Activity-based protein profiling (ABPP) of the Jumonji domain protein JMJD5
Lead Research Organisation:
University of Oxford
Abstract
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.
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.
Planned Impact
This programme is focused on a new cohort-driven approach to the training of next-generation doctoral scientists in the practice of novel and efficient chemical synthesis coupled with an in-depth appreciation of its application to biology and medicine.
This collaborative academic-industrial SBM CDT will have long-term benefit to the chemical industry, including the pharmaceutical, agrochemical and fine chemical sectors. These industries will benefit through: (i) the potential to employ individuals trained with broad and relevant scientific and transferable skills; (ii) new approaches to the investigation of complex biological and medical problems through novel chemistry; and (iii) better and more efficient synthetic methods.
We will link the work of DSTL, and our pharmaceutical and agrochemical partners (GSK, UCB, Vertex, Evotec, Eisai, AstraZeneca, Syngenta, Novartis, Takeda, Sumitomo and Pfizer) through a common theme of synthesis training. The design and synthesis of new compounds is essential for disease treatment and prevention, and for maintaining food security. Synthesis contributes significantly to UK tax revenue and results in sustained employment across a number of sectors. Employers in the finance, law, health, academic, analytical, government, and teaching professions, for example, also recognise the value of the translational skill-sets possessed by synthesis postgraduates, which this programme will provide.
The SBM CDT training programme will adopt an IP-free model to enable completely free exchange of information, know-how and specific expertise between students and supervisors on different projects and across different industrial companies. This will lead to better knowledge creation through unfettered access to information from all academics, partners and students involved in the project. By focussing on basic science, we will engender genuine collaboration leading to enabling technology that will be of use across a wide range of industries.
We will train the next generation of multidisciplinary synthetic chemists with an appreciation of the impact of synthesis in biology and medicine. Their unconstrained view of synthesis will aid in new scientific discoveries leading to new products, which (with appropriate inward investment), can lead to the formation of new companies and new UK employment.
We will, in part through an alliance with the Defence, Science and Technology Laboratory, engage with policy-makers to influence future policy issues involving chemistry such as food security and the rise of antibiotic resistance (both of which are relevant to our programme and are important for society as a whole).
Outreach and public engagement will be a key aspect of our programme; and all students in the proposed SBM CDT will take part in at least one outreach activity. Typical activities include: open days in the Chemistry Department through the 'Outreach Alchemists', engaging with the Oxfordshire Science Festival and participation in the various other activities already in place through the public engagement programme of the Department of Chemistry.
The research output of the students will be disseminated via high impact international publications and lectures; these will be of value to other academics in relevant fields and will be of value in the development of further research funding applications. Outreach activities and research output will also be advertised on a website dedicated to the proposed SBM programme.
This collaborative academic-industrial SBM CDT will have long-term benefit to the chemical industry, including the pharmaceutical, agrochemical and fine chemical sectors. These industries will benefit through: (i) the potential to employ individuals trained with broad and relevant scientific and transferable skills; (ii) new approaches to the investigation of complex biological and medical problems through novel chemistry; and (iii) better and more efficient synthetic methods.
We will link the work of DSTL, and our pharmaceutical and agrochemical partners (GSK, UCB, Vertex, Evotec, Eisai, AstraZeneca, Syngenta, Novartis, Takeda, Sumitomo and Pfizer) through a common theme of synthesis training. The design and synthesis of new compounds is essential for disease treatment and prevention, and for maintaining food security. Synthesis contributes significantly to UK tax revenue and results in sustained employment across a number of sectors. Employers in the finance, law, health, academic, analytical, government, and teaching professions, for example, also recognise the value of the translational skill-sets possessed by synthesis postgraduates, which this programme will provide.
The SBM CDT training programme will adopt an IP-free model to enable completely free exchange of information, know-how and specific expertise between students and supervisors on different projects and across different industrial companies. This will lead to better knowledge creation through unfettered access to information from all academics, partners and students involved in the project. By focussing on basic science, we will engender genuine collaboration leading to enabling technology that will be of use across a wide range of industries.
We will train the next generation of multidisciplinary synthetic chemists with an appreciation of the impact of synthesis in biology and medicine. Their unconstrained view of synthesis will aid in new scientific discoveries leading to new products, which (with appropriate inward investment), can lead to the formation of new companies and new UK employment.
We will, in part through an alliance with the Defence, Science and Technology Laboratory, engage with policy-makers to influence future policy issues involving chemistry such as food security and the rise of antibiotic resistance (both of which are relevant to our programme and are important for society as a whole).
Outreach and public engagement will be a key aspect of our programme; and all students in the proposed SBM CDT will take part in at least one outreach activity. Typical activities include: open days in the Chemistry Department through the 'Outreach Alchemists', engaging with the Oxfordshire Science Festival and participation in the various other activities already in place through the public engagement programme of the Department of Chemistry.
The research output of the students will be disseminated via high impact international publications and lectures; these will be of value to other academics in relevant fields and will be of value in the development of further research funding applications. Outreach activities and research output will also be advertised on a website dedicated to the proposed SBM programme.
