Recruiting the autophagy-lysosome pathway to engineered condensate-forming proteins.

Abstract

Biomolecular condensates (BCs) are membraneless, subcellular compartments segregated from the cytoplasm by liquid-liquid phase separation, a thermodynamic process driven by the chemical properties of the constituent biomolecules. BCs are involved in a range of cellular processes such as signalling and transcriptional regulation, as well as the autophagy-lysosome pathway, one of the cell's main degradation mechanisms. Selective autophagy can recognise BCs containing cargo proteins through direct interactions with LC3, a protein present on the inner membrane of phagophores. Consensus tetratricopeptide repeat proteins (CTPRs) are highly stable proteins composed of repeated helix-turn-helix subunits that are useful in protein engineering as a result of their modular architecture and amenability to rational design. In this project, the CTPR scaffold that has been engineered to bind LC3 through grafted LIR motifs is fused to low complexity domains (LCDs) to induce BC formation. Here we investigate the effects of different LIR motifs within the engineered BCs to understand how LC3 interactions can change the physico-chemical properties of the condensates. Using these BCs we hope to gain a better understanding of how and why the autophagy-lysosome pathway utilises BCs for client degradation, with the potential for adapting the engineered proteins as targeted protein degradation therapeutics.