Structural Studies of the c-myc Gene Regulation

c-myc 基因调控的结构研究

• 批准号: 6432658
• 负责人: NICO TJANDRA
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432658
• 关键词: apoptosis; binding proteins; cytosine; dipole moment; genetic regulation; genetic regulatory element; genetic transcription; mathematical model; molecular dynamics; nuclear magnetic resonance spectroscopy; protein binding; protein structure function; protooncogene

The transcription of the human c-myc proto-oncogene is regulated by multiple cis-elements upstream as well as downstream of the promoter sites. One cis-elements upstream from the c-myc promoters is the CT element. This is a CT rich sequence which is located100 bases upstream from the P1 promoter. One protein that binds the coding strand of the CT element is hnRNPk. Binding of hnRNPk to the CT element upregulates c-myc transcription. There are three homology repeats in hnRNPk. These are called the KH domains. The 86 residue C-terminal segment of the hnRNPk protein comprises the third KH motif (KH3) in hnRNPk. The three-dimensional structure of the C-terminal domain of hnRNPk protein in solution has been determined by NMR spectroscopy. The structure of KH3 of hnRNPk was determined using the liquid crystal technique in addition to conventional protein NMR method. The liquid crystal environment produces a slight order in the system. The ordering of the molecule reintroduces dipolar coupling which contain useful structural information. The final family of structures calculated using the addition of dipolar coupling information results in root mean square deviation (RMSD)for the family of 0.17 Angstrom. In contrast, calculation carried out without the dipolar couplings has an RMSD of 0.32 Angstrom. The RMSD value provides a quantitative evaluation of the precision of the structures with smaller RMSD value being higher precision. This comparison was made under ideal conditions, i.e. : good signal to noise for all of the NMR experiments, some NMR experiments were repeated twice for error estimates as well as consistency check, all distance estimates were done conservatively to take into account any possible systematic error. Thus for a typical NMR structure under non-ideal conditions the increase of the RMSD value with the addition of dipolar coupling information should be much greater. In parallel to the KH3 structure determination we also developed a straightforward protocol of structure refinement using the dipolar coupling information. This protocol is being provided to any laboratory which wishes to take advantage of dipolar coupling information. A dynamic study of KH3 domain has also been carried out. There are two different purposes for carrying out the dynamic study. One is to get the detail information regarding the overall tumbling of the molecule as well as the local microscopic motion. Two is to use the hydrodynamic parameters derived from the relaxation data to cross check the quality of protein structure. The overall tumbling rate of the KH3 has been determined to be 6.87 ns. There is one flexible loop (L52-R56) in the KH3 domain which undergoes rapid (ps) fluctuation. In contrast to previous study of a similar KH domain of FMR1 the conserved loop (G30-G33) do not show any flexibility. This is suspected due to the different in pH under which the two studies were carried out. Thus points to the possibility of this particular loop undergoing chemical exchange which diminishes at low pH where the KH3 study was done. In addition to this local information the overall fit of the relaxation rates to the KH3structure has revealed that the molecule is non spherical with the ratio of the long diffusion axis to the short diffusion axis of 1.37 and the asymmetry (ratio of diffusion along the x and they axis) of 1.12. We pointed out that the quality of the fit of the relaxation data to the structure provides an independent quality assessment of the structure it self. When the dynamics data were fitted to the structure without the dipolar coupling one observed a residual error (chi squared) of 3.0 while using the dipolar refined structure the chi-squared is 1.5. This is suggesting that the structure refined using dipolar coupling is twice as accurate as the one without dipolar coupling. The next steps in the structural study of hnRNPk has been initiated. The construct of KH1+KH2 of hnRNPk has been made. The protein expression is currently being characterized. Single stranded DNA binding constant for the KH1+KH2 domain is also under investigation. In addition the C-terminal domain of hnRNPk contains 3 SH3 binding sites. A plasmid containing the Vav-SH3domain is being constructed to produce the target peptide for KH3domain.In order to get a better understanding of c-myc regulation through the CT element a parallel project has been initiated. In this particular project we would like to determine the structure of cellular nucleic acid binding protein (CNBP). This protein binds the non-coding strand of the CT element, thus the complimentary site to the hnRNPk binding site. CNBP upregulates the CT element activity. A construct of the full length (176residues) CNBP has been made. It consists of seven zinc fingers. A minimum construct of CNBP which still retains the nucleotide binding affinity is being probed. We have completed the NMR backbone dynamic studies of mutant(Gly26-Arg) KH3, wild type KH3, and KH3+ss-DNA complex. We have shown that there is no ss-DNA binding activity for the mutantKH3. We have also done titration study on the KH3+DNA complex to map the DNA binding site. At this point we have access to all dynamic parameters and map of the DNA binding site. We are currently comparing all of these parameters to characterize theKH3 ss-DNA interaction based on structure as well as dynamic information. We also have initiated a study on side chain dynamics using KH3as a model system. We have developed an experiment where we can probe NH2 moiety on the side chain of Gln and Asn. Comparison of the dynamic of this NH2 group in the free and bound form would provide information on side chain interaction with the ss-DNA target. This methodology will be extended to look at CH, CH2, andCH3 moieties in the protein.

