Dynamic post-translational histone modifications studied by NMR spectroscopy

人类细胞的DNA分子比细胞直径长很多倍，因此DNA被包裹在一个叫做染色质的紧密结构中。染色质由DNA分子以一种非常系统的方式缠绕在组蛋白（粘性滑轮）周围。细胞利用几种机制来精确控制DNA分子中哪些可遗传信息被转化为功能性产品（细胞机器）。细胞利用的一种机制是改变某些组蛋白的电荷（削弱或加强滑轮的粘性），从而将DNA的特定部分暴露或限制在细胞的基因生产装置中。HDAC是一种负责改变组蛋白电荷的酶（一种粘性增强剂），将是拟议研究项目的重点。HDAC酶就像一把剪刀，从组蛋白上剥离负电荷，从而使组蛋白尾部带正电荷，从而加强与带负电荷的DNA的相互作用。特别是，拟议项目的重点是HDAC酶的动力学和分子运动（剪刀是如何切割的），动力学将主要通过核磁共振（NMR）光谱学进行研究。因此，该研究的关键目标之一是在原子分辨率上描述HDAC酶改变组蛋白电荷的机制。目标也是表征HDAC酶如何与抑制剂（药物）和组蛋白相互作用。HDAC酶与癌症有关，据信它们抑制肿瘤抑制因子的产生。HDAC酶的抑制剂已经显示出抗肿瘤活性，因此，拟议的研究结果可能会导致设计特定的HDAC酶抑制剂，最终导致更有效的癌症治疗。要了解组蛋白修饰，需要对所涉及的酶的三维结构有详细的了解，并了解这些结构如何随时间变化和波动（剪刀因其打开和关闭运动而切割）。在过去的十年中，人们已经确定了HDAC酶的静态结构，然而，关于这些调节分子的灵活性和动力学的研究很少发表。提议的项目侧重于使用核磁共振波谱作为主要的生物物理工具来阐明分子的灵活性和相互作用，因为核磁共振具有在原子分辨率上提供动力学和相互作用描述的潜力。目标是核磁共振测量与其他实验技术和计算机模拟将创建酶功能的连贯特征。拟议研究的另一个主要目标是开发新的核磁共振方法来表征分子动力学和柔韧性。这些发展旨在对酶运动进行时间分辨描述，也就是说，酶运动随时间的可视化-作为电影-与先前主要提供蛋白质运动幅度的方法相反。这项研究将在结构和分子生物学研究所（ISMB）进行，该研究所是伯克贝克学院和伦敦大学学院（UCL）的合资企业。伦敦大学学院和ISMB提供了一个最先进的和刺激的研究环境，有适合拟议项目的专用核磁共振仪器。此外，ISMB和UCL的高度协作环境和世界一流的专业知识为富有成果的合作开辟了可能性。例如，为了增加新HDAC抑制剂开发成功的可能性，我已经开始与伦敦大学学院的Charles Marson教授合作，他是HDAC抑制剂生产方面的专家。在我看来，这次合作将使通过核磁共振波谱获得的HDAC酶动力学结果在新药设计方面迈出重要的一步。

DNA molecules of human cells are many times longer than the diameter of the cell and consequently the DNA is packed into a compact structure called the chromatin. The chromatin consists of DNA molecules coiled around histone proteins (sticky pulleys) in a very systematic manner. The cell utilises several mechanisms to control exactly what inheritable information from the DNA molecule that is turned into functional product (cellular machines). One mechanism that the cell exploits is to change the charge of certain histone proteins (weaken or strengthen the stickiness of the pulleys) and thus expose or restrict a specific part of the DNA to the cells gene production apparatus. HDAC, an enzyme that is responsible for changing the charge of histone proteins (a stickiness enhancer) will be the focus of the proposed research project. The HDAC enzyme works as a scissors that strips a negative charge off the histone proteins, thereby rendering the histone tails positively charged which strengthen the interaction with the negatively charged DNA. In particular, the focus of the proposed project is the dynamics and molecular motions of the HDAC enzyme (how does the scissors cut) and the dynamics will be studied primarily with nuclear magnetic resonance (NMR) spectroscopy. Thus, one of the key objectives of the research is to characterise, at atomic resolution, the mechanism by which the HDAC enzyme alter the histone charges. The goal is also to characterise how HDAC enzymes interact with inhibitors (drugs) and histones. HDAC enzymes are involved in cancers where they are believed to suppress the production of tumour suppressors. Inhibitors of HDAC enzymes have shown anti-tumour activity and it is therefore likely that the outcome of the proposed research will lead to the design of specific inhibitors of HDAC enzymes ultimately resulting in more efficient cancer therapy. An understanding of histone modifications requires a detailed picture of the three-dimensional structure of the involved enzymes and an appreciation of how these structures vary and fluctuate with time (a scissors cuts due to its opening and closing motions). Static structures of HDAC enzymes have been determined over the last decade, however, very few studies on the flexibility and dynamics of these regulatory molecules have been published. The proposed project focuses on the use of NMR spectroscopy as the primary biophysical tool to elucidate molecular flexibility and interactions since NMR has the potential to provide a description of the dynamics and interactions at atomic resolution. It is the goal that the NMR measurements together with other experimental techniques and computer simulations will create a coherent characterisation of the enzyme function. Another major objective of the proposed research is to develop new NMR methods to characterise molecular dynamics and flexibility in general. These developments aim at a time-resolved description of enzyme motions, that is, a visualisation of the enzyme motions over time - as a movie - as opposed to previous methods that primarily provides the amplitudes of protein motions. The research will be carried out at the Institute of Structural and Molecular Biology (ISMB), a joint venture between Birkbeck and University College London (UCL). UCL and ISMB provide a state-of-the-art and stimulating research environment with dedicated NMR machines suitable for the proposed project. Also, the highly collaborative environment and world-class expertise at ISMB and UCL open up the possibility for fruitful collaborations. For example, to increase the likelihood of success in the development of new HDAC inhibitors, I have initiated a collaboration with Prof Charles Marson, UCL, who is an expert on the productions of HDAC inhibitors. In my opinion this collaboration will allow the results about the HDAC enzyme dynamics, obtained by NMR spectroscopy, to be taken one important step further towards the design of new medicine.