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Butler, Kevin (2021) Biophysical studies of ligand selectivity for the hATG8 family of autophagy proteins. PhD thesis, University of Nottingham.

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Abstract

Selective autophagy is a process by which potentially toxic material such as aggregated proteins, organelles and pathogens are specifically targeted and transported to autophagosomes and subsequent degradation by the lysosome. The process relies on selective autophagy receptor proteins (SARs) recognising cargo and then interacting with the six human autophagy related receptor proteins (hATG8) anchored to the membrane of the autophagosome. The interaction between SARs and hATG8 proteins requires a short amino acid sequence called an LC3 interacting region (LIR). LIRs consist of a core motif [W/F/Y]0-X1-X2-[L/V/I]3 with the aromatic residue often preceded by acidic residues. The LIR interacts with ATG8 proteins at a conserved binding site consisting of two hydrophobic pockets with aromatic and hydrophobic residues sitting in hydrophobic pockets one and two respectively. Neighbour of BRAC1 gene 1 (NBR1), unlike most SARs contains two LIRs and whilst both contain the core LIR motif they vary in the amino acids at the aromatic and hydrophobic position and the composition of amino acids preceding the aromatic residue. Using Biophysical methods the interaction of the two LIRs of NBR1 (Sequences containing 11-12 amino acids) with the hATG8 proteins were studied to gain a better understanding of the effect the different LIR sequence has on binding.

NMR titration experiments with subsequent binding site mapping indicated that whilst both LIRs interact with LC3B WT at the hATG8 canonical binding site there are variations in how the LIR interacts with LC3B. The interaction of NBR1 LIR1 (ASSEDYIIILPE) containing more acidic amino acids preceding the aromatic forms more interactions with the face of LC3B than that of NBR1 LIR2 (AQDLLSFELLD).

An LC3B binding site mutant that altered the charge on the face of LC3B WT showed preferential binding for NBR1 LIR2. NMR binding site studies indicated that this change allowed NBR1 LIR2 to fit tighter against the binding surface of LC3B.

Using NMR binding site mapping a phosphomimetic mutant showed that phosphorylation of LC3B at threonine 29 made very little difference to how NBR1 LIR1 interacted with LC3B. However, it made a large difference in the interaction with LIR2 with the aromatic residue of the LIR no longer sitting in HP1.

The LIRs of NBR1 showed a preference to binding to LC3A over the other members of the LC3 subfamily. NMR binding site mapping showed the LIRs of NBR1 bound to LC3A at the canonical binding site. However, the structures generated in HADDOCK (a molecular docking program) for the interaction of LC3A and LIR1 showed the aromatic binding into HP2 rather than HP1. For NBR1 LIR2 Phe563 was seen to sit more across the face of LC3A and changes around the hydrophobic pocket on LC3A when compared to LC3B allowed a more favourable interaction of the LIR with the face of LC3A.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Searle, Mark
Keywords:	Biophysical studies, Ligand selectivity, hATG8 family, Autophagy proteins
Subjects:	Q Science > QD Chemistry > QD450 Physical and theoretical chemistry
Faculties/Schools:	UK Campuses > Faculty of Science > School of Chemistry
Item ID:	66737
Depositing User:	Butler, Kevin
Date Deposited:	31 Dec 2021 04:41
Last Modified:	31 Dec 2023 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/66737

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