弓形虫棒状体蛋白ROP16的致病机制及TFPI-2与PSAP相互作用研究

发布时间：2018-04-19 09:24

本文选题：弓形虫 + 蛋白质相互作用　；参考：《复旦大学》2012年硕士论文

【摘要】：第一部分弓形虫棒状体蛋白16的致病机制研究刚地弓形虫是一种专性细胞内寄生原虫,可感染几乎所有的恒温动物。近年来,随着城市的发展、人口的增加,宠物饲养队伍不断地扩大,加上忽略饮食卫生等因素,弓形虫感染潜在危险性极度上升。孕妇感染弓形虫后,通常能将这种感染传给胎儿,如果感染发生在胚胎发育早期可导致智障、眼和脑疾病、其它多脏器疾病等严重后果,影响胎儿的发育,导致流产、畸胎、死胎、早产、出生缺陷等先天性弓形虫病。调查显示,我国先天性出生缺陷患儿当中弓形虫的感染率达到22%,弱智和精神病患者的发病与弓形虫感染相关。弓形虫入侵人体细胞过程,棒状体蛋白发挥了极其重要的作用。迄今仅发现两种弓形虫棒状体蛋白可侵入人体细胞核内,分别是棒状体蛋白16(ROP16)和PP2C-Hn。ROP16可快速侵入人体细胞核,业已证明ROP16可影响人体细胞的信号转导和转录激活因子(STAT)信号传导途径从而干扰人体细胞的增殖、分化、凋亡等功能。但该蛋白入核后发挥的具体功能并不知晓。我们的假设是：1、既然ROP16可以进入人体细胞核,对于异源性蛋白入侵,就可影响人体细胞的生理功能；2、既然弓形虫对脑细胞有倾向性,那么侵入脑细胞的弓形虫就可能影响这些细胞的增殖、分化、凋亡等生理功能,同样ROP16入核后,可影响细胞核功能。3、既然弓形虫感染胚胎脑细胞,那么就有可能影响早期大脑的发育。因此我们旨在从ROP16功能的基础研究着手,探索它与人体相互作用的核蛋白,获取下游靶基因的信息从中寻找与生长发育相关的因子,以及ROP16与相互作用的蛋白结合后对其功能的影响,揭示弓形虫脑病的形成以及弓形虫感染导致出生缺陷和智障的机制。本研究工作主要集中于ROP16与人体相互作用核蛋白的筛选和鉴定及蛋白相互作用之后对功能的影响,主要内容及结果如下： (1)成功构建表达ROP16混合克隆细胞株：采用国际公认的弓形虫强毒株RH株感染C57小鼠,待其出现明显的弓形虫感染症状后从中抽取腹水提RNA,RT-PCR扩增目的基因ROP16,构建真核表达载体PEXPR-IBA-105IP-ROP16,重组载体经lip2000瞬时转染入293T细胞,并用嘌呤霉素进行筛选得到混合克隆细胞株,Western-Blot检测目的蛋白成功表达。 (2)筛选出与ROP16相互作用的人体核蛋白：培养稳定表达ROP16的293T细胞,以免疫沉淀技术获得与ROP16存在相互作用的人体核蛋白,质谱分析后得到7个可信的核蛋白,可能与ROP16存在相互作用。 (3)以免疫共沉淀技术分别证实ROP16与7个候选核蛋白中的5个(Histone H2B, Histone H4, lamin B1,hnRNP A2/B1,STMN1)之间的相互作用,证明lamin B1(LMNB1)和histone H2B (HIST1H2B)与ROP16存在相互作用。 (4)与ROP16相互作用蛋白LMNB1及HIST1H2B进一步证实和定位：以免疫共沉淀双向验证ROP16与LMNB1和HIST1H2B之间相互结合,经激光共聚焦证实ROP16与LMNB1共定位于细胞核膜,ROP16与HIST1H2B共定位于细胞核。 (5)ROP16对神经细胞生长、分化的影响：将ROP16基因cDNA克隆入慢病毒载体PWPI,用293T细胞包装病毒颗粒,将构建好的慢病毒感染人脑星型胶质母细胞瘤细胞(u-87MG)(?)口神经母细胞瘤细胞(SH-SY5Y),real-time PCR方法检测得到ROP16下调星型胶质细胞和神经元标志基因。 (6)ROP16对神经细胞凋亡影响：TUNEL和流式细胞术检测ROP16对u-87MG和SH-SY5Y细胞凋亡的影响,证明ROP16促进二者的凋亡,CCK-8实验证实ROP16对二者增殖无显著影响。 (7)ROP16与LMNB1相互作用后对其功能的影响：以全反式维甲酸(RA)诱导SH-SY5Y细胞向神经元分化,电镜检测显示少量神经元细胞核仁增多、核膜空泡化、大量的微管成簇排列。结论：ROP16分别与LMNB1和HIST1H2B存在相互作用。ROP16抑制u-87MG细胞的生长和SH-SY5Y细胞向神经元的分化。ROP16促进u-87MG和SH-SY5Y细胞的凋亡。ROP16与LMNB1相互作用后可能影响LMNB1维持核膜形态等功能,使神经元细胞形态发生异常。