Bioinformatic analysis of the origins of ABCA and ABCG families and development of the thermostability assay for ABCG2

Abstract

The ATP-binding cassette super-family G member 2(ABCG2); also known as the breast cancer resistant protein) mapped on chromosome 4q22 is known to have important physiological roles in many tissues such as mammary gland, blood brain barrier and gastrointestinal tract. Furthermore, ABCG2 is involved in the transportation of chemically diverse substrates such as steroids and other organic ions. Most importantly, ABCG2 has been suggested to be involved in multidrug resistance in chemotherapy. Understanding the thermostability of the ABCG2 protein, will allow a better understanding of how drugs and inhibitor interact with the protein. The first aim in this thesis was to express, characterise and purify ABCG2 using Pichia pastoris, hence establish whether the thermostability cellular thermal shift assay (CETSA) showed proof of principle on ABCG2. Results concluded that Pichia pastoris is an excellent expression system which can be used to express and characterise ABCG2. The thermostability CETSA assay performed on ABCG2 showed that the melting temperature was 55°C, thus, proving that the CETSA assay could be used in future to examine the thermostability of ABCG2 in the presence of a ligand. Understanding the structure and mechanism of ABCG2 starts by looking at the evolutionary relationship with a common ancestor. The second aim of this thesis was to find potential evolutionary relationship between ABCA and ABCG families to bacterial ABC transporter such as: mechanotransducer MacB, WzmWzt, MlaE and MlaF. Results showed MacB transmembrane domain (TMD), and nucleotide binding domain (NBD) had significant alignment to TMD and NBD of ATP-binding cassette super-family A member 1 (ABCA1), ABCG2 and ATP-binding cassette super-family G member 5 and 8 (ABCG5/G8). Henceforth, indicating ABCA and ABCG families may have originated from MacB transporters. Similarly, the TMD and NBD of WzmWzt, MlaE and MlaF are seen to align significantly well to the TMD and NBD of ABCA and ABCG families, this suggest that convergence evolution may be taking place between the two families.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Robert Ford (Supervisor) & Martin Pool (Supervisor)