亨廷顿蛋白出核转运机制和聚集物影响因素的研究
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摘要
亨廷顿舞蹈病(Huntington Disease, HD),一种神经变性疾病,常染色体显性遗传,首先由George Huntington在1872年时报道,其临床特征为三主征：运动异常、认知障碍和精神异常。1993年HD致病基因成功克隆,被命名为IT15基因,正常人该基因的1号外显子内多态性三核苷酸[胞嘧啶-腺嘌呤-鸟嘌呤(CAG)]重复序列的重复数低于27次；当CAG重复次数介于27到35次之间时其本人不发病,但子代的CAG重复数有可能进一步扩增导致发病,尤其是在父系遗传的情况下；介于36到39次时为不完全外显状态,即患者可能有症状也可能完全无症状；当CAG重复数大于40次则为完全外显。到目前为止,还没有任何有效的治疗方法。HD具有家族性、致残性、致死性和终生性的特点,患者多在患病后12到15年间死亡。其病理表现主要是细胞内聚集物形成,该聚集物主要分布于细胞核,但也可见于神经突起内,这可能与多聚谷氨酰胺(polyglutamine, polyQ)之间的相互作用及亨廷顿蛋白(huntingtin, htt)出核困难有关。
近年来关于htt出核转运机制的研究主要在于发现了羧基端的经典的出核信号和氨基端与胞浆定位相关的氨基酸序列(cytoplasmic localization-related domain, CLRD)。htt羧基端存在一富含亮氨酸(leucine, L)的出核信号(nuclear export signal,NES),该序列介导htt通过CRM-1途径出核。同时htt氨基端也存在一个CLRD,其介导htt出核不依赖于经典出核途径。由于htt在513、552或586氨基酸位点被半胱氨酸蛋白酶剪切,而聚集物主要由含polyQ的氨基端构成,所以氨基端的CLRD对于htt聚集物的分布可能具有重要意义。因此我们对这段17个氨基酸构成的CLRD进行细致的研究：通过缺失或突变该序列的个别氨基酸,明确影响出核功能的关键氨基酸位点；研究该定位信号功能缺失对聚集物的影响。
HD发病主要取决于CAG重复数,但有研究显示IT15基因CAG附近的CCG多态和羧基端的A2642多态也与发病相关。为了研究CCG多态与HD发病的相关性,我们对中国大陆地区的HD家系中CCG多态进行分析,并深入研究该多态以及脯氨酸富含区对该蛋白在细胞内的定位和所形成的聚集物的影响。
聚集物成分复杂,目前的研究结果显示其包括p53、mdm-2、HSP70、TFIID、actin、neurofilament、syntaxin 1A和核孔复合物等。对其成分的深入研究将有助于揭示它的形成和作用机制,因此,我们从已知与htt相关的蛋白质入手对聚集物成分进行初步探索。
我们的前期工作包括以下几个方面：
1.收集了两大HD家系,在明确其临床、脑影像学特征的基础上,完成了家系所有成员的IT15基因的突变检测。结果显示,在家系1中18名成员中有9名(其中5名病人,4名症状前患者)CAG重复数大于40次,家系2中24名成员中有5名(其中3名病人,2名症状前患者)CAG重复数大于50次；
2.构建了IT15基因片段pEGFP-C1-19Q(野生型)和pEGFP-C1-70Q (突变型)的真核表达载体,瞬时转染非神经元Hela细胞和神经元SH-SY5Y细胞,成功构建了HD的细胞模型；
3.验证了丁酸钠并不抑制突变htt聚集物的形成,但能保护神经元对抗突变htt的N端片段的毒性作用,突变htt聚集物与神经元死亡并无必然联系；
4.缺失htt前3个氨基酸后N端片段仍具有出核功能,而缺失前5个氨基酸后其出核功能丧失,因此我们推测IT15基因的第4至17个氨基酸参与了htt蛋白N端片段的出核。
第一部分亨廷顿舞蹈病致病蛋白氨基端出核序列的研究及其对聚集物的影响
目的：
明确htt氨基端胞浆定位相关的关键氨基酸位点,确定该定位信号所介导的亚细胞定位,并观察其对舞蹈病病理表征-聚集物的影响。
方法：
1.构建前17个氨基酸、核定位信号(nuclear localization sequence, NLS)与绿色荧光蛋白(green fluorescent protein, GFP)的融合蛋白真核表达载体,在此基础上缺失或突变个别氨基酸,构建一系列表达载体,瞬时转染细胞,观察融合蛋白在细胞核和细胞浆的分布,从而明确CLRD上的关键氨基酸位点；
2.用免疫荧光染色方法对线粒体、内质网和高尔基体等细胞器的标记物进行染色,观察融合蛋白的亚细胞分布；
3.构建野生型(CAG重复20次)和突变型(CAG重复59次)的1号外显子与GFP相融合的真核表达载体,在此基础上定点突变CLRD上的关键氨基酸位点,瞬时转染细胞,通过荧光显微镜下直接观察和western blot的实验方法研究胞浆定位功能的缺失对所形成的聚集物在细胞内的分布及其量的影响。
结果：
1.1-17氨基酸、NLS和GFP的融合蛋白分布在细胞浆,而NLS-GFP分布于细胞核,GFP在胞浆和胞核均匀分布,说明1-17氨基酸具有胞浆定位功能；缺失前3个氨基酸后融合蛋白仍分布于细胞浆,说明其不是该CLRD的关键氨基酸位点；缺失第4、第17个氨基酸后融合蛋白分布于细胞核,说明其对维持该序列的胞浆定位功能具有重要作用；将第4、第7位氨基酸由疏水氨基酸亮氨酸(leucine, L)突变为亲水的精氨酸(arginine, R)后,融合蛋白分布于细胞核,而突变为疏水氨基酸甲硫氨酸(methionine, M)后其仍分布于细胞浆,说明关键位点氨基酸的性质对于胞浆定位具有重要意义;
2.1-17氨基酸、NLS和GFP的融合蛋白与线粒体标记物共定位,而非内质网、高尔基体等其他细胞器；
3.野生型1号外显子与GFP的融合蛋白主要分布于胞浆,而突变第4个氨基酸后该融合蛋白在细胞内呈胞浆胞核均匀分布,进一步验证了第4个氨基酸是介导胞浆定位的关键位点；第4个氨基酸突变导致突变型1号外显子形成的核内聚集物增多,而聚集物总量减少。
结论：
1.在氨基端1-17个氨基酸中前3个氨基酸的缺失对该蛋白的胞浆定位无影响；而缺失第4个或第17个氨基酸或将第4个或第7个氨基酸由L突变为R(由疏水氨基酸变为亲水氨基酸)导致胞浆定位功能缺失,因此我们认为CLRD的关键氨基酸序列为4-17氨基酸；
2.氨基端CLRD介导蛋白定位于线粒体；
3.氨基端CLRD的缺失影响形成聚集物的量和聚集物的分布：该序列功能缺失引起异常增多的CAG重复所致的聚集物总量减少而定位于核内的比例增高。
第二部分对中国大陆地区舞蹈病家系中CCG多态情况的分析及脯氨酸富含区对该蛋白的细胞内定位和聚集物形成的研究
目的：
完善家系收集工作,研究中国大陆地区舞蹈病家系中CCG多态与HD发病的相关性；研究包含该多态的脯氨酸富含区对htt蛋白细胞内定位和聚集物形态和大小的影响。
方法：
1.家系收集工作包括：a.家系成员临床资料的获取;b.外周血采集后用酚氯仿法抽取DNA;c对部分家系内的家系成员(包括患者和症状前患者)进行外周血淋巴细胞建株；
2.测序检测CCG重复序列多态,与临床资料(包括性别比例、发病年龄、病程、家族史、临床症状等)结合后行基因型表型关联分析；
3.构建分别含CCG10和CCG7的突变体(CAG重复46次)与GFP的融合蛋白的真核表达载体,瞬时转染细胞,通过荧光显微镜和western blot技术观察这两种多态对聚集物分布和量的影响；
4.构建一系列含相同CAG重复(20次)但不同CCG重复(5P、7P和9P)的GFP融合蛋白表达载体,瞬时转染细胞,荧光显微镜下观察不同CCG重复对融合蛋白分布的影响；
5.构建一系列含20次和59次CAG重复的、脯氨酸片段被不同程度截短的htt蛋白与GFP融合蛋白表达载体,瞬时转染细胞,通过荧光显微镜和western blot技术观察脯氨酸片段对该蛋白在细胞内的定位和聚集物的影响。