第二部分TFPI-2与PSAP相互作用的研究组织因子途径抑制物-2(tissue factor pathway inhibitor-2, TFPI-2)又称胎盘蛋白—5(placenta protein-5, PP-5)和基质相关丝氨酸蛋白酶抑制剂(matrix associated serine protease inhibitor, MSPI),是一种带有Kunitz型结构域的蛋白酶抑制剂,属于丝氨酸蛋白酶抑制物超家族成员。成熟的人TFPI-2由富含酸性氨基酸的N端、三个串联的Kuntiz (?)吉构域和富含碱性氨基酸的C端组成。众所周知,人TFPI-2能够抑制胰蛋白酶、纤溶酶、糜蛋白酶、血浆缓激肽释放酶、组织蛋白酶G和多种基质金属蛋白酶(MMPs)的活性,从而对抗多种恶性肿瘤的侵袭和转移,如前列腺癌、肺癌、纤维肉瘤、黑色素瘤和神经胶质瘤等。此外,人TFPI-2还可以抑制动脉粥样硬化斑块的脱落。但迄今为止,对TFPI-2的生理功能尚不清楚,TFPI-2与其它蛋白质相互作用的信息也知之甚少。在前期研究中,我们从酵母双杂交技术筛选出与TFPI-2相互作用的候选蛋白,其中之一为鞘脂激活蛋白原rosaposin (PSAP)。PSAP是一个具有多种神经营养作用的多功能糖蛋白,最初被认为是4种鞘脂激活蛋白A、B、C和、D的前体。鞘脂激活蛋白A、B、C和、D功能是作为鞘脂糖水解途径中所涉及到溶酶体酶的激活剂。成熟的鞘脂激活蛋白由PSAP通过蛋白水解途径产生,再运送至溶酶体。作为完整的分泌性蛋白,PSAP存在于多种生物体液中,如人乳汁、血清、脑脊液和精液浆等,也常见于神经及其它质膜中。本项研究是对前期工作的进一步延续和拓宽,主要集中于TFPI-2与PSAP内源性相互作用的进一步确认、及二者蛋白相互作用后对各自功能的影响。通过酵母共转化、GST-pulldown等实验TFPI-2和PSAP在酵母及细胞内外的相互作用；在通过细胞侵袭和迁移实验验证二者相互作用后在体外功能的影响,进一步阐明TFPI-2的生理功能。主要内容及结果如下： (1)确定了TFPI-2和PSAP内源性相互作用。 (2)证明PSAP可以促进人纤维肉瘤细胞的侵袭及迁移能力、TFPI-2可以抑制PSAP作用；PSAP可以增强HT1080细胞分泌的MMP-2的活性并呈剂量依赖性；同样,TFPI-2也可以抑制PSAP。 (3) PSAP对MMP-2和MMP-9的表达量没有影响。结论：TFPI-2与PSAP存在内源性相互作用,并对其促进纤维肉瘤侵袭和迁移功能具有显著的抑制作用。
[Abstract]:Study on the pathogenesis of the first part of the Toxoplasma gondii rod protein 16
Toxoplasma gondii is an obligate intracellular protozoan parasite can infect almost all warm blooded animal. In recent years, with the development of the city, the population increase, pets team continues to expand, and ignore the health diet and other factors, the potential risk of Toxoplasma infection was increased. Toxoplasma infection in pregnant women. Usually can be the infection to the fetus, if the infection occurs in the early embryo development can lead to mental retardation, eye and other brain diseases, multiple organ diseases and other serious consequences, affect fetal development, leading to miscarriage, stillbirth, premature birth, fetus, birth defects and congenital toxoplasmosis. The survey shows that China's congenital birth defects in children among the Toxoplasma infection rate reached 22%, the incidence of retarded and psychotic patients infected with Toxoplasma gondii.
