Synthesis and Preliminary Evaluation of enlarged SAM analogues for Reverse Chemical Genetic Studies on Protein Methylation

用于蛋白质甲基化反向化学遗传学研究的放大 SAM 类似物的合成和初步评价

• 批准号: BB/E014089/1
• 负责人: Neil Thomas
• 金额: $ 50.9万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE014089%2F1
• 关键词: Synthesis; Preliminary; Evaluation; enlarged; SAM

The fewer than expected number of genes (30,000) identified by the human genome project indicates that the complexity of human biology is not due to the number of genes alone but to the molecular mechanism by which these genes are switched on or expressed and on how their protein products function. Inside nuclei genes that encode proteins are by winding the DNA around proteins called histones. These genes must be switched on so that they can cause the important changes required for cells to function inside the body. To do this the DNA has to be unwound from the histone proteins and there are special enzymes that chemically modify the histones in order do this. The study of these modifications is called epigenetics Enzymes called methylases can alter the histone proteins and start a sequence of events that can either switch genes on (transcribe genes) or off (repress genes). The project described here aims to investigate a novel strategy for studying how these methylases work. The difficulty in investigating these is that there are hundreds of similar enzymes inside cells and therefore studying particular ones is very difficult. Also it has been shown that these enzymes modify other proteins apart from histones eg p53. Therefore it is important that we can survey the contents of the whole cell for substrates of one individual enzyme. In this proposal we wish to use what has become known as the 'knobs & holes' approach. This involves modifying the coenzyme called S adenosyl methionine (SAM) to make it larger through the addition of a suitable chemical group called an alkyl group and concurrently modifying the enzyme under investigation. This increases the size of the binding pocket of the enzyme so that it can accommodate the enlarged coenzyme and so allow the protein target to be modified and hence utilised as a substrate. One methylase called histone methylase SET7/9 with an important function in activating the expression of genes will be modified so that it can be easily identified inside cells. In this way its function in causing changes to the expression of genes can be investigated. How these events occur is currently not understood and it is important to explain the events involved as this will shed light on how genes are switched on and off, processes which are vital to human health. In this proposal we descibe a number of enlarged S-adenosylmethionine analogues based on a reactive chemical group called an aziridinoadenosine. Because the aziridinoadenosines are highly reactive, we will prepare a new photoactivatable version that should minimise non-specific activity of these compounds. The aziridinoadenosines have been designed to form permanent covalent bonds between the protein target and the SAM analogue allowing it to be identified by the mass spectrometry. In order for the SAM analogues to be accommodated mutant forms of SET7/9 will be prepared and examined for catalytic activity with both natural S-adenosylmethionine and the aziridinoadenosine analogues. Currently a PhD student funded through a BBSRC strategic studentship is making several other s-adenosylmethionine analogues that should be utilised by the mutated SET7/9 enzymes. In order to assist in the separation of the aziridinoadenosine-protein adducts from complex biological solutions, these have azido- or alkynyl groups attached that can be selectively reacted with fluorescent groups or biotin to allow the adducts to be readily identified. Preliminary experiments with the prepared SAM analogues and complementary mutant forms of SET7/9 will performed to demonstrate that this approach will allow its specific protein targets to be identified.

人类基因组计划确定的基因数量少于预期（30,000个），这表明人类生物学的复杂性并不仅仅取决于基因的数量，而是取决于这些基因开启或表达的分子机制，以及它们的蛋白质产物如何发挥作用。在细胞核内，编码蛋白质的基因是通过将DNA缠绕在被称为组蛋白的蛋白质上。这些基因必须被激活，这样它们才能引起细胞在体内功能所需的重要变化。要做到这一点，DNA必须从组蛋白上解开，有一种特殊的酶可以对组蛋白进行化学修饰。对这些修饰的研究被称为表观遗传学。一种叫做甲基化酶的酶可以改变组蛋白，并开始一系列可以开启基因（转录基因）或关闭基因（抑制基因）的事件。这里描述的项目旨在研究一种新的策略来研究这些甲基化酶是如何工作的。研究这些酶的困难在于，细胞内有数百种类似的酶，因此研究特定的酶非常困难。此外，这些酶还能修饰组蛋白以外的其他蛋白质，如p53。因此，重要的是，我们可以调查整个细胞的一种酶的底物的含量。在这个建议中，我们希望使用被称为“旋钮和孔”的方法。这包括修改辅酶S腺苷蛋氨酸（SAM），通过添加一个合适的称为烷基的化学基团使其更大，同时修改正在研究的酶。这增加了酶的结合袋的大小，以便它可以容纳扩大的辅酶，从而允许蛋白质靶标被修饰，从而用作底物。一种被称为组蛋白甲基化酶SET7/9的甲基化酶在激活基因表达方面具有重要功能，它将被修改，以便在细胞内容易识别。通过这种方式，可以研究其引起基因表达变化的功能。这些事件是如何发生的目前还不清楚，解释所涉及的事件是很重要的，因为这将阐明基因是如何开启和关闭的，这是对人类健康至关重要的过程。在这个提议中，我们描述了一些基于反应性化学基团称为aziridinoadenosine的扩大s -腺苷蛋氨酸类似物。由于叠氮二腺苷具有高活性，我们将制备一种新的光激活版本，以最大限度地减少这些化合物的非特异性活性。aziridinoadenosine被设计成在蛋白质靶和SAM类似物之间形成永久共价键，使其能够通过质谱法识别。为了适应SAM类似物，将制备SET7/9的突变形式，并检测其对天然s -腺苷蛋氨酸和叠氮二腺苷类似物的催化活性。目前，一名通过BBSRC战略奖学金资助的博士生正在制造其他几种s-腺苷蛋氨酸类似物，这些类似物应该被突变的SET7/9酶利用。为了帮助从复杂的生物溶液中分离叠氮二腺苷-蛋白加合物，这些加合物带有叠氮或炔基，可以选择性地与荧光基团或生物素反应，使加合物易于识别。将对制备的SAM类似物和SET7/9的互补突变形式进行初步实验，以证明该方法将允许识别其特定的蛋白质靶点。

项目成果

Accelerated 19F·MRI Detection of Matrix Metalloproteinase-2/-9 through Responsive Deactivation of Paramagnetic Relaxation Enhancement.

通过顺磁弛豫增强的响应失活加速 19F·MRI 检测基质金属蛋白酶-2/-9。

• DOI: 10.1155/2019/4826520
• 发表时间: 2019
• 期刊: Contrast media & molecular imaging
• 作者: Faas HM