgi body (Golgi), the Flynn (Furin) protease was cut into 2 fragments at the S1 cracking site and was transported to the surface of the cell membrane after the formation of the hetero two polymer. When combined with the ligand, the Notch3 protein was located at the extracellular S2 cracking site of the tumor necrosis factor alpha transase (TNF- a converting enzyme, TACE). The cutting is followed by the cleavage of the gamma secretase (gamma -secretase) at the S3 cleavage site in the cell membrane, releasing the Notch3 intracellular region (Notch intracellular domain, NICD) into the nucleus and combining with the CLS transcription factor to initiate the expression of the target gene (such as HES).
The sequence alignment analysis of Notch3 and other members of the Notch protein family (Notch1,2 and 4) found that the N terminal sequence of the Notch3 protein contains a number of successive arginine, proline and leucine, which are significantly different from other members of the Notch family, and are highly conserved in different species of animals, suggesting that they have important biology. Significance.
The missense mutation of the Notch3 gene can lead to autosomal dominant hereditary cerebral arteriopathy associated with subcortical infarction and leukemic encephalopathy (Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, CADASIL). The clinical manifestations of this disease are migraine, ischemic stroke and progressive dementia and other.CADASIL patients The typical pathological changes are multiple subcortical infarcts, the thickening of the vascular wall of the arterioles, the loose gap between the arterial smooth muscle cells and the gradual degradation of the cells with the course of the disease. The electron microscopic examination shows that the granular osmium like substance (Granular electron dense osmiophilic material, GOM) is deposited around SMC.
The related mutation of CADASIL occurs in the EGFR of Notch3 protein, causing a cysteine to change into other amino acids or other amino acids to cysteine, resulting in the number of cysteine in the corresponding EGFR from even number to odd number, which may change the conformation of Notch3 protein and induce the disease, but its specific pathogenesis is not so far.
Based on the above background, this study has two main purposes: (1) to elucidate the biological significance of the special N terminal sequence of Notch3 protein; (2) to explore the potential pathogenesis of CADASIL. Therefore, the deletion mutants of the N terminal sequence of the Notch3 protein and the CADASIL related missense mutants were constructed, and their expression to the Notch3 protein was studied. Work, intracellular distribution, signal transduction and biological functions. The above study lays the foundation for a comprehensive understanding of the biological characteristics of Notch3 protein and the elucidation of the pathogenesis of CADASIL.
1 the deletion of N terminal sequence of Notch3 gene affects its biological characteristics.
Construction of 1.1 Notch3 protein N terminal deletion mutant 14-Notch3, 39-Notch3 and Sec- 39-Notch3
According to the characteristics of the N terminal sequence of the Notch3 receptor, we constructed three mutant N terminated sequences. The delta 14-Notch3 lacks the first 14 amino acids containing multiple arginine and uses second methionine as the starting site of the translation. Compared with the wild type Notch3, the clone is constructed to elucidate the potential biological work of the first 14 amino acids. Yes. The delta 39-Notch3 lacks the first 39 amino acids, deleting the complete potential signal peptide sequence (bioinformatics software analysis) and multiple continuous leucine near the signal peptide cleavage site. Compared with the wild type Notch3, the clone is used to study the effect of the first 39 amino acids on the target secretion and signal transduction of the egg white. After that, we constructed Sec- Delta 39-Notch3 using the IgKappa secretory peptide instead of the first 39 amino acids. The aim was to compare the function of the N terminal signal peptide sequence of Notch3 itself with the known high efficient secretory signal peptide. The results of enzyme digestion and sequencing showed that all the above mutants were constructed successfully.
1.2 the effect of N terminal sequence of Notch3 protein on its expression, processing and cytoplasmic distribution
The results of indirect immunofluorescence staining (Indirect immunofluorescent assay, IFA), Western blot and cytoplasmic extraction showed that compared with WT-Notch3, Delta 14-Notch3 did not affect protein expression level and processing efficiency, but significantly increased the cytoplasmic distribution of protein, and delta 39-Notch3 not only significantly reduced protein expression and processing efficiency, but also significantly reduced protein expression and processing efficiency. Sec- 39-Notch3 did not affect the level of protein expression, but could improve the processing efficiency and inhibit the distribution of protein cytoplasm.
1.3 the effect of N terminal sequence of Notch3 receptor on signal transduction, smooth muscle cell growth and anti apoptotic activity
We used a co culture of L cells expressing ligand Jagged1 or Delta1 to activate Notch3 receptors, and the signal transduction activity of Notch3 receptors was analyzed by a reporting system (HES-luciferase) regulated by the promoter of the Notch3 target gene (HES-luciferase). The results showed that the activity of the delta 14-Notch3 and Sec- Delta 39-Notch3 transfected groups showed a similar activity to WT-Notch3, but the signal of the delta 39-Notch3 The transduction ability was significantly reduced.
In view of the effect of normal Notch3 signal on promoting the growth and anti apoptosis of vascular smooth muscle cells, we studied the effect of N terminal sequence mutation of Notch3 receptor on these biological activities. The results showed that WT-Notch3, Delta 14-Notch3 and Sec- Delta 39-Notch3 could significantly promote cell growth, but the effect of delta 39-Notch3 on cell growth was significantly reduced.
The apoptosis analysis was carried out by Propidium iodide (PI) staining. We compared the sensitivity of vascular smooth muscle cells transfected with wild type and mutant Notch3 to the Fas ligand (Fas ligand, Fas-L). The results showed that WT-Notch3, Delta 14-Notch3 and Sec- Delta 39-Notch3 could effectively resist apoptosis induced by the cells. CH3 lost the corresponding protective effect.
