Dynamic post-translational histone modifications studied by NMR spectroscopy

人类细胞的DNA分子比细胞直径长许多倍，因此DNA被包装成一种紧凑的结构，称为染色质。染色质由DNA分子以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转化为功能产品(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基因生产装置中。HDAC是一种负责改变组蛋白(一种粘性增强剂)电荷的酶，将是拟议研究项目的重点。HDAC酶起到剪刀的作用，剥离组蛋白的负电荷，从而使组蛋白尾部带正电，从而加强与带负电的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)，动力学将主要用核磁共振光谱学来研究。因此，这项研究的关键目标之一是在原子分辨率下表征HDAC酶改变组蛋白电荷的机制。其目标也是描述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，在癌症中，它们被认为可以抑制肿瘤抑制因子的产生。HDAC酶的抑制剂已显示出抗肿瘤活性，因此，拟议的研究结果很可能导致设计特定的HDAC酶抑制剂，最终导致更有效的癌症治疗。要理解组蛋白的修饰，需要详细了解所涉及的酶的三维结构，并了解这些结构如何随时间变化和波动(由于其打开和关闭的运动而剪刀切割)。在过去的十年里，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。由于核磁共振有可能提供原子分辨率下的动力学和相互作用的描述，因此拟议的项目侧重于使用核磁共振光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。我们的目标是，核磁共振测量与其他实验技术和计算机模拟一起，将创建一个连贯的酶功能特征。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一般的分子动力学和灵活性。这些发展旨在对酶的运动进行时间分辨的描述，即以电影的形式可视化酶随时间的运动，而不是以前的方法，主要提供蛋白质运动的幅度。这项研究将在伯克贝克和伦敦大学学院(UCL)的合资企业结构和分子生物学研究所(ISMB)进行。伦敦大学学院和ISMB提供了一个最先进和刺激的研究环境，配备了适合拟议项目的专用核磁共振仪。此外，ISMB和UCL高度协作的环境和世界级的专业知识为富有成效的合作打开了可能性。例如，为了增加成功开发新的HDAC抑制剂的可能性，我与伦敦大学学院的Charles Marson教授展开了合作，他是HDAC抑制剂生产方面的专家。在我看来，这一合作将使通过核磁共振光谱学获得的关于HDAC酶动力学的结果朝着新药的设计进一步迈出重要的一步。

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.