结果：
1.自2002年开始我们共收集舞蹈病家系53个,其中病人60人；外周血淋巴细胞建株8人。上述家系成员的临床资料、DNA模板和所建立的淋巴细胞株均保存完好；
2.经测序证实各真核表达载体均构建成功；
3.在所收集的53个家系中,CCG10和CCG7的分别有29个和24个(分别占了54.72%和45.28%),在56个正常对照(112条染色体)中,CCG10和CCG7分别占46.43%和35.71%,余CCG9、CCG8和CCG6分别占了10.71%、1.79%和5.36%；携带CCG1O和CCG7两种多态的病人在性别比例、发病年龄、病程、家族史、临床症状等方面均无统计学差异。
4.携带CCG7和CCG10多态的突变体形成的聚集物在定位和数目上无显著差异；
5.当CAG重复序列的3’端不加或加入5个CCG重复时,融合蛋白在胞浆和胞核接近均匀分布,而当加入大于7个CCG重复时,融合蛋白分布明显以胞浆为主,提示CCG重复数目影响其蛋白的分布；
6.含20/59个CAG重复的1号外显子与GFP的融合蛋白以胞浆分布为主,缺失羧基端的脯氨酸富含区(含2个脯氨酸片段)后则胞浆胞核弥散分布,而第一个脯氨酸片段(CCG多态存在的区域)的添加能逆转这一趋势。含59个CAG重复的1号外显子与GFP的融合蛋白在核周形成大而单一的聚集物,缺失羧基端的脯氨酸富含区(含2个脯氨酸片段)后则形成多个较小且散在分布的聚集物,而第一个脯氨酸片段(CCG多态存在的区域)的添加仍能使其形成大而单一的聚集物;western blot结果显示缺失脯氨酸富含区后其形成的SDS不溶的聚集物显著减少,而第一个脯氨酸片段的添加能逆转这一趋势。
结论：
1.在中国大陆地区舞蹈病家系CCG重复多态以CCG10和CCG7两种形式为主,分别占54.72%和45.28%；
2.携带CCG10和CCG7两种多态的病人在临床表型上无明显差异,在细胞模型中这两种多态对所形成的聚集物的量和分布无显著影响,提示该多态在舞蹈病发病过程中不具有显著作用；
3.本实验验证了脯氨酸富含区对该蛋白细胞内胞浆分布的作用,在此基础上首次揭示了脯氨酸多态存在区域起了关键作用；而且,精确说明其中起作用的是当脯氨酸个数达到7个时其辅助出核功能完善；
4.和文献报道一致,本实验验证了脯氨酸富含区缺失后聚集物减小、且散在存在,首次提出脯氨酸多态存在区域添加后即已逆转了上述趋势。
第三部分聚集物内在成分的研究
目的：探究聚集物内在成分,为阐明其形成和在HD发病过程中的作用机制提供线索。
方法：
将在第二部分中构建好的突变型1号外显子与GFP融合蛋白的表达载体转染细胞后,采用免疫荧光染色法鉴定聚集物与一些细胞内成分(ubiquitin、vimentin、caspase-3、caspase-9和HSP-70)的关系。
结果：
ubiquitin、vimentin、caspase-3、caspase-9和HSP-70等均与聚集物共定位,表明聚集物内成分复杂,囊括了细胞内的很多成分。
结论：
1)验证了聚集物内在成分包括ubiquitin、vimentin和HSP-70等；
2)首次提出caspase-3和caspase-9也是聚集物的成分。
Huntington's Disease (HD), a neurodegenerative disease with dominant inheritance, was first reported in 1872 by George Huntington. It is characterized by motor symptoms, cognitive decline as well as neuropsychiatric abnormalities. In 1993, the gene for HD was successfully cloned and designated as IT15 gene. In normal individuals, the range of the CAG repeat is below 27; between 27 and 35, the carrier with no symptoms may transmit the disease to their children, especially in the case of paternal inheritance; people who take the CAG repeat in the range of 36 to 39 may present with an incomplete penetrance:when the repeats number is larger than 40, it is fully penetrant. Till now, there is no effective therapy. Once suffered from the disease, the patients may become disabled, or even die during the later period of 12-15 years after onset. Additionally, several family members may have the disease through their lifetime. The pathology is marked by the presence of neuronal intranuclear inclusions, as well as dystrophic neurites inclusions. The interaction between the polyglutamines (polyQ) and possibly the nuclear export deficit of huntingtin (htt) may be the culprit.
With regard to study of mechanism of nuclear export of htt, the classical nuclear export signal (NES) in the carboxy-terminus and the cytoplasmic localization-related domain (CLRD) in the N-terminus of htt were both discovered. A carboxy-terminal conserved NES leucine (L)-rich sequence was detected, and further study suggested that it mediated the nuclear export of htt through a potent CRM-1/exportin export pathway. Subsequently, a CLRD, independent of the classical nuclear export pathway, was also confirmed to be located in the N-terminus of htt. Since full-length htt is cleaved at residue 513,552 or 586, and the aggregation is mainly composed of the N-terminal truncated htt fragment, suggesting that the N-terminal CLRD is more important for its cytoplasmic localization. Thus, we focus on this N-terminal CLRD in the present study. We introduced deletion and substitution to explore the essential sequence that is required for its cytoplasmic localization as well as its effect on aggregates.
