Understanding supply and demand for heme in cells
Lead Research Organisation:
University of Leicester
Department Name: Chemistry
Abstract
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Technical Summary
Heme is essential for the survival of virtually all living systems - from bacteria, fungi and yeast, through plants to animals. In the last few years, heme has been shown to have an important regulatory role in cells, in processes such as transcription, regulation of the circadian clock, and the gating of ion channels.
To act in a regulatory capacity, heme needs to move from its place of synthesis (in mitochondria) to other locations in cells. Whilst this concept is broadly acknowledged, how it happens is has remained unknown. Hence, while we know in detail how the heme lifecycle begins (heme synthesis), and how it ends (heme degradation) what happens in between is almost completely blank. This is important if we are to understand, and then to control, heme-dependent regulatory process. New approaches are needed to precisely quantify heme distributions and patterns of heme movements across sub-cellular compartments.
Our hypothesis, based on considerable preliminary data, is that a proportion of the total heme complement of the cell (which we have named "exchangeable heme") can be mobilised discretely and specifically, with a level of control that provides a mechanism for heme-dependent regulation as well as protection against the deleterious effects of excess heme at high concentrations. We have designed a new fluorescent heme-responsive sensor (mAPXmEGFP) that can precisely quantify heme concentrations - in different cellular locations and in real time. We will use this sensor along with fluorescent lifetime imaging and other fluorescent heme-binding probes to build a detailed picture of cellular-heme dynamics and mobilisation. We will identify where heme is located, what the concentrations are, how and when heme moves around in cells, and how heme distributions vary in response to local changes in heme concentration.
These are ambitious questions at the forefront of the discipline. It will change what we understand about the role of heme in biology.
To act in a regulatory capacity, heme needs to move from its place of synthesis (in mitochondria) to other locations in cells. Whilst this concept is broadly acknowledged, how it happens is has remained unknown. Hence, while we know in detail how the heme lifecycle begins (heme synthesis), and how it ends (heme degradation) what happens in between is almost completely blank. This is important if we are to understand, and then to control, heme-dependent regulatory process. New approaches are needed to precisely quantify heme distributions and patterns of heme movements across sub-cellular compartments.
Our hypothesis, based on considerable preliminary data, is that a proportion of the total heme complement of the cell (which we have named "exchangeable heme") can be mobilised discretely and specifically, with a level of control that provides a mechanism for heme-dependent regulation as well as protection against the deleterious effects of excess heme at high concentrations. We have designed a new fluorescent heme-responsive sensor (mAPXmEGFP) that can precisely quantify heme concentrations - in different cellular locations and in real time. We will use this sensor along with fluorescent lifetime imaging and other fluorescent heme-binding probes to build a detailed picture of cellular-heme dynamics and mobilisation. We will identify where heme is located, what the concentrations are, how and when heme moves around in cells, and how heme distributions vary in response to local changes in heme concentration.
These are ambitious questions at the forefront of the discipline. It will change what we understand about the role of heme in biology.
People |
ORCID iD |
| Andrew Hudson (Principal Investigator) |
| Description | We have made three significant advances in our methods for measuring exchangeable haem in live cells. Exchangeable haem are molecules of the small organo-transition complex that are not associated with the following protein classes: the globins (haemoglobin and myoglobin), the cytochromes, the peroxidases and other enzymes. Recent discoveries have been made that implicate exchangeable haem in cellular regulation and signalling. However, the abundance of exchangeable haem in different cellular compartments was poorly understood until recently. Before the project started, we had developed a haem sensor that could be used to measure the distribution of exchangeable haem using fluorescence live-cell imaging. Our haem sensor is a fusion of two different proteins to make a chimeric construct; these are a fluorescent protein and a haem recognition motif. The binding of haem to the recognition motif leads to quenching of the emission intensity of the fluorescent protein and a reduction of its fluorescence lifetime. In our previous work, we used the the change in fluorescence lifetime in order to quantify the abundance of haem in different cellular compartments. Images of the cellular distribution of haem were obtained by fluorescence lifetime imaging. In the current funded project, we have made the following four advances: (1) Previously, the fluorescence lifetime images were obtained by exciting the fluorescent protein (in this case, green fluorescent protein) by blue laser light. This particular wavelength of light is highly damaging to