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

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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.