Abnormal CAG repeat contributes to the onset of HD, and other genetic polymorphisms in IT15 gene, like the CCG polymorphism in the N-terminus and the△2642 glutamic acid polymorphism in the C-terminus, have also been reported to be modifiers. To study the correlation between the CCG polymorphism and HD, we analyzed the CCG repeats in mainland Chinese HD families and its effect on the aggregate. Subsequently, we further investigated the effects of the proline-rich domain on the sub-cellular distribution of the protein and its aggregate formation.
The composition of aggregate is complex, consisting of p53、mdm-2、HSP70、TFIID、actin、neurofilament、syntaxin 1A and nuclear pore complex as revealed by previous studies. Fully exploration of the aggregates' components may help us understand the formation process and the associated mechanism. Therefore, the co-localization analysis of htt-associated proteins and aggregates were performed preliminarily in the present study as well.
Our previous work includes:
1 We have collected two large HD pedigrees. On the basis of acquisition of their clinical and neuroimaging features, we further finished the IT15 gene mutation analysis in all the remaining family members. The result showed that in pedigree 1, the CAG repeat number of 9 of the 18 family members (5 patients and 4 asymptomatic subjects) reached 40; in pedigree 2, the CAG repeat number of 5 of the 24 family members (3 patients and 2 asymptomatic subjects) was more than 50;
2 We constructed the plasmids carrying the wide-type and mutant-type of truncated htt fragment (pEGFP-Cl-19Q and pEGFP-Cl-70Q), both of which were transfected into non-neuronal Hela cells and neuronal SH-SY5Y cells.
3 We found that sodium butyrate protected SH-SY5Y cells against death induced by mutant htt fragment. However, it did not decrease mutant htt aggregates formation, suggesting there is no essential association between htt aggregates and neuron death.
4 Our previous experiment revealed that the nuclear export function of N-terminal htt fagment was eliminated in the absense of the first 5 amino acids, while still preserved in the deletion of the first 3 amino acids, indicating that Htt4-17 might be involved in the nuclear export process.
Part 1 Essential sequence of the N-terminal CLRD of huntingtin and its effect on aggregates
Objectives:
To confirm the key sequence of the N-terminal CLRD of htt that is required for its cytoplasmic localization; to determine which cellular apparatus the sequence directed to, and its contribution to the pathology.
Methods:
1 Plasmids of Htt1-17-NLS-GFP、Htt4-17-NLS-GFP、Htt5-17-NLS-GFP. Htt4-16-NLS-GFP、Htt(L4M.1-17)-NLS-GFP、Htt(M8L.1-17)-NLS-GFP、Htt(L4R.1-17)-NLS-GFP、Htt(L7R.1-17)-NLS-GFP、HttEx1P(20Q)-GFP、 HttEx1p(L4R,20Q)-GFP、HttEx1P(59Q)-GFP and HttEx1P(L4R,59Q)-GFP were all constructed by recombinant DNA technology. To confirm the key residue that is required for its cytoplasmic localization, we observed the protein's intracellular distribution after transient transfection.
