Notch3基因N端序列的缺失突变以及CADASIL相关的错义突变对其相关生物学特性的影响

发布时间：2018-05-20 05:45

本文选题：Notch3 + N端序列缺失突变　；参考：《扬州大学》2007年博士论文

【摘要】： Notch3信号通路在机体发育过程中调控细胞生长、分化和凋亡等多种重要生物学过程。在成年人，Notch3受体特异性表达于动脉血管平滑肌细胞(Smooth muscle cell，SMC)。Notch3是保守的Ⅰ型跨膜受体，结构上可以分为细胞外的34个表皮生长因子样重复序列(Epidermal growth factor-like repeats，EGFR)和胞浆中的多个功能结构域，前者与配体结合，后者介导细胞内的信号传导。Notch3受体合成后要经过三次蛋白酶切割作用才能发挥其生物学活性。首先，Notch3前体蛋白在表达后被运输到内质网(Endoplasmic reticulum，ER)，继而转运到高尔基体(Golgi)内被弗林(Furin)蛋白酶在S1裂解位点切割为2个片断，形成异二聚体后被转运到细胞膜表面。当与配体结合后，Notch3蛋白被肿瘤坏死因子-α-转化酶(TNF-αconverting enzyme，TACE)在胞膜外S2裂解位点切割，接着在胞膜内被γ-促分泌酶(γ-secretase)在S3裂解位点切割，释放Notch3胞内区(Notch intracellular domain，NICD)进入细胞核，与CLS转录因子结合，启动靶基因(如HES)的表达。通过对Notch3与Notch蛋白家族的其它成员(Notch1，2和4)进行序列比对分析后发现，Notch3蛋白N端序列包含多个连续的精氨酸、脯氨酸和亮氨酸，该特征显著不同于Notch家族其它成员，而且在不同种属动物中高度保守，提示其具有重要的生物学意义。 Notch3基因的错义突变可以导致伴皮质下梗死及白质脑病的常染色体显性遗传性脑动脉病(Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy，CADASIL)。该病临床表现为偏头痛、缺血性脑卒中和进行性痴呆等。CADASIL病人的典型病理学变化是多发性皮质下梗死、小动脉血管壁增厚，动脉平滑肌细胞之间间隙疏松，随病程发展细胞逐渐降解，电镜检测发现在SMC周围有颗粒状嗜锇样物质(Granular electron dense osmiophilic material，GOM)沉积。 CADASIL的相关突变发生于Notch3蛋白的EGFR内，造成一个半胱氨酸变为其它氨基酸或者其它氨基酸变为半胱氨酸，导致相应EGFR内半胱氨酸的数量由偶数变成奇数，进而可能改变Notch3蛋白构象并诱发该病，但其具体致病机制迄今不明。基于以上背景，本研究主要有两个目的：(1)阐明Notch3蛋白特殊的N端序列的生物学意义；(2)探讨CADASIL的潜在致病机制。为此，本文构建了Notch3蛋白N端序列的缺失突变体以及CADASIL相关的错义突变体，并研究了它们对Notch3蛋白的表达、加工、胞内分布、信号转导和生物学功能的影响。以上研究为全面认识Notch3蛋白的生物学特性和阐明CADASIL的致病机制奠定了基础。 1 Notch3基因N端序列的缺失突变对其相关生物学特性的影响 1．1 Notch3蛋白N端序列缺失突变体Δ14-Notch3、Δ39-Notch3和Sec-Δ39-Notch3的构建根据Notch3受体N端序列的特征，我们构建了三个N端序列缺失的突变体。Δ14-Notch3缺少含有多个精氨酸的前14个氨基酸，并用第二个甲硫氨酸作为翻译起始位点。与野生型的Notch3相比，，该克隆的构建是为了阐明前14个氨基酸的潜在生物学功能。