Novel Cap Analogs and Interactions with Target Proteins

描述(由申请人提供) 本次申请Fogarty国际研究合作奖(FIRCA)是授予R01GM20818的延伸，题为“真核蛋白质合成启动的调节”。这项研究将主要在波兰华沙大学与爱德华·达尔津凯维奇合作完成。母公司Grant和FIRCA项目的长期目标都是了解真核细胞mRNA招募到核糖体过程中发生的步骤以及它们是如何受到调控的。这一过程既决定了蛋白质合成的总体速度，也决定了翻译的mRNAs的谱。FIRCA应用的四个特定目标涉及含有7-甲基鸟苷的mRNA帽及其在帽依赖的蛋白质合成启动过程中与目标蛋白相互作用的elF4E。线虫为研究蛋白质合成等复杂的生化过程提供了无可比拟的优势。令人惊讶的是，它表达五种eIF4E亚型，称为IFE蛋白，具有不同的帽结合特性。在具体目标1中，我们将合成并在生物系统中测试新的二核苷酸和寡核苷酸帽类似物，特别是一系列新的反反向帽类似物，旨在抵抗焦磷酸酶降解的二核苷酸帽类似物，以及被封顶的三核苷酸。在特定的目标2中，我们将基于能够穿越质膜的CAP类似物来合成和测试新的翻译抑制剂，试图将CAP依赖的翻译作为一种抗癌策略。在具体目标3中，我们将分析帽与线虫elF4E亚型结合的热力学和溶剂方面。在特定目标4中，我们将使用化学合成的人Ser-209磷酸化的elF4E来研究elF4E的磷酸化作用。由于elF4E的过度表达会导致细胞的恶性转化，而且自然发生的肿瘤含有大量的elF4E，这些研究与癌症相关，特别是针对2。它们也与由微小核糖核酸病毒引起的疾病相关，如脊髓灰质炎病毒(脊髓灰质炎)、鼻病毒(普通感冒)和柯萨奇病毒(心力衰竭)，因为微小病毒通过蛋白水解性切割elF4G来关闭帽依赖的翻译，elF4G是一种蛋白质，在翻译开始时将eIF4E拴在40秒的核糖体亚单位上。

DESCRIPTION (provided by applicant) This application for a Fogarty International Research Collaboration Award (FIRCA) is an extension of Grant R01GM20818, entitled "Regulation of Eukaryotic Protein Synthesis Initiation." The research will be done primarily in Poland at Warsaw University in collaboration with Edward Darzynkiewicz. The long-term goals of both the Parent Grant and the FIRCA project are to understand the steps that occur during the recruitment of eukaryotic mRNA to the ribosome and how they are regulated. This process determines both the overall rate of protein synthesis and also the spectrum of mRNAs translated. The four Specific Aims of the FIRCA application involve the 7-methylguanosine-containing cap of mRNA and the target protein which it interacts during cap-dependent initiation of protein synthesis, elF4E. The nematode C. elegans provides unparalleled advantages for studying such a complex biochemical process as initiation of protein synthesis. Surprisingly, it expresses five eIF4E isoforms, termed IFE proteins, that have differing cap-binding properties. In Specific Aim 1, we will synthesize and test in biological systems new classes of di- and oligonucleotide cap analogues, specifically a new series of anti-reverse cap analogues, dinucleotide cap analogues intended to be resistant to degradation by pyrophosphatases, and capped trinucleotides. In Specific Aim 2, we will synthesize and test new inhibitors of translation based on cap analogues that are able to cross the plasma membrane, in an attempt to target cap-dependent translation as an anticancer strategy. In Specific Aim 3, we will analyze thermodynamiC and solvent aspects of cap binding to elF4E isoforms of C. elegans. In Specific Aim 4, we will investigate the role of elF4E phosphorylation using chemically synthesized human Ser-209 phospho-elF4E. Because overexpression of elF4E causes malignant transformation of cells, and because naturally occurring tumors contain greatly elevated levels of elF4E, these studies have relevance to cancer, especially Specific Aim 2. They also have relevance for diseases caused by picornaviruses such as poliovirus (polio), rhinovirus (the common cold) and Coxsackievirus (heart failure), since picomaviruses shut down cap-dependent translation by proteolytically cleaving elF4G, the protein that tethers eIF4E to the 40S ribosomal subunit during initiation of translation.