人c-myc原癌基因的转录受启动子上游和下游多个顺式元件的调控。c-myc启动子上游的一个顺式元件是CT元件。这是一个富含CT的序列，位于P1启动子上游100个碱基。结合CT元件的编码链的一种蛋白质是hnRNPk。hnRNPk与CT元件的结合上调c-myc转录。hnRNPk中有三个同源重复序列。这些被称为KH域。hnRNPk蛋白的86个残基的C-末端区段包含hnRNPk中的第三个KH基序（KH 3）。通过核磁共振光谱法测定了溶液中hnRNPk蛋白C-末端结构域的三维结构。除了常规的蛋白质NMR方法之外，还使用液晶技术确定了hnRNPk的KH 3的结构。液晶环境在系统中产生轻微的秩序。分子的有序性重新引入偶极耦合，偶极耦合包含有用的结构信息。使用添加偶极耦合信息计算的最终结构族导致该族的均方根偏差（RMSD）为0.17埃。相比之下，在没有偶极耦合的情况下进行的计算具有0.32埃的RMSD。RMSD值提供了结构精度的定量评估，RMSD值越小，精度越高。该比较是在理想条件下进行的，即：所有NMR实验的信噪比良好，一些NMR实验重复两次以进行误差估计以及一致性检查，所有距离估计都是保守进行的，以考虑任何可能的系统误差。因此，对于非理想条件下的典型NMR结构，随着偶极耦合信息的增加，RMSD值的增加应该大得多。在平行的KH 3结构的测定，我们还开发了一个简单的协议，使用偶极耦合信息的结构细化。该方案提供给任何希望利用偶极耦合信息的实验室。对KH 3结构域进行了动力学研究。开展动态研究有两个不同的目的。一种是获得分子整体翻滚和局部微观运动的详细信息。二是利用弛豫数据得到的流体动力学参数对蛋白质结构的质量进行交叉检验。KH 3的整体翻滚速率已被确定为6.87 ns。在KH 3结构域中存在一个柔性环（L52-R56），其经历快速（ps）波动。与先前对FMR 1的类似KH结构域的研究相反，保守环（G30-G33）不显示任何柔性。怀疑这是由于进行两项研究的pH值不同。因此，指出了这种特殊回路进行化学交换的可能性，这种化学交换在进行KH 3研究的低pH值下减少。除了这种局部信息的整体拟合的弛豫速率的KH 3结构揭示了该分子是非球形的长扩散轴与短扩散轴的比率为1.37和不对称性（比率扩散沿着x和y轴）的1.12。我们指出，弛豫数据与结构的拟合质量提供了结构本身的独立质量评估。当动力学数据拟合到没有偶极耦合的结构时，观察到3.0的残余误差（卡方），而使用偶极精细结构，卡方为1.5。这表明使用偶极耦合细化的结构是没有偶极耦合的结构的两倍准确。hnRNPk结构研究的下一步已经开始。构建了hnRNPk的KH_1 + KH_2结构。目前正在对蛋白质表达进行表征。KH 1 + KH 2结构域的单链DNA结合常数也在研究中。此外，hnRNPk的C-末端结构域含有3个SH 3结合位点。正在构建含有Vav-SH 3结构域的质粒以产生HH 3结构域的靶肽。为了更好地了解通过CT元件调节c-myc，已经启动了一个平行项目。在这个特定的项目中，我们想确定细胞核酸结合蛋白（CNBP）的结构。该蛋白结合CT元件的非编码链，因此是hnRNPk结合位点的互补位点。CNBP上调CT元件活性。构建了全长（176个残基）CNBP。它由七个锌指组成。正在探索仍然保留核苷酸结合亲和力的CNBP的最小构建体。我们完成了突变型（Gly 26-Arg）KH_3、野生型KH_3和KH_3 +ss-DNA复合物的NMR主链动力学研究。我们已经表明，有没有ss-DNA结合活性的muplastin KH 3。我们还对KH ~（3+）DNA络合物进行了滴定研究，以绘制DNA结合位点。此时，我们可以访问所有的动态参数和DNA结合位点的地图。我们目前正在比较所有这些参数来表征theh 3 ss-DNA相互作用的基础上的结构以及动态信息。我们还开始了侧链动力学研究使用KH 3作为模型系统。我们已经开发了一个实验，我们可以探测谷氨酰胺和天冬酰胺侧链上的NH 2部分。比较游离和结合形式的该NH 2基团的动力学将提供关于侧链与ss-DNA靶标相互作用的信息。这种方法将被扩展到看看CH，CH 2，和CH 3部分的蛋白质。