Investigating a key autophagy protein-protein interaction and its modulation by phosphorylation and an ALS-related mutation

Autophagy is a cellular degradation pathway which removes cytoplasmic material including protein aggregates, damaged organelles and pathogens. The interaction of the autophagy adaptor p62 with lipid-anchored hAtg8 receptor proteins is a key step in this process whereby cargo bound to p62 is sequestered to the autophagosomal membrane. Here it is engulfed for transport to the lysosome where it is enzymatically degraded. Recruitment of p62 filaments to the autophagosome membrane also plays an important role in autophagosome expansion. The p62-hAtg8 interaction is mediated by the Atg8-interacting motif (AIM) which is a short sequence present in many autophagy adapter/receptor proteins. For the biophysical characterisation required in this work, seven hAtg8 proteins were produced and characterised; the six hAtg8 proteins and one phosphomimetic mutant. In vitro biophysical characterisation has shown a fivefold range of binding affinities for a WT p62 AIM peptide in its binding to the six hAtg8 proteins, with GABARAPL1 bound with the highest affinity. The binding of GABARAPL2 with the WT p62 AIM peptide has been structurally characterised by NMR chemical shift mapping as an investigation into the entropically driven nature of this interaction, which is unique amongst the hAtg8 proteins. This identified a novel binding patch for the peptide on the GABARAPL2 surface, particulaly in relation to Leu341 of p62. Regulation of this interaction by phosphorylation has been investigated. Phosphorylation of p62 by TBK1 has been shown to increase the binding affinity of the protein for LC3B. A previously known phosphorylation site on p62 has then been characterised which showed that a pS342 p62 AIM peptide binds to all six hAtg8 proteins with higher affinity than the WT peptide. NMR-based strutural characterisation of the pS342 p62 AIM-LC3B interaction indicated that this interaction occurs in a related but slightly altered manner to the WT p62 AIM-LC3B interaction. The phosphomimetic T50E LC3B mutant has been shown to bind with a weaker affinity to the WT p62 AIM peptide than WT LC3B. Dysregulation of autophagy has become a key area of research in a number of neurodegenerative disorders including amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). Previous work has shown that an ALS-associated L341V mutation of the p62 AIM is defective in recognition of LC3B. This work expands upon this observation and looks at the interaction of L341V p62 with all hAtg8 proteins which produces a more complex picture where different hAtg8 proteins are affected differently by the mutation. However, the average binding affinity and selectivity of the hAtg8 proteins with the p62 AIM is decreased by the mutation which can be clearly associated with ALS-related neurodegeneration