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长牡蛎Caspase家族蛋白调控细胞凋亡的机制研究
徐家超
学位类型硕士
导师宋林生
2016-05-13
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词长牡蛎 细胞凋亡 Cgcaspase-3 Cgcaspase-8 Lps 免疫应答
摘要细胞凋亡是细胞程序性死亡的一种,它主要由半胱氨酸蛋白酶介导执行,这类半胱氨酸蛋白酶被称为Caspase。细胞凋亡在细胞内稳态的维持,组织与器官发育以及免疫防御等方面都发挥着重要作用。目前在高等生物中,Caspase家族蛋白及其介导的凋亡调控通路已研究得较为透彻,而对无脊椎动物,尤其是软体动物的研究还十分有限。本研究以长牡蛎(Crassostrea gigas)为研究对象,利用分子生物学、细胞生物学以及生物信息学等相关技术手段,对长牡蛎Caspase-3(命名为CgCaspase-3)、Caspase-8(命名为CgCaspase-8)的功能及其介导的凋亡调控机制进行了初步的研究。
CgCaspase-3基因的开放阅读框(ORF)为924 bp,编码307个氨基酸,CgCaspase-8基因ORF全长为1455bp,编码484个氨基酸。CgCaspase-3和CgCaspase-8分子皆为酶原形式,包含典型的CASc结构域及酶活性位点。CgCaspase-3和CgCaspase-8 mRNA在各组织中皆呈组成性表达,且都在外套膜及血淋巴细胞中表达量较高,性腺中较低。在长牡蛎个体发育各个阶段(2细胞期-壳顶期),CgCaspase-3和CgCaspase-8 mRNA皆有表达,但表达模式不同。细胞内定位显示,CgCaspase-3分子少量分布于细胞核中,而CgCaspase-8分子仅分布于细胞质中。
CgCaspase-3酶原形式已具有催化活性,倾向于Asp-Xaa-Xaa-Asp (DXXD)形式底物。而Caspase-3特异性抑制剂Ac-DEVD-CHO及Caspase家族蛋白酶泛抑制剂Z-VAD-FMK都对rCgCaspase-3酶活性有显著的抑制效果。胞内实验同样表明CgCaspase-3可以诱导细胞凋亡。此外,CgCaspase-3具有模式识别受体(PRR)的功能,LPS亲和层析、基于ELISA原理的LPS结合实验以及胞内共定位检测都表明CgCaspase-3可以直接结合LPS,而表面等离子共振(SPR)检测发现与LPS结合具有高度特异性,其解离常数(KD)为1.08×10-6 M,未检测到CgCaspase-3与磷壁酸、甘露聚糖及葡聚糖的结合活性。CgCaspase-3截短体重组蛋白rCgCas-3 (NT)及rCgCas-3 (ΔN58)与LPS结合能力均显著降低。体外检测环境下,LPS可以抑制rCgCaspase-3的活性,且呈现剂量效应。而胞内LPS同样可以抑制CgCaspase-3对底物(PARP)的水解活性,抑制细胞凋亡。
CgCaspase-8分子对Caspase-8的底物(Ac-IETD-pNA)水解活性微弱,激活后,其活性有一定程度提高,并且rCgCaspase-3可在rCgCaspase-8作用下发生水解,形成约20 kDa和11 kDa小片段。CgCaspase-8可抑制细胞增殖,诱导细胞凋亡,其Caspase-8特异性抑制剂Z-IETD-FMK对CgCaspase-8酶活性具有显著抑制作用。胞内共转染CgCaspase-3与CgCaspase-8实验表明,CgCaspase-3与CgCaspase-8完全共定位,两者可能存在相互作用。CgCaspase-8能够响应LPS、PGN及灿烂弧菌刺激,其中对LPS和灿烂弧菌刺激响应强烈。LPS刺激后,CgCaspase-8 mRNA及蛋白表达水平皆显著上升,后逐渐下降。而灿烂弧菌刺激,CgCaspase-8 mRNA表达量同样在3 h达到峰值,其后下降,而蛋白表达量则从0 h后逐渐降低,mRNA表达水平变化与蛋白水平不同。
上述研究结果表明,与脊椎动物相比,长牡蛎Caspase家族成员CgCaspase-3和CgCaspase-8分子在进化上高度保守,具有典型的CASc结构域及半胱氨酸水解酶活性,皆可介导细胞凋亡,并可响应不同的免疫刺激。同时研究发现长牡蛎存在特殊的凋亡调控机制,LPS可以抑制CgCaspase-3介导的细胞凋亡,这在其他高等生物及无脊椎生物中从未报道。这种特殊的调控机制可能在长牡蛎免疫防御中发挥重要作用。研究结果为深入研究无脊椎动物Caspase家族蛋白功能及凋亡调控机制奠定了重要基础。
其他摘要Apoptosis is a form of programmed cell death process controlled by a family of cysteine proteases called caspases, which plays crucial roles in homeostasis, development of tissues and organs and immune defense. The apoptosis and the detailed regulation mechanism have been well studied in vertebrate, but the information in invertebrate, especiallymollusc, is still very limited. In the present study, two new members of caspase family including CgCaspase-3 and CgCaspase-8 were cloned and identified from Pacific oyster Crassostrea gigas. Their function and the regulation mechanism of caspase-mediated apoptosis were conducted by using the method of molecular biology, cell biology and bioinformatics.