1.4 cytoplasmic Notch3 interacts with proteasome PSMA1 subunit and inhibits proteasome activity.
Previous studies have found that the first 14 amino acid sequences rich in arginine have an important role in reducing the cytoplasmic distribution of Notch3 protein. In order to study the potential role of the protein cytoplasm distribution, we confirmed the Notch3 protein energy expressed in the cytoplasm by yeast two hybrid (Yeast two-hybrid, YTH) and immunoprecipitation (Coimmunoprecipitation, Co-IP). The PSMA1 subspecific binding of proteasome; through proteasome activity analysis, it was found that Notch3 could inhibit proteasome function. Enhancing the cytoplasmic expression of Notch3 protein could significantly enhance the interaction with PSMA1 subunits and significantly inhibit the interaction of proteasome activity,.Notch3 and PSMA1, to represent a new type of interaction. The regulation of proteasome activity mediated by translocation of membrane proteins.
To sum up, the main findings are as follows: (1) the 1 to 39 amino acid residues of the Notch3 protein N end (including a number of continuous arginine, proline and leucine) are essential for their expression, processing, intracellular distribution and biological activity; (2) 1 to 14 amino acid residues in the N terminal (including seven continuous arginine) can inhibit the cell of the Notch3 protein. Pulp distribution, but does not affect its expression, processing and biological activity; (3) Notch3 protein in the cytoplasm can be associated with the PSMA1 subunit of proteasome and significantly inhibits the activity of proteasome. The above study shows that the conservative sequence of the N terminal of Notch3 protein has multiple functions, besides promoting the protein targeting to the secretory pathway, it can also be reduced. The cytoplasm distribution of protein can avoid the inhibition of proteasome.
Effects of 2 CADASIL related missense mutations on biological characteristics of Notch3 protein
Construction of 2.1 CADASIL related mutants
In order to explore the potential pathogenesis of CADASIL, we constructed two clinically existing CADASIL related Notch3 mutant C49Y (Notch3 forty-ninth bits amino acids from C to Y) and R90C (Notch3 ninetieth amino acids from R mutation to C), which represent the loss of the EGFR or a cysteine mutation, leading to cysteine. We studied the effects of the above mutants on protein expression, processing, intracellular distribution, cell growth and anti apoptotic activity. In order to highly specifically and sensitively reflect the effect of CADASIL related mutations on signal transduction, we applied a new type of GV fusion protein (GAL4 binding region - VP16 live) In this system, the intracellular region of WT-Notch3, C49Y and R90C is replaced by GV fusion protein, respectively. When the Notch3 signal is activated, Notch3-GV undergo S3 site cutting, release the GV fusion peptide segment into the nucleus, specifically bind to the GAL4 sequence contained in the report carrier (pFR-luciferase), and activate the expression of the reporter gene. Sequencing and sequencing showed that the above mutants were successfully constructed.
2.2 the effects of R90C and C49Y on the expression level, processing efficiency and intracellular distribution of Notch3 protein.
The expression of WT-Notch3, C49Y and R90C was compared by IFA. The results showed that the fluorescence intensity of the three was the same; Western blot analysis further confirmed that the mutant protein did not affect the expression level. Then, we evaluated the processing efficiency of the protein by calculating the ratio of the intracellular and unprocessed total protein after processing, and found the mutant protein. In the end, we extracted cytoplasmic proteins from WT-Notch3, C49Y and R90C transfected cells, and found that the mutant protein did not affect the cytoplasmic distribution of the protein.
Effects of 2.3 R90C and C49Y on signal transduction of Notch3 protein
We constructed a highly specific Notch-GV new reporting system and evaluated the effect of CADASIL related mutations on the signal transduction ability of Notch3 on the target cells (vascular smooth muscle cells) of CADASIL. The results showed that the signal transduction activity of C49Y-GV and R90C-GV was significantly lower than that of WT-Notch3-GVP., when WT-Notch3-GVP and WT-Notch3, R90C were respectively. After CO transfection of C49Y into vascular smooth muscle cells, R90C and C49Y can significantly inhibit the signal transduction activity of Notch3-GV. This discovery provides clues for the elucidation of the pathogenesis of CADASIL.
Effects of 2.4 R90C and C49Y on the growth and anti apoptotic activity of Notch3 protein in smooth muscle cells
The cell total analysis by LDH activity assay showed that WT-Notch3 significantly promoted the growth of A7r5 in vascular smooth muscle cells, while R90C and C49Y reduced the cell growth. PI staining was used to determine the sensitivity of WT-Notch3, R90C and C49Y vascular smooth muscle cells A7r5 to Fas-L. The results showed that WT-Notch3 could effectively protect the cells. The cells resisted apoptosis induced by Fas-L, but R90C and C49Y did not produce obvious protective effects. The above results showed that the CADASIL related mutants not only weakened the Notch3 signal transduction ability, but also directly reduced the protective effect of the arterial vascular smooth muscle cells.
To sum up, the main findings are as follows: (1) CADASIL mutants (R90C and C49Y) do not affect the expression of Notch3 protein, processing and cytoplasm distribution; (2) R90C and C49Y reduce the signal transduction activity of Notch3 protein, and when the wild type Notch3 exists simultaneously, the mutant protein inhibits the signal transduction activity of the wild protein and plays the dominant inhibition. (3) (3) the growth of vascular smooth muscle cells (R90C and C49Y) decreased and the activity of anti apoptosis was lost. The above study laid the foundation for the exploration of the pathogenesis of CADASIL.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 汪志云;;常染色體顯性遺傳腦動(dòng)脈病合并皮質(zhì)下梗死和白質(zhì)腦病[J];醫(yī)學(xué)綜述;2006年15期

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