The invasion of Toxoplasma gondii rhoptry protein in human cells, play a very important role. So far only found two kinds of Toxoplasma gondii rhoptry protein can invade the human body and the nucleus are rhoptry protein 16 (ROP16) and PP2C-Hn.ROP16 can quickly invade the human nucleus, proving that ROP16 can activate human cell signal transduction and transcription factor (STAT) signal transduction pathway which interfere with human cell proliferation, differentiation, apoptosis and other functions. But the specific function of protein into the nucleus after the play is not known.
Our hypothesis is: 1, since ROP16 can enter the cell nucleus of the human body, for heterologous protein invasion, can affect the physiological function of human cells; since 2, Toxoplasma on brain cells have the inclination, then invade the brain cells of Toxoplasma gondii may influence the cell proliferation, differentiation, apoptosis and other physiological functions, the same ROP16 into the nucleus, nucleus can affect the function of.3, the embryo brain cells of Toxoplasma infection, so it may affect early brain development. Therefore we aimed to research from the ROP16 function to explore the interaction between human and nuclear protein, obtaining information from the downstream target genes for growth and development of the and the ROP16 factor, and the interaction of protein binding on the function, reveal the formation of Toxoplasma encephalopathy and Toxoplasma infection mechanisms leading to birth defects and mental retardation.
This work focuses on the screening and identification of nucleoprotein interacting with human body and the effect of protein interaction on function after ROP16. The main contents and results are as follows:
(1) the successful construction of the expression of ROP16 hybrid cell clone: adopt internationally recognized Toxoplasma virulent strain RH. C57 mice were infected, the apparent Toxoplasma infection symptoms after extracted from ascites RNA, RT-PCR amplification of ROP16 gene and construct the eukaryotic expression vector PEXPR-IBA-105IP-ROP16, the recombinant vector was transiently transfected into 293T by lip2000 cells, and by puromycin screening hybrid cell clone, Western-Blot to detect protein expressed successfully.
(2) to screen human nucleoprotein interacting with ROP16: to cultivate 293T cells that stably express ROP16, to obtain human nucleoprotein interacting with ROP16 by immunoprecipitation technology, and to obtain 7 credible nucleoproteins by mass spectrometry, which may interact with ROP16.
(3) the interaction between ROP16 and 5 candidate nuclear proteins (Histone H2B, Histone H4, lamin B1, hnRNP A2/B1, STMN1) was confirmed by CO immunoprecipitation technology. It was proved that the interaction between lamin and H2B was existed.
(4) the interaction proteins LMNB1 and HIST1H2B with ROP16 were further confirmed and located: the combination of ROP16 and LMNB1 and HIST1H2B was confirmed by CO immunoprecipitation. Confocal laser scanning showed that ROP16 and LMNB1 were located in the cell nuclear membrane, and ROP16 and HIST1H2B were located in the nucleus.
(5) ROP16 on the growth of nerve cells, differentiation effect: ROP16 cDNA gene was cloned into the lentiviral vector PWPI with 293T cells to package virus particles, the constructed lentivirus infected human glioblastoma cells (u-87MG) (?) in neuroblastoma cells (SH-SY5Y), the detection of real-time method of PCR ROP16 down-regulation of astrocyte and neuron marker genes.