cells, and precluded time-lapse imaging over extended period in order to observe cellular changes in the abundance of cellular haem. In the most recent work, we have adapted the method to enable for multiphoton fluorescence imaging microscopy (which uses IR radiation that is considerably less harmful to cells) in place of single-photon fluorescence imaging microscopy (which uses the aforementioned blue laser light at a wavelength that is near to the UV region and damaging to cells). This development will be described in a future publication. (2) We have designed a new protein using the same principal as the original construct, however, the new design is sensitive to the oxidation state of haem and it is able to allow us to discriminate between ferrous and ferric haem. This particular design was one of the main objectives of the funded-project work. (3) We have also designed a new protein construct that can be used to deplete haem in specific cellular compartments. (4) We have designed a variants of the sensor using different colours of fluorescent protein. This has enabled multiplexed measurements of haem in different cellular compartments. |
| Exploitation Route | Our designs for haem sensors would be valuable tools for anyone interested in the regulatory role of haem in cells. We also anticipate that, in the years to come, these sensors will be important tools to determine the efficacy of drug compounds designed to mitigate against diseases that lead to excesses or deficiencies of haem in cell assays. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| Description | Electron paramagnetic resonance studies of heme sensor proteins |
| Organisation | Liverpool John Moores University |
| Department | School of Pharmacy and Biomolecular Sciences |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Preparation of heme sensor proteins using expression protocols in bacteria. Spectroscopic analysis of these proteins by UV-visible, fluorescence and NMR spectroscopy. |
| Collaborator Contribution | Intellectual input, training, and support in electron paramagnetic resonance spectroscopy. |
| Impact | The collaboration started in November 2024, and we are planning the first experiments in March 2025. Currently, there are no outputs or outcomes. |
| Start Year | 2024 |
| Description | Heme sensor design, development and deployment |
| Organisation | University of Bristol |
| Department | School of Chemistry |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This collaboration was fully articulated in the case for support made in the grant application, and we have followed this plan in the research activities. Our collaboration with Prof Emma Raven's group is long standing. We have expertise in sensor design and the interpretation of quantitative sensing, with the Raven group providing expertise in protein expression and purification, along side the preparation of cell lines for imaging. Our work has focussed on understanding the mechanism of heme supply and demand through the design, development and deployment of heme sensors. |
| Collaborator Contribution | This collaboration was fully articulated in the case for support made in the grant application, and we have followed this plan in the research activities. Our collaboration with Prof Emma Raven's group is long standing. We have expertise in sensor design and the interpretation of quantitative sensing, with the Raven group providing expertise in protein expression and purification, along side the preparation of cell lines for imaging. Our work has focussed on understanding the mechanism of heme supply and demand through the design, development and deployment of heme sensors. |
| Impact | This is a multidisciplinary collaboration: biophysics, imaging and spectroscopy (Leicester) and molecular biology (Bristol) |
| Start Year | 2022 |
| Description | Small angle X Ray scattering studies of heme sensor proteins |
| Organisation | Max IV Laboratory |
| Country | Sweden |
| Sector | Academic/University |
| PI Contribution | Preparation of heme sensor proteins using expression protocols in bacteria, and experimentation on the heme sensors using a X Ray beamline at the MAX IV synchrotron. |
| Collaborator Contribution | Training and technical support in small angle X Ray scattering on the CoSAXS beamline at MAX IV synchrotron, Lund, Sweden. |
| Impact | The collaboration started in December 2024, and there are no outputs at present. |
| Start Year | 2024 |
| Description | Work experience (three separate activities in June 2023, August 2023 and August 2024) |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | In June 2023, the Hudson group at the University of Leicester hosted two pupils from Robert Smyth Academy in Market Harborough for 5 working days. They were given their own research project, and they had the opportunity to shadow other members of the research group. At the end of the 5 working days, they presented their findings by giving a short talk to the group. The activity was intended to provide experience of scientific research and careers in STEM. In August 2023, Andrew Hudson at the University of Leicester hosted two pupils from Leicester schools for 1 day. During this time, they worked alongside Andrew to answer a question directly related to the BBSRC-funded project. The activity was intended to provide experience of the research components of a Chemistry degree course at a UK University. |
| Year(s) Of Engagement Activity | 2023,2024 |