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

Jumonji 结构域蛋白 JMJD5 的基于活性的蛋白分析 (ABPP)

• 批准号: 2112240
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2112240
• 关键词: Activity; based; protein; profiling; ABPP

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.

Jumonji（JmjC）酶是2-酮戊二酸（2-OG）双加氧酶，其使用2-OG和亚铁作为共底物/辅因子进行氧化反应。在包括精神障碍和癌症的疾病中，JmjC家族的20 G依赖性加氧酶通常通过突变、易位而改变，并且在某些情况下可以完全缺失。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。JMJD 5是一种精氨酸羟化酶，是一种存在于植物和动物细胞核和细胞质中的含有JmjC结构域的蛋白质。化学探针是一种小分子，设计用于选择性地结合目标并改变其功能;通常通过抑制。通过抑制或刺激靶标（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使用，以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称为基于活性的蛋白质分析（ABPP）的技术。ABPP将在植物酶JMJD 5上进行; ABPP设计将涉及使用结构信息和探针的合成以及体外测试。然后，新型JMJD 5抑制剂将用于探测JMJD 5在人类和植物细胞以及长期完整植物中的功能。我们还将在采用质谱法的测定开发过程中研究植物和人类JMJD 5的动力学，这是以前从未进行过的。在合作中，我还将努力获得植物JMJD 5的第一个晶体结构（包括与抑制剂/探针复合以告知设计过程）。探针还将用于捕获JMJD 5底物。JMJD 5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JMJD 5的研究协同作用; JMJD 5与癌症有关。将研究植物JMJD 5的抑制以观察产生的效果，特别是关于昼夜节律;这一途径在植物模型中比在人类/动物中更容易进行（并且从伦理角度来看更好）。设想植物JMJD 5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1年用于化学生物学工作。合成和利用植物JMJD 5的探针应该有助于使我们能够产生植物JMJD 5的第一个晶体结构，并将使植物细胞中的蛋白质谱分析能够检测植物中JMJD 5的内源水平并鉴定其底物。这项工作将开启对植物信号传导的研究，并从生理学相关的角度更好地了解2 OG依赖性加氧酶。在植物JMJD 5上进行的工作将与在人类JMJD 5上进行的工作协同。由于植物JMJD 5与人类JMJD 5如此相似，因此应该可以将使用植物酶获得的知识转移到人类酶中，并改进所创建的探针以制备选择性人类JMJD 5探针，从而使我们能够了解内源性人类JMJD 5与癌症的联系。JMJD 5探针的化学工作有可能对昼夜节律/植物细胞信号传导的知识和加氧酶在癌症中的作用产生实质性影响。该项目福尔斯属于EPSRC“化学生物学和生物化学”研究领域，并符合“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MS在开发JMJD 5新检测方法中的应用。蜂窝工作将与学术和工业合作者合作进行。