(6) the effect of ROP16 on neuronal apoptosis: TUNEL and flow cytometry were used to detect the effect of ROP16 on the apoptosis of u-87MG and SH-SY5Y cells. It was proved that ROP16 promoted the apoptosis of the two. CCK-8 test confirmed that ROP16 had no significant effect on the proliferation of the two cells.
(7) the interaction of ROP16 and LMNB1 on its function: the induction of SH-SY5Y cells into neurons by all trans retinoic acid (RA). Electron microscopy showed that a small number of neurons increased nucleolus, vacuolated the nuclear membrane, and a large number of microtubules were arranged in clusters.
Conclusion: ROP16 interaction between.ROP16 inhibition promotes neuronal apoptosis of.ROP16.ROP16 and LMNB1 u-87MG and SH-SY5Y cell interactions may influence the function of LMNB1 to maintain the nuclear morphology of cell growth and SH-SY5Y of u-87MG cells with LMNB1 and HIST1H2B, the neural cell morphology abnormalities.
The study of the interaction between second parts TFPI-2 and PSAP
Tissue factor pathway inhibitor -2 (tissue factor pathway inhibitor-2, TFPI-2) and placental protein 5 (placenta, protein-5, PP-5) and matrix associated serine protease inhibitor (matrix associated serine protease inhibitor, MSPI), is a Kunitz type domain with protease inhibitors belonging to the serine protease inhibitor superfamily. The mature man TFPI-2 is rich in acidic amino acids N terminal, three tandem Kuntiz (?) and Kyrgyzstan domains rich in basic amino acids C terminal. As everyone knows, TFPI-2 can inhibit plasmin, trypsin, chymotrypsin, plasma kallikrein, cathepsin G and a variety of matrix metalloproteinase (MMPs) activity against a variety of malignant in order to tumor invasion and metastasis, such as prostate cancer, lung cancer, fibrosarcoma, melanoma and glioma. In addition, people can also inhibit TFPI-2 The loss of atherosclerotic plaque. But to date, the physiological function of TFPI-2 is not clear, and the information of the interaction between TFPI-2 and other proteins is little known.
In our previous study, we screened candidate proteins interacting with TFPI-2 by yeast two hybrid technique, which is one of the prosaposin rosaposin (PSAP).PSAP is a variety of neurotrophic effect of multifunctional glycoprotein, was initially thought to be 4 kinds of sphingolipid activated protein A, B, and C, before the body of the D. The sphingolipid activator protein A, B, C and D, function as sphingolipid hydrolysis pathways involved in the activation of lysosomal enzymes. The mature protein produced by sphingolipid activator PSAP through proteolytic pathway, and then transported to the lysosomes. As a secretory protein complete, PSAP exists in many kinds of biological fluids such as, human milk, serum, cerebrospinal fluid and seminal plasma, are also common in other nerve and plasma membrane.
This research is a preliminary work on the further extension and widening, mainly concentrated in the TFPI-2 and PSAP further confirmed the endogenous interaction, and effects of the two protein interaction on their functions. Through yeast CO transformation, the interaction between GST-pulldown and PSAP in the TFPI-2 and yeast cells and in the cell invasion and; the migration experiments in vitro effects of functional interaction between the two, to further clarify the physiological function of TFPI-2. The main contents and results are as follows:
(1) endogenous interaction between TFPI-2 and PSAP was determined.
(2) it is proved that PSAP can promote the invasion and migration of human fibrosarcoma cells. TFPI-2 can inhibit the effect of PSAP. PSAP can enhance the activity of MMP-2 secreted by HT1080 cells in a dose-dependent manner. Similarly, TFPI-2 can also inhibit PSAP..
(3) PSAP has no effect on the expression of MMP-2 and MMP-9.
Conclusion: there is an endogenous interaction between TFPI-2 and PSAP, and it has a significant inhibitory effect on promoting the invasion and migration of fibrosarcoma.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R382.5

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