人源蛋白质ARAP3选择性识别RhoA的结构基础以及拟南芥蛋白质APUM23特异性识别18S核糖体RNA的结构基础

发布时间：2018-04-23 11:50

本文选题：ARAP3 + GTP酶激活蛋白质　；参考：《中国科学技术大学》2017年博士论文

【摘要】：本论文的第一章介绍了我们关于含有两个GTP酶激活蛋白质结构域的人源蛋白质ARAP3选择性识别RhoA的结构与功能研究。ARAP3是一个多结构域的蛋白质,它含有1个ArfGAP结构域、1个RhoGAP结构域、1个SAM结构域、5个PH结构域、以及1个RA结构域。ARAP3的特点是同时拥有两个具有活性的GTP酶激活蛋白质结构域,其中ArfGAP可以选择性地识别并加速Arf6-GTP水解为Arf6-GDP,RhoGAP可以选择性地识别并加速RhoA-GTP水解为RhoA-GDP。PtdIns(3,4,5)P3与 ARAP3 的结合可以提高 ArfGAP 的活性,Rap1-GTP与ARAP3的结合可以提高RhoGAP的活性。ARAP3很多生物学功能都与它的RhoGAP结构域的活性有关,如参与板状伪足的形成、参与血管生成、抑制硬胃癌细胞的腹膜扩散、调节中性粒细胞的趋化性和粘附相关过程、以及参与神经突生长等。为了更清晰的了解ARAP3是如何特异性识别RhoA,我们解析了 ARAP3的RhoGAP结构域与RhoA·GDP·AlF4-在反应过渡态的复合物晶体结构。ARAP3的RhoGAP结构域由9个长的α螺旋和2个短的α螺旋反向平行折叠组成。我们对复合物结构提供的作用界面上的氨基酸进行突变,并通过细胞外1TC实验以及酶动力学实验研究了这些氨基酸对RhoGAP结构域识别RhoA以及加速RhoA-GTP水解能力的影响。我们发现对ARAP3的氨基酸的Arg-942、Arg-949、Arg-982或Arg-985的单点突变都可以显著的降低ARAP3-RhoGAP结构域与RhoA-GTP的结合能力以及GAP活性。我们还发现ARAP3是通过RhoA的α3螺旋中的氨基酸Asp-90和Glu-97来选择RhoA作为特异性作用底物的。本论文的第二章介绍了我们关于拟南芥中的PUF蛋白质APUM23特异性识别18S核糖体RNA中11个核苷酸的5'-GGAAUUGACGG-3'序列的结构与功能研究。多年来PUF蛋白质得到了广泛的结构与功能研究,但是我们对植物中的PUF蛋白质仍然知之甚少。大部分的PUF蛋白质都存在于细胞质中,它们通过结合信使RNA的3'非翻译区来调控信使RNA的翻译及定位。APUM23是少数的存在于核仁中的PUF蛋白质,它参与调节前核糖体RNA的加工过程,然而其中的分子机制目前尚不清楚。我们解析了 APUM23和目的RNA的复合物晶体结构,这是第一个解析出的植物中的PUF蛋白质结构,APUM23是由10个Puf repeat串联结构域折叠成的C型结构,APUM23有一段插入序列并在C型结构内侧折叠成α螺旋。这段插入序列参与RNA的识别。最后,我们还解析了不含插入氨基酸序列的APUM23的突变体和RNA 5'-GGAAUUGACGG-3'的复合物晶体结构。这个复合物结构显示,Go的碱基不再与APUM23的第十个Puf repeat结构域结合,而是经过大角度的翻转和A-3的碱基形成堆积相互作用稳定A+3碱基的构象。插入氨基酸序列的存在会通过空间位阻限制Go碱基的翻转,因此APUM23的插入氨基酸序列既识别RNA的碱基,又可以稳定RNA的构象。这项工作揭示了APUM23特异性识别18S核糖体RNA的结构基础。
[Abstract]:In the first chapter of this thesis, we introduce our research on the structure and function of ARAP3 selective recognition of RhoA with two GTP enzyme activated protein domains. ARAP3 is a multidomain protein. It contains one ArfGAP domain, one RhoGAP domain, one SAM domain, five PH domains, and one RA domain. ARAP3 is characterized by two active GTP enzyme activated protein domains. ArfGAP can selectively recognize and accelerate the hydrolysis of Arf6-GTP to Arf6-GDPN RhoGAP, which can selectively recognize and accelerate the hydrolysis of RhoA-GTP to RhoA-GDP.PtdInsf3 and ARAP3. It can improve the activity of ArfGAP, Rap1-GTP and ARAP3, which can improve the activity of RhoGAP. ARAP3. The function is related to the activity of its RhoGAP domain, They are involved in the formation of platelike pseudopodia, angiogenesis, inhibition of peritoneal diffusion, regulation of neutrophil chemotaxis and adhesion related processes, and involvement in neurite growth. In order to understand more clearly how ARAP3 specifically recognizes RhoA, we have analyzed the RhoGAP domain of ARAP3 and RhoA GDP AlF4- the RhoGAP domain of the complex in transition state. The RhoGAP domain of ARAP3 is composed of 9 long 伪 helix and 2 short 伪 helix reverse parallel folding. We mutated the amino acids at the interaction interface provided by the complex structure and studied the effects of these amino acids on the RhoA recognition and RhoA-GTP hydrolysis ability of the RhoGAP domain by extracellular 1TC and enzyme kinetics experiments. We found that the single point mutation of Arg-942Ag-949 Arg-982 or Arg-985 could significantly decrease the binding ability of ARAP3-RhoGAP domain to RhoA-GTP and the activity of GAP. We also found that ARAP3 selects RhoA as the specific substrate through the amino acid Asp-90 and Glu-97 in the 伪 3 helix of RhoA. In the second chapter of this thesis, we introduce the structure and function of PUF protein APUM23 in Arabidopsis thaliana, which specifically recognizes 11 nucleotides in 18s ribosomal RNA. PUF proteins have been widely studied in structure and function for many years, but we still know little about PUF proteins in plants. Most of the PUF proteins are present in the cytoplasm. They regulate the translation and localization of messenger RNA by binding the 3'untranslated region of messenger RNA. APUM23 is a small number of PUF proteins in nucleoli. It is involved in the processing of pre-ribosomal RNA. However, the molecular mechanism is unclear. We have elucidated the crystal structure of the complex of APUM23 and RNA. This is the first elucidated PUF protein structure in plants, APUM23 is a C-type structure folded from 10 Puf repeat tandem domains with an insert sequence and folded into a helix inside the C-type structure. This insert sequence is involved in the identification of RNA. Finally, the crystal structure of the APUM23 mutant and the complex of RNA 5G GGAAUGACGG-3 'without the insertion amino acid sequence were analyzed. The structure of the complex shows that the base of Go is no longer bound to the tenth Puf repeat domain of APUM23, but the conformation of A 3 is stabilized by large angle reversal and stacking interaction of A-3 bases. The existence of inserted amino acid sequence will limit the turnover of go base by steric hindrance, so the inserted amino acid sequence of APUM23 can not only recognize the base of RNA, but also stabilize the conformation of RNA. This work reveals the structural basis of APUM23 specific recognition of 18 S ribosomal RNA.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q51;Q78

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