2 Immunofluorescence was used to detect the expression and subcellular distribution of eukaryotic expression plasmids. The mitochondria, ER and Golgi were all stained by their corresponding markers.
3 The effect of this sequence on the aggregate was evaluated by immunofluorescence and immunoblotting analysis after site-specific mutagenesis of both wide-type and mutant-type plasmids.
Results:
1 we found that the fused protein of the first 17 amino acids、NLS and GFP distributed in the cytoplasm, in contrary to either NLS-GFP or GFP, suggesting this sequence has a role of cytoplasmic localization. Deletion of the first 3 amino acids did not affect its distribution, which indicated they were not the key residues. The subcellular distribution changed when it was mutated to include amino acids of different charges (L4R or L7R), or deletion of one more amino acid from either the N-terminus or the C-terminus of Htt4-17, suggesting a structural requirement of the first 17 amino acids for the cytoplasmic localization of htt.
2 The fused protein co-localized with mitochondria, rather than ER or golgi.
3 L4R mutation of truncated htt fragment resulted in altered intracellular distribution and a reduction of total SDS-insoluble htt aggregates and increased nuclear aggregates.
Conclusions:
1 We demonstrated that the essential sequence for htt cytoplasmic localization was Htt4-17, and deletion of the first 3 amino acids did not affect the process.
2 We found that this CLRD co-localized with mitochondria.
3 We prove that the absence of the first 17 amino acids resulted in a reduction of total htt aggregates and increased nuclear aggregates, further supporting its effect on aggregate formation.
Part 2 CCG repeats among mainland Chinese HD families and effects of the CCG polymorphism as well as the proline-rich region on intracellular distribution of the protein and aggregates
Objectives:
To improve the work of genetic family data collection, explore the CCG repeats among mainland Chinese HD families and analyze its correlation with HD; to determine the effect of the proline-rich region on intracellular distribution of the N-terminal htt fragment and its aggregates.
Methods:
1 Genetic family collection work includes:a. acquisition of clinical data of family members; b, phenolic alcohol/chloroform method was carried out to isolate genomic DNA from peripheral blood; c, cultured colonies of peripheral blood lymphocyte from HD family members were conducted.
2 The correlation analysis was conducted between genotype (CCG polymorphisms) and phenotype (the clinical data, including sex ratio, age of onset, disease duration, family history and symptoms).
3 Plasmids of exon 1 of IT15 gene with the same CAG repeat and different CCG repeat were constructed, and effects of the two polymorphisms on the intracellular distribution and quantity of the aggregates were observed after transient trasfection by both immunostaining and immunoblotting analysis.
4 To determine the requirement of the CCG repeat number for htt cytoplasmic distribution, we constructed plasmids with the same CAG repeat number and different CCG repeat number (5P、7P and 9P) and observed their subcellular distributions after transient transfection.
5 Plasmids with the same CAG repeat number and differently-truncated proline-rich fragment were constructed, and immunostaining and immunoblotting analysis were used to detect their effects on its intracellular distribution and aggregate formation.
Results:
1 53 HD families, including 60 patients, were gathered; 8 cultured colonies of peripheral blood lymphocyte from HD family members were successfully constructed. The clinical data, DNA samples as well as lymphocyte colonies were all conserved well.
2 DNA sequencing confirmed the successful construction of all the above plasmids.
3 In our 53 HD families studied,54.72% had 10-repeat alleles, and the left 45.28% had 7-repeat alleles; 56 normal controls (112 unrelated normal chromosomes) were also examined. The percentage of CCG(IO) and CCG(7) is 46.43% and 35.71% respectively, while CCG(9)、CCG(8) and CCG(6) is 10.71%、1.79%and 5.36%. In terms of clinical data, there was no significant difference between these two groups.
4 Two plasmids of Exonl-46Q-10P-GFP and Exonl-46Q-7P-GFP were constructed. Similar intracellular distribution and aggregate quantity were observed after their expression.
5 We also found that the protein presented a cytoplasmic distribution only with 7 CCG repeats, rather than with 5 CCG repeats, indicating that the CCG repeat number affected its intracellular distribution.
6 Compared with Exonl-20Q/59Q-GFP, the distribution of 1-17-20Q/59Q-GFP was more in the nucleus, which could be reversed by adding of the first proline-rich region, further supporting the role of this region in its intracellular localization. Similarly, compared with Exonl-59Q-10P-GFP, aggregates formed by 1-17-59Q-GFP were smaller and more scattered, which could be reversed by adding the first proline-rich region. Western blot shows that the ratio of aggregate to soluble protein is significantly lowest in cells transfected by 1-17-59Q-GFP.
Conclusion:
1 We find that the 7-CCG repeat and the 10-CCG repeat are the most frequent (45.28% and 54.72%, respectively) types of mutant alleles in mainland Chinese HD families;
2 We find that the two polymorphisms did not differ from the clinical information to the cell model, which suggests that this polymorphism is not associated with HD.
3 In addition to confirmation of the role of the proline-rich region in the protein's distribution, our study suggests the key role that the first proline-rich region plays for the first time; we also point out the requirement of 7 CCG repeats for its cytoplasmic distribution.
4 In addition to confirming that the aggregates become smaller and more scattered after deletion of the proline-rich region, we also find that it can be reversed by adding the first proline-rich region
Part 3 Exploration of the component of the aggregate
Objectives:
To explore the component of the aggregate, and to provide some preliminary data for future study of HD.