Δ39-Notch3缺少前39个氨基酸，删除了完整的潜在信号肽序列(经生物信息学软件分析)以及靠近于信号肽切割位点的多个连续亮氨酸。与野生型的Notch3相比，该克隆用于研究前39个氨基酸对该蛋白的靶向性分泌以及信号转导的影响。最后，我们用IgKappa的分泌信号肽代替前39个氨基酸构建了Sec-Δ39-Notch3，其目的是将Notch3自身的N端信号肽序列与已知的高效分泌信号肽的功能进行比较。酶切及测序结果表明以上突变体均构建成功。 1．2 Notch3蛋白N端序列对其表达、加工和胞浆分布的影响间接免疫荧光染色(Indirect immunofluorescent assay，IFA)、Western blot以及胞浆抽提等分析结果显示，与WT-Notch3相比，Δ14-Notch3不影响蛋白表达水平和加工效率，但显著增高了蛋白的胞浆分布；Δ39-Notch3不仅显著降低蛋白表达水平和加工效率，而且极显著地提高了蛋白胞浆分布；Sec-Δ39-Notch3不影响蛋白表达水平，但可提高加工效率，抑制蛋白胞浆分布。 1．3 Notch3受体N端序列对其信号转导、促平滑肌细胞生长和抗凋亡活性的影响我们用与表达配体Jagged1或Delta1的L细胞共培养来活化Notch3受体，用受Notch3靶基因启动子调控的报告系统(HES-luciferase)分析Notch3受体的信号转导活性。结果表明，Δ14-Notch3和Sec-Δ39-Notch3转染组显示出与WT-Notch3相似的活性，但Δ39-Notch3的信号转导能力显著降低。鉴于正常Notch3信号具有促进血管平滑肌细胞生长和抗凋亡的作用，我们研究了Notch3受体N端序列突变对这些生物学活性的影响。结果表明，WT-Notch3、Δ14-Notch3和Sec-Δ39-Notch3可显著促进细胞生长，但Δ39-Notch3对细胞生长促进效果显著降低。通过碘化丙啶(Propidium iodide，PI)染色进行细胞凋亡分析，我们比较了转染野生型以及突变型Notch3的血管平滑肌细胞对Fas配体(Fas ligand，Fas-L)的敏感性。结果表明，WT-Notch3、Δ14-Notch3和Sec-Δ39-Notch3能有效抵抗由Fas-L所诱导的细胞凋亡，而Δ39-Notch3则丧失了相应的保护效果。 1．4胞浆Notch3与蛋白酶体PSMA1亚基相互作用并抑制蛋白酶体活性前面研究发现，富含精氨酸的前14个氨基酸序列对降低Notch3蛋白的胞浆分布具有重要作用。为研究该蛋白胞浆分布的潜在作用，我们通过酵母双杂交(Yeast two-hybrid，YTH)和免疫共沉淀(Coimmunoprecipitation，Co-IP)证实了胞浆表达的Notch3蛋白能与蛋白酶体的PSMA1亚基特异性结合；通过蛋白酶体活性分析发现了Notch3可以抑制蛋白酶体功能。提高Notch3蛋白的胞浆表达水平可以显著增强与PSMA1亚基的相互作用并更加显著地抑制蛋白酶体活性。Notch3和PSMA1相互作用代表了一种新型的、由膜蛋白胞浆转位所介导的蛋白酶体活性调节方式。综上所述，该研究主要发现为：(1) Notch3蛋白N端1—39位氨基酸残基(包含多个连续的精氨酸、脯氨酸和亮氨酸)是其表达、加工、细胞内分布以及生物活性所必需的；(2) N端1—14位氨基酸残基(包含七个连续的精氨酸)可抑制Notch3蛋白的胞浆分布，但并不影响其表达、加工以及生物活性；(3)胞浆内Notch3蛋白能与蛋白酶体的PSMA1亚基特异性结合，并显著抑制蛋白酶体的活性。以上研究表明，Notch3蛋白N端保守序列具有多重功能，除了促进蛋白靶向到分泌通路以外，还可以减少蛋白的胞浆分布，从而避免对蛋白酶体的抑制作用。 2 CADASIL相关的错义突变对Notch3蛋白相关生物学特性的影响 2．