【作者】 李蕊；

【导师】 黄东阳；

【作者基本信息】 汕头大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 维甲酸(retinoic acid，RA)是哺乳动物体内重要的激素类物质，RA由维生素A(也称视黄醇)经两步氧化脱氢酶促反应产生，中间产物是视黄醛。辅酶Ⅱ依赖性视黄醛脱氢／还原酶(NADP(H)-dependent retinol dehydrogenase／reductase，NRDR)是黄东阳教授从兔肝中纯化出的一种酶，该酶普遍存在于哺乳动物肝脏中，显示出较强的视黄醇氧化与视黄醛还原活性。此外，NRDR还催化视黄醛以外的许多醛、酮类化合物的还原反应。生物信息学分析显示NRDR蛋白N末端含有线粒体定位信号(mitochondrial targeting signal，MTS)，C末端含有过氧化物酶体定位信号(peroxisomal targeting sequence type 1，PTS1)。hNRDRA2是从人神经母细胞瘤细胞SK-N-SH中发现的一种亚型，简称A2。它相对于NRDR缺第4、6外显子，从第5外显子开始读码框架前移1bp，导致蛋白翻译提前终止，并失去C端PTS1，而在C端附近出现核定位信号(nuclear localization signal，NLS)。根据课题组前期研究结果和序列分析，兔NRDR因翻译起始位点选择及翻译后加工的不同，可能存在三种蛋白形式，所含氨基酸数目分别为279、260及256。蛋白在细胞内的定位与其体内活性直接相关，蛋白定位的改变通常会导致功能相应的改变。因此，研究NRDR及其选择性剪接亚型在细胞内的定位对了解其功能具有重要意义。本研究构建了A2的原核表达质粒，在大肠杆菌中重组表达A2，重组蛋白免疫家兔得到抗血清。利用融合绿色荧光蛋白(green fluorescent protein，GFP)的真核表达载体，外源重组表达A2及兔NRDR蛋白(279／260／256)，观察不同融合方式下A2及兔NRDR的定位。同时外源表达天然状态(native)A2，利用制备的抗血清进行免疫荧光定位。为深入了解A2的定位信号功能及定位机制，本研究还构建了NLS序列及A2的多种截短片段的GFP融合表达质粒，来分析A2定位信号的功能及各区段对定位的影响。原核表达A2重组蛋白作为免疫原，制备得到了高滴度的兔抗血清。虽然NLS序列与GFP的融合蛋白集中分布在细胞核，GFP融合的A2并没有定位到核，而是分布于细胞质。分析A2各区段对定位的影响，结果表明A2与人NRDR的一致序列会影响A2的定位。利用兔抗血清进行免疫荧光检测，外源表达native A2呈点状分布在细胞质，与Flag融合A2的分布一致。N端融合GFP的兔NRDR均分布于过氧化物酶体，而C端融合GFP的兔NRDR260则均匀分布于细胞质。更多还原

【Abstract】 Retinoids are important excitatory autacoid in mammals. As the active form of retinoids,retinoic acid (RA) can be drived from retinol by two-step oxidation with retinal as intermediatein vivo. The change from retinol to retinal is the rate-limited step in this reaction. Lots ofenzymes are considered to take part in these two metabolism processes. NADP(H)-dependentretinol dehydrogenase/reductase, NRDR, was purified from rabbit liver by Huang DY. NRDRexists in mammalian livers universally, and shows high activity in retinol oxidation and retinalreduction. In addition, NRDR can catalyse the reduction of many aldehydes and ketones.The results of bioinformatics analysis show NRDR contains an N terminal mitochondrialtargeting signal (MTS) and a C terminal peroxisomal targeting sequence type 1 (PST1)containing three amino acids, Ser-Arg-Lys (SRL).Human NRDR A2, abbreviated to A2, is a novel alternative splicing isoform of NRDRdetected from human neuroblastoma cell line SK-N-SH. A2 differs from NRDR in that it lacksthe fourth and sixth exons, and the reading frame has shift forward for 1 base pair beginning withthe fifth exon. The frame shift mutation terminates protein translation in advance, and a novellocalization signal, nuclear localization signal (NLS), takes the place of PST1.Accoding to our previous experimental results and NRDR sequence analysis, it was suggestedthere are three forms of rabbit NRDR generated by alternating translational initiation site orpost-translation processing, which contain 279aa, 260aa and 256aa respectively.Functions of protein are tightly related with its localization in vivo. The changes of proteinlocalization usually result in the function alteration, accordingly. Therefore, identification thesubcellular localization of NRDR and its isoforms has great significance for the function study ofNRDR.A2 was expressed in E. coli, and the recombination protein was purified for immunizing rabbitto prepare rabbit anti human A2 antiserum. Green fluorescent protein (GFP) fusion plasmidswere constructed to demonstrate the distribution of A2 and NRDR (279/260/256) in mammaliancells. Native A2 was also expressed in mammalian cells and its subcellular location was detectedby immtmofluorescence. To study the mechanisms by which A2 is distributed, GFP fusion expression plasmids were constructed containing NLS sequence and different A2 fragmentsrespectively.Though NLS could import GFP into nuclear, A2 fused with GFP was distributed thoughout thecell, and exogenous expressed native A2 was localized in cytoplasm. Results of recombinationexpression experiment about different A2 fragments indicate that the homologous sequence withhumNRDR may impact A2 localization. All the forms of rabbit NRDR with GFP fused to their Nterminals are localized in peroxisome, and rabNRDR260 with GFP fused to its C terminal isdiffused in cytoplasm.更多还原