Methods:
Laser scanning confocal microscope was used to observe the relationship between the aggregate and ubiquitin、vimentin、caspase-3、caspase-9、HSP-70.
Results:
Ubiquitin、vimentin、caspase-3、caspase-9 and HSP-70 were all co-localized with the aggregates, suggesting that the aggregate composition is complex.
Conclusions:
1) We confirmed that ubiquitin、vimentin and HSP-70 were all included in the aggregate.
2) For the first time we point out that caspase-3 and caspase-9 are also the composition of aggregate.
近年来关于htt出核转运机制的研究主要在于发现了羧基端的经典的出核信号和氨基端与胞浆定位相关的氨基酸序列(cytoplasmic localization-related domain, CLRD)。htt羧基端存在一富含亮氨酸(leucine, L)的出核信号(nuclear export signal,NES),该序列介导htt通过CRM-1途径出核。同时htt氨基端也存在一个CLRD,其介导htt出核不依赖于经典出核途径。由于htt在513、552或586氨基酸位点被半胱氨酸蛋白酶剪切,而聚集物主要由含polyQ的氨基端构成,所以氨基端的CLRD对于htt聚集物的分布可能具有重要意义。因此我们对这段17个氨基酸构成的CLRD进行细致的研究：通过缺失或突变该序列的个别氨基酸,明确影响出核功能的关键氨基酸位点；研究该定位信号功能缺失对聚集物的影响。
HD发病主要取决于CAG重复数,但有研究显示IT15基因CAG附近的CCG多态和羧基端的A2642多态也与发病相关。为了研究CCG多态与HD发病的相关性,我们对中国大陆地区的HD家系中CCG多态进行分析,并深入研究该多态以及脯氨酸富含区对该蛋白在细胞内的定位和所形成的聚集物的影响。
聚集物成分复杂,目前的研究结果显示其包括p53、mdm-2、HSP70、TFIID、actin、neurofilament、syntaxin 1A和核孔复合物等。对其成分的深入研究将有助于揭示它的形成和作用机制,因此,我们从已知与htt相关的蛋白质入手对聚集物成分进行初步探索。
我们的前期工作包括以下几个方面：
1.收集了两大HD家系,在明确其临床、脑影像学特征的基础上,完成了家系所有成员的IT15基因的突变检测。结果显示,在家系1中18名成员中有9名(其中5名病人,4名症状前患者)CAG重复数大于40次,家系2中24名成员中有5名(其中3名病人,2名症状前患者)CAG重复数大于50次；
2.构建了IT15基因片段pEGFP-C1-19Q(野生型)和pEGFP-C1-70Q (突变型)的真核表达载体,瞬时转染非神经元Hela细胞和神经元SH-SY5Y细胞,成功构建了HD的细胞模型；
3.验证了丁酸钠并不抑制突变htt聚集物的形成,但能保护神经元对抗突变htt的N端片段的毒性作用,突变htt聚集物与神经元死亡并无必然联系；
4.缺失htt前3个氨基酸后N端片段仍具有出核功能,而缺失前5个氨基酸后其出核功能丧失,因此我们推测IT15基因的第4至17个氨基酸参与了htt蛋白N端片段的出核。
第一部分亨廷顿舞蹈病致病蛋白氨基端出核序列的研究及其对聚集物的影响
目的：
明确htt氨基端胞浆定位相关的关键氨基酸位点,确定该定位信号所介导的亚细胞定位,并观察其对舞蹈病病理表征-聚集物的影响。
方法：
1.构建前17个氨基酸、核定位信号(nuclear localization sequence, NLS)与绿色荧光蛋白(green fluorescent protein, GFP)的融合蛋白真核表达载体,在此基础上缺失或突变个别氨基酸,构建一系列表达载体,瞬时转染细胞,观察融合蛋白在细胞核和细胞浆的分布,从而明确CLRD上的关键氨基酸位点；
2.用免疫荧光染色方法对线粒体、内质网和高尔基体等细胞器的标记物进行染色,观察融合蛋白的亚细胞分布；
3.构建野生型(CAG重复20次)和突变型(CAG重复59次)的1号外显子与GFP相融合的真核表达载体,在此基础上定点突变CLRD上的关键氨基酸位点,瞬时转染细胞,通过荧光显微镜下直接观察和western blot的实验方法研究胞浆定位功能的缺失对所形成的聚集物在细胞内的分布及其量的影响。
结果：
1.1-17氨基酸、NLS和GFP的融合蛋白分布在细胞浆,而NLS-GFP分布于细胞核,GFP在胞浆和胞核均匀分布,说明1-17氨基酸具有胞浆定位功能；缺失前3个氨基酸后融合蛋白仍分布于细胞浆,说明其不是该CLRD的关键氨基酸位点；缺失第4、第17个氨基酸后融合蛋白分布于细胞核,说明其对维持该序列的胞浆定位功能具有重要作用；将第4、第7位氨基酸由疏水氨基酸亮氨酸(leucine, L)突变为亲水的精氨酸(arginine, R)后,融合蛋白分布于细胞核,而突变为疏水氨基酸甲硫氨酸(methionine, M)后其仍分布于细胞浆,说明关键位点氨基酸的性质对于胞浆定位具有重要意义;
2.1-17氨基酸、NLS和GFP的融合蛋白与线粒体标记物共定位,而非内质网、高尔基体等其他细胞器；
3.野生型1号外显子与GFP的融合蛋白主要分布于胞浆,而突变第4个氨基酸后该融合蛋白在细胞内呈胞浆胞核均匀分布,进一步验证了第4个氨基酸是介导胞浆定位的关键位点；第4个氨基酸突变导致突变型1号外显子形成的核内聚集物增多,而聚集物总量减少。
结论：
1.在氨基端1-17个氨基酸中前3个氨基酸的缺失对该蛋白的胞浆定位无影响；而缺失第4个或第17个氨基酸或将第4个或第7个氨基酸由L突变为R(由疏水氨基酸变为亲水氨基酸)导致胞浆定位功能缺失,因此我们认为CLRD的关键氨基酸序列为4-17氨基酸；
2.氨基端CLRD介导蛋白定位于线粒体；
3.氨基端CLRD的缺失影响形成聚集物的量和聚集物的分布：该序列功能缺失引起异常增多的CAG重复所致的聚集物总量减少而定位于核内的比例增高。
第二部分对中国大陆地区舞蹈病家系中CCG多态情况的分析及脯氨酸富含区对该蛋白的细胞内定位和聚集物形成的研究
目的：
完善家系收集工作,研究中国大陆地区舞蹈病家系中CCG多态与HD发病的相关性；研究包含该多态的脯氨酸富含区对htt蛋白细胞内定位和聚集物形态和大小的影响。
方法：
