【作者】 申兴桂；

【导师】 李青山； 付学奇；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 肝再生磷酸酶3(Phosphatase of regenerating Liver 3,PRL3)是蛋白质酪氨酸磷酸酶家族的一个重要成员,分子量为20 KDa,其编码基因位于染色体8q24.3,和其亚家族另外两个成员(PRL1、PRL2)的氨基酸序列同源性超过78%,除了具有蛋白质酪氨酸磷酸酶活性保守序列外,在C端都有一个CCAX结构域。PRL3基因在转移性结肠癌患者中高水平表达,表明其与结肠癌的转移密切相关,因此PRL3可能作为一种阻断结肠癌转移的理想治疗靶点。本研究利用基因工程技术,构建了PRL3及其突变体的融合蛋白表达质粒,在大肠杆菌中表达并分离纯化了这两个蛋白质酪氨酸磷酸酶,就其酶学性质进行对比研究,比较分析了PRL3的第49位Cys突变对其酶活的影响;同时利用分子对接、分子力学、分子动力学等分子模拟方法,得到了酶与底物复合物的最优势构象,比较分析了在Cys49突变前后,活性中心区域氨基酸的静电势分布及其对酶与底物结合过程的贡献,从空间结构上分析了酶和底物的相互作用。在对模型的结构研究和模拟计算中发现,静电相互作用是酶和底物结合是主要的远程作用力,而在它们接近到一定距离后,疏水堆积作用使两者稳定的聚合并维持一定的相对空间关系。另外,在体外进行了PRL3在氧化条件下的初步研究,说明了Cys49能对PRL3的氧化起保护作用。总之,理论计算结果与实验结果相互验证,力图从蛋白结构信息学、动力学等方面,阐明PRL3的Cys49的作用,为进一步研究酶在体内的信号通路奠定了基础。更多还原

【Abstract】 Phosphatase of Regenerating Liver 3 （PRL3） has attracted more and more attentions because it is involved in metastasis of colonic cancer. The three PRL protein tyrosine phosphatase members （PRL1, PRL2 and PRL3） comprise a distinct subclass of protein tyrosine phosphatases with a COOH-terminal prenylation motif. PRL3 mRNA expression is consistently elevated in most metastatic lesion derived from colorectal cancers. PRL3 is expressed at higher levels in human metastatic colon cancers than in the corresponding primary cancers or normal tissue, and cells transfected with PRL3 acquire greater motility, invasion and metastatic antivity. Therefore PRL3 is a potential marker for liver metastasis of colorectal cancer cells, and may a represent novel target for cancer therapy.With the development of the computer technology, the software technology, molecular mechanics, molecular dynamics and theory of the quantum chemistry in recent years, molecular modeling can construct the three-dimensional structures of the biological macromolecule, research biological function, analyze enzyme-catalyzed mechanism, therefore molecular modeling has already become an important method in the fields of biology and medical science In the paper, PRL3 and the mutant were separately expressed and purified by engineering technology, enzyme character was investigated by pNPP substrate. The protection mechanism of PRL3 phosphate activity was assumed in the condition of oxidated experiment in vitro. At the same time, we use docking, molecular mechanics and the molecular dynamics method to separately build the model of three-dimensional structures of the enzyme （mutant） and substrate, and determine the role of each reside in the active domain.1. Molecular dynamics of PRL3 and its mutantThe primary model is obtained by X diffraction crystallization structure of PRL3 （The PDB serial number:1V3A ）. In condition of the ffgmx field offorce and the salvation cycle boundary, the superiority conformation was optimized by the Gromacs 3.3 procedures until RMS reached 10-6 kcal mol-1. PRL3/mutant （C49S） was combined with the substrate pNPP by autodock 3.0.5, the most superiority conformation was obtained by choosing molecular dynamics and the genetics algorithm and screening with the X-score procedure. The superficial electrostatic potential and the role of each residue in the catalytic activity domain are analyzed to the enzyme and substrate complex by the Delphi 2.0 procedure.The results indicate that the electric charge role is playing the vital role in the formation of enzyme and substrate complex. The residue of Arg110, Asp72 and Cys104 are extremely essential to PRL3 phosphate activity. After Cys49 mutated to Ser, role of ALA111 transforms from hydrophobic interaction to electrostatic interaction, greatly promoted formation of the enzyme and substrate complex. There are new discoveries, such as ALA106, Gly109, Gly107 and Pro25, mainly contributing to formation of the complex with electrostatic interaction. Hydrophobic interaction is the crucial factor which