CgCaspase-3 encodes a polypeptide of 307 amino acids with an ORF of 924 bp. CgCaspase-8 encodes a polypeptide of 484 amino acids with an ORF of 1455 bp. CgCaspase-3 and CgCaspase-8 are both zymogens containing characteristic CASc domains in the C terminals and conserved caspase active site motifs. In addition, CgCaspase-3 and CgCaspase-8 mRNA can be examined in all tested tissues including gill, gonad (female), gonad (male), hemocytes, hepatopancrease, mantle and muscle, and the highest expression levels are observed in the mantle and hemocytes. In different development stages (from two cell embryos to umbo larvae), CgCaspase-3 and CgCaspase-8 mRNA can be examined with a different expression patterns. The immunofluorescence analysis indicated that CgCaspase-3 mainly locates in the cytoplasm of hemocytes, while a small amount of CgCaspase-3 appears in the nucleus. CgCaspase-8 mainly locates in the cytoplasm of hemocytes.
In vitro, rCgCaspase-3 prefers an Asp-Xaa-Xaa-Asp (DXXD)-like substrate, which was similar to that of human Caspase-3. Both pan-caspase inhibitor Z-VAD-FMK and caspase-3 inhibitor Ac-DEVD-CHO exhibit significant inhibition on the proteolytic activity of rCgCaspase-3. In vivo, the proliferation of HEK293T cells transfected with CgCaspase-3 decreases significantly, while the apoptosis ratio increases significantly. In addition, CgCaspase-3 functions as a pattern recognition receptor (PRR). LPS pull-down, enzyme-linked immunosorbent assay and confocal microscopic analysis collectively reveal that CgCaspase-3 binds LPS directly and surface plasmon resonance analysis further demonstrate its high binding specificity and moderate binding affinity (KD = 1.08×10-6 M) to LPS. However, rCgCas-3 (ΔN58) displays very weak LPS-binding activity, while rCgCas-3 (NT) did not exhibit LPS-binding capability. The binding of CgCaspase-3 to LPS significantly inhibits its proteolytic activity toward AC-DEVD-pNA in a dose dependent manner in vitro. Compared with the control and vector transfection groups, the cell proliferation of HEK293T cells with CgCaspase-3 significantly decreases, while the transfection of LPS rescues the cells from apoptosis. Intracellular LPS inhibits the proteolytic activity of CgCaspase-3 to PARP and apoptosis induced by CgCaspase-3.
Recombinant CgCaspase-8 shows very weak activity to substrate Ac-IETD pNA. After concentration for 12 h, the proteolytic activity of CgCaspase-8 increases. rCgCaspase-3 can be cleaved into two submits p20 and p11 after incubation with rCgCaspase-8. The proliferation of HEK293T cells transfected with CgCaspase-8 decreases significantly, while the apoptosis ratio increases significantly. The Caspase-8 inhibitor Ac-IETD-FMK exhibits significant inhibition on the proteolytic activity of rCgCaspase-8. In addition, CgCaspase-8 is observed to be completely colocalized with CgCaspase-3, indicating that they may interact with each other in vivo. CgCaspase-8 can respond to the stimulation of LPS, PGN and Vibrio splendidus. The expression levels of CgCaspase-8 mRNA and protein show an obvious increase at 3 h after the treatment of LPS and then decrease to the normal level. In contrast, the expression of CgCaspase-8 mRNA increases at 3 h after the treatment of V. splendidus and then decreases to the normal level. However, the protein expression of CgCaspase-8 declines from 0 h after the treatment. CgCaspase-8 shows a weak response to the PGN treatment, with a minor increase of mRNA expression at 6 h.
In summary, CgCaspase-3 and CgCaspase-8 are highly conserved in evolution. They exhibitproteolytic activity with typical CASc domains and induce cell apoptosis. Meanwhile, they can respondto the treatment of PAMPs and pathogens. In addition, C. gigas has a special regulation mechanism of apoptosis, LPS can inhibit apoptosis mediated by CgCaspase-3, which has never been reported in other vertebrates or invertebrates and probably plays a vital role in the immune defense of C. gigas. The results provide further information for the knowledge of caspase in C. gigas and laid a solid foundation for unveiling the cellular and molecular mechanisms of apoptosis in invertebrates as well.
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/116965
专题实验海洋生物学重点实验室
作者单位中国科学院海洋研究所
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徐家超. 长牡蛎Caspase家族蛋白调控细胞凋亡的机制研究[D]. 北京. 中国科学院大学,2016.
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