1.家系收集工作包括：a.家系成员临床资料的获取;b.外周血采集后用酚氯仿法抽取DNA;c对部分家系内的家系成员(包括患者和症状前患者)进行外周血淋巴细胞建株；
2.测序检测CCG重复序列多态,与临床资料(包括性别比例、发病年龄、病程、家族史、临床症状等)结合后行基因型表型关联分析；
3.构建分别含CCG10和CCG7的突变体(CAG重复46次)与GFP的融合蛋白的真核表达载体,瞬时转染细胞,通过荧光显微镜和western blot技术观察这两种多态对聚集物分布和量的影响；
4.构建一系列含相同CAG重复(20次)但不同CCG重复(5P、7P和9P)的GFP融合蛋白表达载体,瞬时转染细胞,荧光显微镜下观察不同CCG重复对融合蛋白分布的影响；
5.构建一系列含20次和59次CAG重复的、脯氨酸片段被不同程度截短的htt蛋白与GFP融合蛋白表达载体,瞬时转染细胞,通过荧光显微镜和western blot技术观察脯氨酸片段对该蛋白在细胞内的定位和聚集物的影响。
结果：
1.自2002年开始我们共收集舞蹈病家系53个,其中病人60人；外周血淋巴细胞建株8人。上述家系成员的临床资料、DNA模板和所建立的淋巴细胞株均保存完好；
2.经测序证实各真核表达载体均构建成功；
3.在所收集的53个家系中,CCG10和CCG7的分别有29个和24个(分别占了54.72%和45.28%),在56个正常对照(112条染色体)中,CCG10和CCG7分别占46.43%和35.71%,余CCG9、CCG8和CCG6分别占了10.71%、1.79%和5.36%；携带CCG1O和CCG7两种多态的病人在性别比例、发病年龄、病程、家族史、临床症状等方面均无统计学差异。
4.携带CCG7和CCG10多态的突变体形成的聚集物在定位和数目上无显著差异；
5.当CAG重复序列的3’端不加或加入5个CCG重复时,融合蛋白在胞浆和胞核接近均匀分布,而当加入大于7个CCG重复时,融合蛋白分布明显以胞浆为主,提示CCG重复数目影响其蛋白的分布；
6.含20/59个CAG重复的1号外显子与GFP的融合蛋白以胞浆分布为主,缺失羧基端的脯氨酸富含区(含2个脯氨酸片段)后则胞浆胞核弥散分布,而第一个脯氨酸片段(CCG多态存在的区域)的添加能逆转这一趋势。含59个CAG重复的1号外显子与GFP的融合蛋白在核周形成大而单一的聚集物,缺失羧基端的脯氨酸富含区(含2个脯氨酸片段)后则形成多个较小且散在分布的聚集物,而第一个脯氨酸片段(CCG多态存在的区域)的添加仍能使其形成大而单一的聚集物;western blot结果显示缺失脯氨酸富含区后其形成的SDS不溶的聚集物显著减少,而第一个脯氨酸片段的添加能逆转这一趋势。
结论：
1.在中国大陆地区舞蹈病家系CCG重复多态以CCG10和CCG7两种形式为主,分别占54.72%和45.28%；
2.携带CCG10和CCG7两种多态的病人在临床表型上无明显差异,在细胞模型中这两种多态对所形成的聚集物的量和分布无显著影响,提示该多态在舞蹈病发病过程中不具有显著作用；
3.本实验验证了脯氨酸富含区对该蛋白细胞内胞浆分布的作用,在此基础上首次揭示了脯氨酸多态存在区域起了关键作用；而且,精确说明其中起作用的是当脯氨酸个数达到7个时其辅助出核功能完善；
4.和文献报道一致,本实验验证了脯氨酸富含区缺失后聚集物减小、且散在存在,首次提出脯氨酸多态存在区域添加后即已逆转了上述趋势。
第三部分聚集物内在成分的研究
目的：探究聚集物内在成分,为阐明其形成和在HD发病过程中的作用机制提供线索。
方法：
将在第二部分中构建好的突变型1号外显子与GFP融合蛋白的表达载体转染细胞后,采用免疫荧光染色法鉴定聚集物与一些细胞内成分(ubiquitin、vimentin、caspase-3、caspase-9和HSP-70)的关系。
结果：
ubiquitin、vimentin、caspase-3、caspase-9和HSP-70等均与聚集物共定位,表明聚集物内成分复杂,囊括了细胞内的很多成分。
结论：
1)验证了聚集物内在成分包括ubiquitin、vimentin和HSP-70等；
2)首次提出caspase-3和caspase-9也是聚集物的成分。
Huntington's Disease (HD), a neurodegenerative disease with dominant inheritance, was first reported in 1872 by George Huntington. It is characterized by motor symptoms, cognitive decline as well as neuropsychiatric abnormalities. In 1993, the gene for HD was successfully cloned and designated as IT15 gene. In normal individuals, the range of the CAG repeat is below 27; between 27 and 35, the carrier with no symptoms may transmit the disease to their children, especially in the case of paternal inheritance; people who take the CAG repeat in the range of 36 to 39 may present with an incomplete penetrance:when the repeats number is larger than 40, it is fully penetrant. Till now, there is no effective therapy. Once suffered from the disease, the patients may become disabled, or even die during the later period of 12-15 years after onset. Additionally, several family members may have the disease through their lifetime. The pathology is marked by the presence of neuronal intranuclear inclusions, as well as dystrophic neurites inclusions. The interaction between the polyglutamines (polyQ) and possibly the nuclear export deficit of huntingtin (htt) may be the culprit.