1 CADASIL相关突变体的构建为了探讨CADASIL的潜在致病机制，我们构建了两个临床存在的CADASIL相关的Notch3突变体C49Y(Notch3第49位氨基酸由C突变为Y)和R90C(Notch3第90位氨基酸由R突变为C)，它们分别代表所在EGFR失去或获得一个半胱氨酸的突变，导致EGFR内半胱氨酸的数量由偶数变成奇数。我们研究了以上突变体对蛋白的表达、加工、细胞内分布以及促细胞生长和抗凋亡活性的影响。为了高度特异且敏感地反映CADASIL相关突变对信号转导能力的影响，我们应用了新型的GV融合蛋白(GAL4结合区域—VP16活化区域)报告系统。在该体系中，WT-Notch3、C49Y和R90C的胞内区分别被GV融合蛋白所取代。当Notch3信号活化后，Notch3-GV经历S3位点切割，释放GV融合肽段进入细胞核，与报告载体(pFR-luciferase)所含有的GAL4序列特异性结合并活化报告基因的表达。酶切及测序结果表明以上突变体均构建成功。 2．2 R90C和C49Y对Notch3蛋白表达水平、加工效率和细胞浆内分布的影响通过IFA比较了WT-Notch3、C49Y和R90C的表达，结果发现三者荧光强度相当；Western blot分析也进一步证实突变蛋白不影响表达水平。继而，我们通过计算蛋白加工后形成的胞内区和未加工全长蛋白的比值评价了蛋白的加工效率，发现突变蛋白与正常蛋白水平相当。最后，我们抽提了WT-Notch3、C49Y和R90C转染细胞的胞浆蛋白，发现突变蛋白也不影响蛋白的胞浆分布。 2．3 R90C和C49Y对Notch3蛋白信号转导的影响我们构建了高特异性的Notch-GV新型报告系统，并在CADASIL的靶细胞(血管平滑肌细胞)上评价了CADASIL相关突变对Notch3信号转导能力的影响。结果表明，C49Y-GV和R90C-GV的信号转导活性均显著低于WT-Notch3-GVP。而且，当WT-Notch3-GVP分别与WT-Notch3、R90C和C49Y共转染入血管平滑肌细胞后，R90C和C49Y能显著抑制Notch3-GV的信号转导活性。该发现为CADASIL致病机制的阐明提供了线索。 2．4 R90C和C49Y对Notch3蛋白促平滑肌细胞生长和抗凋亡活性的影响利用LDH活性检测进行细胞总数分析显示，WT-Notch3可显著促进血管平滑肌细胞A7r5的生长，而R90C和C49Y对细胞生长的促进作用减弱。通过PI染色确定转染WT-Notch3、R90C和C49Y的血管平滑肌细胞A7r5对Fas-L的敏感性。结果表明，WT-Notch3能有效保护细胞抵抗由Fas-L所诱导的细胞凋亡，但R90C和C49Y不能产生明显的保护效应。以上结果表明，CADASIL相关突变体不仅减弱Notch3信号转导能力，而且直接降低了对动脉血管平滑肌细胞的保护作用。综上所述，该研究主要发现为：(1)CADASIL突变体(R90C和C49Y)不影响Notch3蛋白的表达、加工和胞浆分布；(2)R90C和C49Y降低Notch3蛋白信号转导活性，而且当与野生型Notch3同时存在时，突变蛋白可抑制野生蛋白的信号转导活性，发挥显性抑制作用；(3)R90C和C49Y促血管平滑肌细胞生长的活性下降，同时抗凋亡的活性丧失。以上研究为CADASIL致病机制的探索奠定了基础。
[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.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341

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