Yeast-expression and molecular properties of human mitochondrial Uncoupling Protein 2

Dela Rosa, Margeoux Angelique Siscar (2023) Yeast-expression and molecular properties of human mitochondrial Uncoupling Protein 2. Doctoral thesis, University of East Anglia.

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Abstract

Uncoupling protein 2 (UCP2) is a mitochondrial carrier protein implicated in various pathophysiological conditions, such as diabetes and cancers. Its involvement in numerous dysregulated metabolic pathways make it an attractive target for therapeutic intervention. However, the exact function of the protein remains unclear. UCP2 has been proposed to function as an uncoupling protein to ‘mildly uncouple’ mitochondria mitigating the production of reactive oxygen species by mitochondria, or to transport 4-carbon metabolites across the mitochondrial inner membrane, which provides metabolic flexibility to the cell under conditions of nutrient stress. Mitochondrial carriers are inherently unstable, which makes them a particularly challenging class of membrane proteins to study. UCP2 is expressed in a variety of tissues, though at low abundance, where extraction from native sources is impractical. The heterologous expression of UCP2 in recombinant systems, such as bacteria, can produce misfolded protein, leading to incorrect conclusions on the protein’s properties. To date, the biochemical function of UCP2 remains widely contested.

This work describes optimisation of human UCP2 protein expression in a protease-deficient S. cerevisiae strain, to yield folded protein suitable for extraction, purification and functional characterisation. It was observed that substitution of leucine at position 2 produced ~4-fold more UCP2 protein compared to WT protein. Quantification of UCP2 expression in yeast mitochondria indicates that this system produces ~20-fold more protein than native sources, with ~75% of protein produced extractable with non-ionic detergents, indicative of folded material. Purification attempts using a combination of ion exchange and covalent chromatography were able to enrich UCP2 in an intact form, but not purify the protein to homogeneity.

To probe the ligand binding properties of UCP2 without the need to purify, a novel thermostability assay, termed the “gel blot protein thermostability” assay, was developed using a polyethylene glycol-conjugated, maleimide-based thiol-reactive probe. The assay was able to successfully detect the pH-dependent binding of inhibitory nucleotides to human UCP1 in solubilised yeast mitochondrial membranes, through significant stabilising shift in relative protein thermostability.

Equivalent tests with UCP2 revealed that the protein is less stable than UCP1, where purine nucleotides do not induce any changes in stability, consistent with lack of any binding. These findings suggest that UCP2 is not regulated by these ligands and has different functional properties to UCP1, implying a distinct biochemical role.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Chris White
Date Deposited:	04 Nov 2024 14:37
Last Modified:	04 Nov 2024 14:37
URI:	https://ueaeprints.uea.ac.uk/id/eprint/97461
DOI:

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