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

Jumonji （JmjC）酶是2-氧葡萄糖酸酯（2-OG）双加氧酶，它以2-OG和亚铁作为辅物/辅因子进行加氧反应。在包括精神障碍和癌症在内的疾病中，JmjC家族的2OG依赖性加氧酶经常通过突变、易位而改变，在某些情况下可以完全删除。这些改变被认为是致癌事件，因为这些酶被认为是癌蛋白和肿瘤抑制因子。JMJD5是存在于植物和动物细胞的细胞核和细胞质中的精氨酸羟化酶和含JmjC结构域的蛋白。化学探针可用于定义生物功能，但尚未确定JMJD5的化学探针。化学探针是一种小分子，可以选择性地与目标结合并改变其功能；通常是通过抑制。通过抑制或刺激目标（例如酶）来改变其功能，化学探针可以帮助确定蛋白质在生命系统中的作用。化学探针与遗传方法一起用于发现和验证蛋白质或酶在疾病中的作用。生产探针来分析新蛋白质是一种被称为基于活性的蛋白质分析（ABPP）的技术。ABPP将在植物酶JMJD5上进行；ABPP的设计将涉及到结构信息的使用和探针的合成以及体外测试。新的JMJD5抑制剂将用于探测JMJD5在人类和植物细胞中的功能，以及在长期完整植物中的功能。我们还将利用质谱法在分析开发过程中表征植物和人类JMJD5动力学，这是以前从未进行过的。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/探针的复合物，以告知设计过程）。这些探针还将用于捕获JMJD5底物。JMJD5在植物中的特性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；这与癌症有关。将对植物JMJD5的抑制进行研究，观察产生的效果，特别是对昼夜节律的影响；这种方法在植物模型中比在人类/动物模型中更容易实施（从伦理角度来看更好）。预计植物JMJD5的化学和（初步）结构工作可以在2年内完成，化学生物学工作将在1年内完成。植物JMJD5探针的合成和利用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分析能够检测内源性JMJD5水平并识别其在植物中的底物。这项工作将开启对植物信号传导的研究，并将从生理学相关的角度更好地理解依赖于20g的加氧酶。在植物JMJD5上开展的工作将与人类JMJD5的工作协同进行。由于植物JMJD5与人类JMJD5非常相似，应该有可能将利用植物酶获得的知识转移到人类酶上，并改进创建的探针，以制造选择性的人类JMJD5探针，从而使我们能够了解内源性的人类JMJD5与癌症的联系。JMJD5探针的化学工作有可能对昼夜节律/植物细胞信号传导和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。在这个博士项目中进行的工作将主要是有机合成和利用质谱技术开发新的JMJD5检测方法。蜂窝工作将与学术和工业合作者合作进行。

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.