Activity-based protein profiling (ABPP) of the Jumonji domain protein JMJD5

Jumonji(JmjC)酶是2-氧代戊二酸(2-OG)双加氧酶，它使用2-OG和亚铁作为辅基/辅因子进行氧合反应。在包括精神障碍和癌症在内的疾病中，JmjC家族的2OG依赖的氧合酶通常会通过突变、易位而改变，在某些情况下可以完全删除。这些变化被认为是致癌事件，因为这些酶被认为是癌蛋白和肿瘤抑制因子。JMJD5是一种精氨酸羟化酶，是一种含有JMjC结构域的蛋白质，存在于动植物细胞的胞核和细胞质中。化学探针可以用来确定生物学功能，但尚未发现JMJD5的任何生物学功能。化学探针是一种小分子，旨在选择性地与目标结合并改变其功能；通常是通过抑制。通过抑制或刺激来改变靶标(例如酶)的功能，化学探针可以帮助确定蛋白质在生命系统中的作用。在化学中，探针与遗传方法一起使用，以发现和验证蛋白质或酶在疾病中的作用。生产探针以分析新的蛋白质是一种被称为基于活性的蛋白质图谱(ABPP)的技术。ABPP将在植物酶JMJD5上进行；ABPP设计将涉及利用结构信息、合成探针和体外测试。然后，新的JMJD5抑制剂将被用于探索JMJD5在人类和植物细胞中的功能，以及在长期完好的植物中的功能。我们还将利用质谱仪在分析开发过程中表征植物和人类JMJD5的动力学，这是以前从未进行过的事情。在合作中，我还将努力获得植物JMJD5的第一个晶体结构(包括与抑制剂/探针的复合体，以告知设计过程)。这些探头还将用于捕获JMJD5底物。在植物中表征JMJD5将为昼夜节律系统研究开辟新的途径，并将与人类JMJD5的工作协同；人类JMJD5与癌症有关。将研究对植物JMJD5的抑制，以观察所产生的影响，特别是在昼夜节律方面；这一途径在植物模型中比在人类/动物模型中更容易实施(从伦理的角度来看也更好)。预计植物JMJD5的化学和(初始)结构工作可以在2年的时间尺度内完成，其中化学生物学工作为1年。合成和利用植物JMJD5的探针将有助于我们产生植物JMJD5的第一个晶体结构，并将使植物细胞中的蛋白质谱能够检测植物中内源JMJD5的水平并确定其底物(S)。这项工作将开启对植物信号转导的研究，并将使人们能够从生理相关的角度更好地理解依赖于2OG的加氧酶。在JMJD5植物上进行的工作将与在人类JMJD5上进行的工作协同进行。由于植物JMJD5与人类JMJD5如此相似，应该有可能将使用植物酶所获得的知识转移到人类酶上，并改进探针以制作选择性的人类JMJD5探针，从而使我们能够理解内源性人类JMJD5‘S与癌症的联系。JMJD5探针的化学工作可能对昼夜节律/植物细胞信号转导的知识以及加氧酶在癌症中的作用产生重大影响。该项目属于EPSRC‘化学生物学和生物化学’研究领域，属于‘表观遗传学功能探针’SBM项目领域。DPhil项目开展的工作将主要是有机合成MS，并将其用于开发JMJD5的新分析方法。蜂窝工作将在学术和工业合作者的合作下进行。

The Jumonji (JmjC) enzymes are 2-oxoglutarate (2-OG) dioxygenases that carry out oxygenation reactions using 2-OG and ferrous iron as a cosubstrate / cofactor. In diseases including mental disorders and cancer, 2OG- dependant oxygenases of the JmjC family are often altered through mutation, translocation and can be in some cases deleted entirely. These alterations are suggested as cancer causing events, as these enzymes are proposed to be oncoproteins and tumor suppressors. JMJD5 is an arginine hydroxylase and a JmjC domain-containing protein present in the cell nucleus and cytoplasm of plant and animal cells.Chemical probes can be useful in defining biological function, but none have been identified for JMJD5. A chemical probe is a small molecule designed to bind selectively to a target and alter its function; normally by inhibition. By changing the function of the target (an enzyme for example) through inhibiting or stimulating it, a chemical probe can help determine the protein's role in living systems. In Chemical probes are used alongside genetic approaches to discover and validate the role of a protein or enzyme in a disease. Producing probes to profile new proteins is a technique known as activity-based protein profiling (ABPP).ABPP will be carried out on the plant enzyme JMJD5; the ABPP design will involve the use of structural information and synthesis of probes and testing in vitro. The novel JMJD5 inhibitors will then be used to probe the function of JMJD5 in human and plant cells, and in the longer-term intact plants. We will also characterise plant and human JMJD5 kinetics during assay development employing mass spectrometry, something that has never previously been carried out before. In collaboration, I will also work to obtain the first crystal structures of plant JMJD5 (including in complex with inhibitors / probes to inform on the design process). The probes will also be used in efforts to capture JMJD5 substrates. Characterisation of JMJD5 in plants will open new pathways into circadian system research and will synergise with work on human JMJD5; which has links to cancer. Inhibition of plant JMJD5 will be studied to observe the effects produced, especially with respect to circadian rhythm; this avenue is much easier to carry out in plant models than humans/animals (and better from an ethical perspective). It is envisaged the chemical and (initial) structural work on plant JMJD5 can be completed within a 2-year timescale, with 1 year for the chemical biology work.Synthesising and utilising probes for plant JMJD5 should help enable us to produce the first crystal structures for plant JMJD5 and will enable protein profiling in plant cells to detect endogenous levels of JMJD5 and identify its substrate(s) in plants. This work will open research into plant signalling and will enable a better understand the 2OG-dependent oxygenases from a physiologically relevant perspective. Work carried out on plant JMJD5 will be synergistic with work on human JMJD5. Due to plant JMJD5 being so similar to human JMJD5, it should be possible to transfer knowledge obtained working with the plant enzyme to the human enzyme and refine the probes created to make selective human JMJD5 probes and therefore enable us to understand endogenous human JMJD5's link to cancer. Chemical work on JMJD5 probes has the potential to have a substantial impact on both knowledge of circadian rhythm / plant cell signalling and the roles of oxygenases in cancer.This project falls within the EPSRC 'Chemical Biology and Biological Chemistry' research area and fits into the 'Functional Probes for Epigenetics' SBM project field. The work carried out in this DPhil project will be mainly organic synthesis and use of MS in development of novel assays for JMJD5. Cellular work will be carried out in collaboration with academic and industrial collaborators.