Development of a novel engineered sensor to detect heme bioavailability within cells.

Heme (Iron protoporphyrin IX) is an essential cofactor.

When bound to a heme dependent protein, the protein is able to participate in a number of functions such as, electron transport, gas transport and metabolism.

An increase in heme unbound to protein (free heme) however can be dangerous as this cofactor is both hydrophobic and cytotoxic. Heme production and degradation requires precise control in order to maintain a healthy concentration of free heme.

It has come to light that heme chaperone proteins can transport heme to its desired destination, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one chaperone protein of interest. Heme chaperones may be another mechanism to ensure the concentration of free heme remains stable. The aim of this project is to understand the bioavailibity of heme for indoleamine 2,3 dioxygenase (IDO1).

IDO1 catalyses the oxidation of the essential amino acid tryptophan to N-formyl kynurenine and in turn activates the kynurenine pathway.

The role of the kynurenine pathway is the formation of nicotinamide adenine dinucleotide (NAD+), an important energy source, however some metabolites of this pathway have been associated with numerous neurological disorders such as Alzheimer, Huntington's disorder and schizophrenia.

Another downside of increased IDO1 activity is the depletion of tryptophan, which can cause a signalling cascade resulting in T reg cell proliferation and ultimately immunosuppression.

This is especially prevalent in tumour microenvironments whereby increased IDO1 activity has been linked to a poor cancer prognosis.

Heme binding to IDO1 is what makes this enzyme able to activate the kynurenine pathway and so is an important step to probe in a physiological environment. This is done using a new engineered sensor, named IDO1mCitrine. This recombinant protein consists of IDO1 tagged with a compatible fluorescent protein named mCitrine.

When heme is bound to IDO1 there is energy transfer from the fluorescent protein to heme by Förster Resonance Energy Transfer (FRET), resulting in a reduction of fluorescence from mCitrine, this is called quenching.

This fluorescence decrease can be quantitatively measured using methods such as Fluorescence Lifetime Imaging Microscopy (FLIM) and therefore the bioavailability of heme can be deduced and compared within different cell types under varying conditions.

This project is supervised by Professor Emma Raven at the University of Bristol and falls within the Chemical Biology and Biological Chemistry EPSRC research area.