the protein stable spatial structure forms. On the long-distance distance, orientation and docking of the enzyme and substrate mainly depends on the electrostatic interaction. After both approaches to the centain distance, hydrophobic interaction becomes very important. These results are consistent with previously literature.2. Construction and site-directed mutationPRL3 gene are amplified from human heart cDNA by PCR, then cloned into the pVluscript KS vector. After confirmed by DNA sequence analysis, the coding region of PRL3 excised from pKS-PRL3 plasmid was subcloned into pGEX-4T vector via the EcoRⅠ-HindⅢsites. Positive recombinant vector named pGEX-4T-PRL3. Moreover, the improved mutation method was used to finish the mutations of Cys49 to Ser, and then pGEX-4T-PRL3 （C49S） vector was obtained. Both recombinant vectors were transformed into competent E.coli BL21 cells.3. Expression and purificationWe expressed recombinant fusion protein of PRL3 and mutant PRL3（C49S） with pGEX-4T as a carrier and E.coli BL21 as a host cell. These PRL3 enzyme was purified by using glutathione-Sepharose 4B affinity column, thrombin digestion and S-100 Sepharose gel filtration column. Purified PRL3 and PRL3 （C49S） reach a band （20Kda） of SDS-PAGE, purity of both are greater than 99% by analysis of HPLC.In order to study the influence of Cys49 to the phosphatase activity, we characterized PRL3 and PRL3 （C49S） with pNPP as a substrate.（1） The optimal pH of PRL3 and PRL3（C49S） respectively are 6.5 and 6.0; The optimal temperature of both is 30℃; Under 30℃conditions, Km respectively is 4.785 and 4.948 m mol/L, Kcat respectively be 2.12×104 and 2.75×104 s^-1, although the mutant slightly has the drop to the substrate affinity, but its transformed number increased, thus directly causes the mutant to be higher than the wild.（2） Pi （catalytic production of PRL3） has the inhibition to PRL3, the apparent the Km value （Km ’） of PRL3 is 8.4 mmol/L and 14.4 mmol/L respectively with the condition of 1 m mol/L and 3 mmol/L K2HPO4-KH2PO4（pH 6.5）, its suppression constant Ki is 1.5 m mol/L.（3） Ion intensity also affects the phosphatase activity of PRL3 and the mutant. When the ion intensity increases, activity decreases progressively, the speed of mutant activity decreasing is faster than that of the wild. Na3VO4, Cu2+ and Co2+ have the inhibition to phosphatase activity of PRL3 and the mutant, while Zn2+, Mg2+, Ca2+ and the Mn2+ have no obvious effection on its activity.4. Activity of oxidated PRL3 in vitroAt present, the regulated mechanism of PTPs remains little understood, the oxidation regulatory is an important mechanism of PTPs. Just like other PTPs, Cys104 of PRL3 has lower pKa because of the microenvironment influence, Cys104 is easily to be oxidized, which results in deactivation, Cys49 can prevent the irreversible oxidation through forming disulfide bond and stable PRL3-S-GSH is producted with the existence of GSH. Both can restore their phosphatase activity by deoxidization of DTT, the thiol transferase.Though the above experiments, we establish a systematic set of methods in studying the biological functions of enzyme by site-directed mutation and molecular modeling, including cloning and expressing of the enzyme gene, site-directed mutation ,isolating and purifying of the enzyme and mutant, molecular mechanics, molecular dynamics, molecular modeling, kinetic study of enzyme, etc. Additionally, we primarily explore the protection of PRL3 under the condition of oxidated effect. In conclusion, the calculations and experimental results validate each other,clarifying the role of Cys49 in PRL3 from structure, dynamics.We expect that our study will contribute to further investigation into the mechanism of PRL3 and provide a drug target for the treatment of diseases.更多还原