Dissection The Role Of Lysine Crotonylation In Leucine Deprivation-induced Autophagy

Leucine served as substrates for protein synthesis and signaling molecules to regulate diverse biological processes.Under various stress conditions,series of adaptive responses are activated,including autophagy.Leucine deprivation induced cellular autophagy.Autophagosomes wrap the substrates to be degraded.Under the catalysis of lysosomal hydrolases,substrates degrade and produce small molecular nutrients,which are recycled and utilized by the body.Therefore,autophagy is important for the maintenance of cell homeostasis and cell survival under the stress condition of nutrient deficiency.Protein posttranslational modification is the process of covalent protein processing.With the development of mass spectrometry,many new kinds of post-translational modifications have been identified including lysine crotonylation.In this study,the relationship between lysine crotonylation and leucine deprivation induced autophagy was found,and the molecular mechanism by which lysine crotonylation regulated autophagy was revealed.When cells are in a state of stress such as deprivation of leucine,autophagy will be activated to degrade and produce small molecule nutrients that can be recycled to maintain cellular homeostasis.This study found that the global crotonylation level was significantly increased during leucine deprivation-induced autophagy.Treatment of the crotonylation activator,sodium crotonate,increased the level of autophagic flux.Through proteomic identification and screening,it was found that 14-3-3ε K73 and K78 crotonylation modifications may play an important role in the regulation of autophagy induced by leucine deletion.Functional experiments found that the expression of the crotonylation deficient mutants,14-3-3ε K73 R and K78 R,significantly inhibited the autophagy process induced by leucine deprivation.Molecular dynamics simulation showed that the crotonylation of14-3-3ε K73 and K78 significantly increased the conformational instability of 14-3-3ε,leading to the disappearance of its interaction domain.Using immunoprecipitation(IP)combined with mass spectrometry assay,we identified that PPM1 B was downstream of 14-3-3ε,and leucine deprivation led to crotonylation of 14-3-3ε and subsequent dissociation of PPM1 B and 14-3-3ε.The dissociated activated PPM1 B had the activity of dephosphorylating the autophagy initiation complex ULK1,which led to the initiation of autophagy.Furthermore,HDAC7 interacted with 14-3-3ε and mediated the decrotonylation of 14-3-3ε.Leucine deprivation significantly suppressed the enzyme activity of HDAC7.Taken together,we propose that leucine deprivation inhibits HDAC7 activity and increases14-3-3ε crotonylation.PPM1 B was then released from 14-3-3ε to dephosphorylate ULK1,which leads to initiation of autophagy.This study analyzed the molecular mechanism of crotonylation regulation of leucineinduced autophagy and provided new insights for the functional study of leucine and crotonylation.In addition,this study highlights the important role of lysine crotonylation in the regulation of cell homeostasis,reveals the new biological function of lysine crotonylation as a protein modification,and provides a theoretical basis for potential application research.