With regard to study of mechanism of nuclear export of htt, the classical nuclear export signal (NES) in the carboxy-terminus and the cytoplasmic localization-related domain (CLRD) in the N-terminus of htt were both discovered. A carboxy-terminal conserved NES leucine (L)-rich sequence was detected, and further study suggested that it mediated the nuclear export of htt through a potent CRM-1/exportin export pathway. Subsequently, a CLRD, independent of the classical nuclear export pathway, was also confirmed to be located in the N-terminus of htt. Since full-length htt is cleaved at residue 513,552 or 586, and the aggregation is mainly composed of the N-terminal truncated htt fragment, suggesting that the N-terminal CLRD is more important for its cytoplasmic localization. Thus, we focus on this N-terminal CLRD in the present study. We introduced deletion and substitution to explore the essential sequence that is required for its cytoplasmic localization as well as its effect on aggregates.
Abnormal CAG repeat contributes to the onset of HD, and other genetic polymorphisms in IT15 gene, like the CCG polymorphism in the N-terminus and the△2642 glutamic acid polymorphism in the C-terminus, have also been reported to be modifiers. To study the correlation between the CCG polymorphism and HD, we analyzed the CCG repeats in mainland Chinese HD families and its effect on the aggregate. Subsequently, we further investigated the effects of the proline-rich domain on the sub-cellular distribution of the protein and its aggregate formation.
The composition of aggregate is complex, consisting of p53、mdm-2、HSP70、TFIID、actin、neurofilament、syntaxin 1A and nuclear pore complex as revealed by previous studies. Fully exploration of the aggregates' components may help us understand the formation process and the associated mechanism. Therefore, the co-localization analysis of htt-associated proteins and aggregates were performed preliminarily in the present study as well.
Our previous work includes:
1 We have collected two large HD pedigrees. On the basis of acquisition of their clinical and neuroimaging features, we further finished the IT15 gene mutation analysis in all the remaining family members. The result showed that in pedigree 1, the CAG repeat number of 9 of the 18 family members (5 patients and 4 asymptomatic subjects) reached 40; in pedigree 2, the CAG repeat number of 5 of the 24 family members (3 patients and 2 asymptomatic subjects) was more than 50;
2 We constructed the plasmids carrying the wide-type and mutant-type of truncated htt fragment (pEGFP-Cl-19Q and pEGFP-Cl-70Q), both of which were transfected into non-neuronal Hela cells and neuronal SH-SY5Y cells.
3 We found that sodium butyrate protected SH-SY5Y cells against death induced by mutant htt fragment. However, it did not decrease mutant htt aggregates formation, suggesting there is no essential association between htt aggregates and neuron death.
4 Our previous experiment revealed that the nuclear export function of N-terminal htt fagment was eliminated in the absense of the first 5 amino acids, while still preserved in the deletion of the first 3 amino acids, indicating that Htt4-17 might be involved in the nuclear export process.
Part 1 Essential sequence of the N-terminal CLRD of huntingtin and its effect on aggregates
Objectives:
To confirm the key sequence of the N-terminal CLRD of htt that is required for its cytoplasmic localization; to determine which cellular apparatus the sequence directed to, and its contribution to the pathology.
Methods:
1 Plasmids of Htt1-17-NLS-GFP、Htt4-17-NLS-GFP、Htt5-17-NLS-GFP. Htt4-16-NLS-GFP、Htt(L4M.1-17)-NLS-GFP、Htt(M8L.1-17)-NLS-GFP、Htt(L4R.1-17)-NLS-GFP、Htt(L7R.1-17)-NLS-GFP、HttEx1P(20Q)-GFP、 HttEx1p(L4R,20Q)-GFP、HttEx1P(59Q)-GFP and HttEx1P(L4R,59Q)-GFP were all constructed by recombinant DNA technology. To confirm the key residue that is required for its cytoplasmic localization, we observed the protein's intracellular distribution after transient transfection.
2 Immunofluorescence was used to detect the expression and subcellular distribution of eukaryotic expression plasmids. The mitochondria, ER and Golgi were all stained by their corresponding markers.
3 The effect of this sequence on the aggregate was evaluated by immunofluorescence and immunoblotting analysis after site-specific mutagenesis of both wide-type and mutant-type plasmids.
