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Understanding the role of haem in the mediation of circadian rhythmicity

Primary Supervisor: Professor Andrew Hudson, School of Chemistry and the Leicester Institute of Structural & Chemical Biology

Secondary supervisor: Dr Ezio Rosato and Prof Charalambos Kyriacou, Department of Genetics & Genome Biology

PhD project title: Understanding the role of haem in the mediation of circadian rhythmicity

University of Registration: University of Leicester

Project outline:

Background

Iron protoporphyrin IX (haem) has long been known to play a pivotal role, as a cofactor or prosthetic group, in the functional activity of many different proteins involved in fundamental biological processes: from transport and storage of oxygen (i.e., the globins), movement of electrons and redox chemistry (i.e., the cytochromes) to chemical catalysis (i.e., the P450s). However, recently haem has been shown to intervene in the circadian clock by regulating the rhythmic transcriptional activity of some clock proteins.

The fundamental nature of the discovery stems from the fact that it highlights a previously unknown interaction of haem with proteins. In this case, the haem complex is not pivotal to the functional activity of the protein. Instead, it is only required intermittently to modulate the protein’s behaviour. This observation suggests that haem is not a static complex locked inside a protein and then degraded when the same protein is recycled. Conversely, haem must be able to roam about the cell, as and when required. Exactly how this can be achieved is a conundrum because free haem (which is not associated with a protein partner) is cytotoxic.

Objectives

This project builds on a breakthrough discovery (see reference below) in which an in-built haem-buffering system was shown to exist in cells. This buffering system sequesters large concentrations of haem, and then releases the haem complexes one-at-a-time for incorporation into regulatory or signaling proteins. The student will have the opportunity to explore exactly how this sophisticated mechanism is utilised by mammalian and Drosophila (fruit fly) cells to modulate circadian rhythms. Our group has designed a fluorescent-protein based sensor that can be expressed in both cell lines (mammalian) and living organisms (fruit fly). By means of fluorescence lifetime imaging (FLIM), the sensor is responsive to heme availability in the cell and can be used to precisely determine the cellular distribution of heme. The aims of this project are:

  • To image the distribution of haem in synchronised (by a serum shock) fibroblasts at different circadian times. This will reveal whether the subcellular distribution/availability of haem varies with the molecular progression of the circadian cycle assessed by parallel monitoring of clock proteins (as markers).
  • To image the distribution of haem, in vivo, in transgenic Drosophila melanogaster flies at different circadian times. We will monitor the subcellular distribution of haem in clock neurons such as pacemaker cells and photoreceptors while in parallel measuring the rhythmic abundance of clock proteins
  • To manipulate the subcellular concentration and distribution of haem in fibroblasts and Drosophila clock cells and measure the impact on molecular (fibroblasts and fly clock neurons) and behavioural (whole flies) rhythmicity. By these means, we will discover the relevance of haem in different cellular compartments, demonstrate how changes in local concentrations of haem lead to clock-related phenotypes, and, by measuring transcription and protein abundance, show how the transcription and the activity of clock genes and proteins are modulated by haem availability.
  • To establish new methodologies to detect binding interactions between haem and clock proteins, such as Rev-erb, E75, NPAS2, and CLOCK. This will involve the design, preparation and testing of new genetically-encoded sensors.

Methodology

The PhD candidate will join an interdisciplinary team of geneticists, chemical biologists and biophysicists, however, prior experience is required in either genetics or molecular biology only. The student will learn techniques in protein preparation and purification, how to culture genetically-modified cell lines and organisms, and fluorescence spectroscopy and cellular imaging methods.

Reference:

Gallio, et al. Understanding the Logistics for the Distribution of Heme in Cells. JACS Au 2021.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Neuroscience and behaviour & Integrated Understanding of Health: Ageing

Techniques that will be undertaken during the project:

  • Design of genetically-encoded biosensors
  • Genetic modification of cell lines and organisms
  • Fluorescence lifetime imaging microscopy

Contact: Professor Andrew Hudson, University of Leicester