A novel mechanism that regulates the interaction of the autophagy receptor p62/SQSTM1 and the autophagosome protein LC3

BIONDI, RM; ALCOBER-BOQUET, L; ZANG, T; PIETSCH, L; SUESS, E; HARTMANN, M; PROSCHAK, E; GROSS, LZF; SACERDOTI, M; ZEUZEM, S; ROGOV, VV; LEROUX, AE; PIIPER, ALBRECHT

Rosario

Congreso; LIX Reunión Científica anual; 2023

Sociedad Argentina de Investigacion Bioquimica y Biologia Molecular (SAIB)

Autophagy is a highly conserved cellular process that allows degradation of large macromolecules. p62/SQSTM1 is a key adaptor protein that interacts both with material to be degraded and with LC3 at the autophagosome, enabling degradation of cargos such as protein aggregates, lipid droplets and damaged organelles by selective autophagy. Dysregulation of autophagy contributes to the pathogenesis of many diseases. y. In collaboration with our Frankfurt network of colleagues, we previously conducted a compound library screening and identified a small compound that binds to LC3, disrupting its interaction with the polypeptide LIRtide, a sequence derived from the LC3 Interacting Region (LIR) of p62/SQSTM1 (Hartmann et al., J Med Chem. 2021, 64(7):3720-3746. doi:10.1021/acs.jmedchem.0c01564). In this study we investigated if the interaction of p62/SQSTM1 with LC3b could be regulated. We purified full-length p62/SQSTM1 and established an in vitro assay that measures the interaction with LC3b. We used the assay to determine the role of the different domains of p62/SQSTM1 in the interaction with LC3. We identified a mechanism of regulation of p62 where the ZZ and the PB1 domains regulate the exposure of the LIR-sequence to enable or inhibit the interaction with LC3b. A mutation to mimic the phosphorylation of a site on the ZZ domain leads to increased interaction with LC3b. Also, a small compound that binds to the ZZ domain enhances interaction with LC3b. We conclude that the exposure of the LIR sequence is negatively regulated by the ZZ domain. We arrived at this conclusion through domain deletion experiments, mutagenesis to disrupt ZZ domain folding, mutagenesis to Glu of a ZZ domain Thr known to become phosphorylated, and further verification using a ZZ-domain binding compound that enhanced the interaction with LC3b. Dysregulation of these mechanisms in p62/SQSTM1 could have implications for diseases where autophagy is affected. In conclusion, our study highlights the regulated nature of p62/SQSTM1 and its ability to modulate the interaction with LC3b through a LIR-sequence Accessibility Mechanism (LAM). Furthermore, our findings suggest the potential for pharmacological modulation of the exposure of LIR, paving the way for future therapeutic strategies.