Results:
1 we found that the fused protein of the first 17 amino acids、NLS and GFP distributed in the cytoplasm, in contrary to either NLS-GFP or GFP, suggesting this sequence has a role of cytoplasmic localization. Deletion of the first 3 amino acids did not affect its distribution, which indicated they were not the key residues. The subcellular distribution changed when it was mutated to include amino acids of different charges (L4R or L7R), or deletion of one more amino acid from either the N-terminus or the C-terminus of Htt4-17, suggesting a structural requirement of the first 17 amino acids for the cytoplasmic localization of htt.
2 The fused protein co-localized with mitochondria, rather than ER or golgi.
3 L4R mutation of truncated htt fragment resulted in altered intracellular distribution and a reduction of total SDS-insoluble htt aggregates and increased nuclear aggregates.
Conclusions:
1 We demonstrated that the essential sequence for htt cytoplasmic localization was Htt4-17, and deletion of the first 3 amino acids did not affect the process.
2 We found that this CLRD co-localized with mitochondria.
3 We prove that the absence of the first 17 amino acids resulted in a reduction of total htt aggregates and increased nuclear aggregates, further supporting its effect on aggregate formation.
Part 2 CCG repeats among mainland Chinese HD families and effects of the CCG polymorphism as well as the proline-rich region on intracellular distribution of the protein and aggregates
Objectives:
To improve the work of genetic family data collection, explore the CCG repeats among mainland Chinese HD families and analyze its correlation with HD; to determine the effect of the proline-rich region on intracellular distribution of the N-terminal htt fragment and its aggregates.
Methods:
1 Genetic family collection work includes:a. acquisition of clinical data of family members; b, phenolic alcohol/chloroform method was carried out to isolate genomic DNA from peripheral blood; c, cultured colonies of peripheral blood lymphocyte from HD family members were conducted.
2 The correlation analysis was conducted between genotype (CCG polymorphisms) and phenotype (the clinical data, including sex ratio, age of onset, disease duration, family history and symptoms).
3 Plasmids of exon 1 of IT15 gene with the same CAG repeat and different CCG repeat were constructed, and effects of the two polymorphisms on the intracellular distribution and quantity of the aggregates were observed after transient trasfection by both immunostaining and immunoblotting analysis.
4 To determine the requirement of the CCG repeat number for htt cytoplasmic distribution, we constructed plasmids with the same CAG repeat number and different CCG repeat number (5P、7P and 9P) and observed their subcellular distributions after transient transfection.
5 Plasmids with the same CAG repeat number and differently-truncated proline-rich fragment were constructed, and immunostaining and immunoblotting analysis were used to detect their effects on its intracellular distribution and aggregate formation.
Results:
1 53 HD families, including 60 patients, were gathered; 8 cultured colonies of peripheral blood lymphocyte from HD family members were successfully constructed. The clinical data, DNA samples as well as lymphocyte colonies were all conserved well.
2 DNA sequencing confirmed the successful construction of all the above plasmids.
3 In our 53 HD families studied,54.72% had 10-repeat alleles, and the left 45.28% had 7-repeat alleles; 56 normal controls (112 unrelated normal chromosomes) were also examined. The percentage of CCG(IO) and CCG(7) is 46.43% and 35.71% respectively, while CCG(9)、CCG(8) and CCG(6) is 10.71%、1.79%and 5.36%. In terms of clinical data, there was no significant difference between these two groups.
4 Two plasmids of Exonl-46Q-10P-GFP and Exonl-46Q-7P-GFP were constructed. Similar intracellular distribution and aggregate quantity were observed after their expression.
5 We also found that the protein presented a cytoplasmic distribution only with 7 CCG repeats, rather than with 5 CCG repeats, indicating that the CCG repeat number affected its intracellular distribution.
6 Compared with Exonl-20Q/59Q-GFP, the distribution of 1-17-20Q/59Q-GFP was more in the nucleus, which could be reversed by adding of the first proline-rich region, further supporting the role of this region in its intracellular localization. Similarly, compared with Exonl-59Q-10P-GFP, aggregates formed by 1-17-59Q-GFP were smaller and more scattered, which could be reversed by adding the first proline-rich region. Western blot shows that the ratio of aggregate to soluble protein is significantly lowest in cells transfected by 1-17-59Q-GFP.
Conclusion:
1 We find that the 7-CCG repeat and the 10-CCG repeat are the most frequent (45.28% and 54.72%, respectively) types of mutant alleles in mainland Chinese HD families;
2 We find that the two polymorphisms did not differ from the clinical information to the cell model, which suggests that this polymorphism is not associated with HD.
3 In addition to confirmation of the role of the proline-rich region in the protein's distribution, our study suggests the key role that the first proline-rich region plays for the first time; we also point out the requirement of 7 CCG repeats for its cytoplasmic distribution.
4 In addition to confirming that the aggregates become smaller and more scattered after deletion of the proline-rich region, we also find that it can be reversed by adding the first proline-rich region
Part 3 Exploration of the component of the aggregate
Objectives:
To explore the component of the aggregate, and to provide some preliminary data for future study of HD.
Methods:
Laser scanning confocal microscope was used to observe the relationship between the aggregate and ubiquitin、vimentin、caspase-3、caspase-9、HSP-70.
Results:
Ubiquitin、vimentin、caspase-3、caspase-9 and HSP-70 were all co-localized with the aggregates, suggesting that the aggregate composition is complex.
Conclusions:
1) We confirmed that ubiquitin、vimentin and HSP-70 were all included in the aggregate.
2) For the first time we point out that caspase-3 and caspase-9 are also the composition of aggregate.
引文
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