Identification And Mechanical Exploration Of Regulators In Amino Acid Deprivation-Induced Autophagy

(Macro)autophagy is an evolutionarily conserved process that survives cell during nutrition starvation and other stress condition.Amino acid deprivation efficiently induces autophagy.There are two ubiquitin-like conjugation systems involved in autophagosome biogenesis: Atg12-Atg5 and Atg8-phosphatidyl-ethanolamine(PE).In the Atg8-PE conjugation system,cysteine protease Atg4 cleaves initially synthesized Atg8(an unprocessed form,pro Atg8)to expose the glycine residue for conjugation with PE(lipidation)or Atg8-PE in autophagosomal membranes to recycle Atg8(delipidation).Among all the Atg4 orthologs(i.e.,ATG4A-D),ATG4 B performs a highly selective preference to microtubule-associated protein 1 light chain 3(LC3)in humans.Several studies proteins functioning upstream of ATG4 B regulates ATG4 B activity,but whether the regulation is affected by amino acid deprivation is unknown.The study was based on the previous results in our lab,by using a high-fidelity technology that combined tandem affinity purification with mass spectrometry,we identified a number of candidates which might interact with ATG4 B.Based on this,we performed two studies:1.PFKP facilitates ATG4 B phosphorylation during amino acid deprivation-induced autophagyIn the present study,we firstly validated that ATG4 B is essential for amino acid deprivation-induced autophagy in HEK293 T cells.We constructed ATG4 B KO cell line using CRISPR/Cas9.Subsequent western blotting(WB),immunofluorescence(IF)and phospholipase D bandshift assays indicated that ATG4 B loss inhibited the cleavage of pro LC3 and formation of LC3-I,while ATG4 B overexpression displayed excess delipidation of LC3-II,both eventually inhibit autophagy.ATG4B-interacting protein phosphofructokinase 1 platelet isoform(PFKP)was firstly focused on.Further immunoprecipitation(IP),GST pull-down and WB assays found that PFKP interacted with ATG4 B in an amino acid-sensitive manner.Loss-of-function assay of PFKP revealed that PFKP is a positive regulator for autophagy.Mechanical explorations using phosphorylation MS,IP,and in vitro phosphorylation assay indicated that PFKP functioning as a protein kinase phosphorylated ATG4 B at S34 upon amino acid deprivation.Furthermore,mutant assay in WT or PFKP KO cell and in vitro ATG4 B activity detection assay demonstrated that the PFKP-mediated phosphorylation of ATG4 B promoted p62 degradation and ATG4 B activity.Finally,due to the identification of phosphorylation of PFKP S386 in phosphorylation MS,mutant assay,IP and WB were performed in PFKP KO cells,and indicated that PFKP itself at S386 is important for its ability to regulating ATG4 B S34 phosphorylation.In sum,our findings for the first time identify PFKP as a protein kinase to phosphorylate Atg4 B at S34,which promotes Atg4 B activity and autophagy.2.LRP130-SLIRP complex regulates amino acid-induced autophagySeveral mitochondrial proteins were identified as ATG4B-interacting protein.ATG4 B IP and WB assays validated that LRP130-SLIRP interacted with ATG4 B in an amino acid sensitive manner.Sequence and domain analysis suggested that LRP130-SLIRP mainly locates to mitochondria,and probably dispersed in cytoplasm.Then rapid immunopurification of mitochondria and WB assay found that ATG4 B has no mitochondrial location.But LRP130-SLIRP could disperse in cytoplasm through WB of crude mitochondria and cytosol prepared by differential centrifugation.These results indicated that the interaction of LRP130-SLIRP with ATG4 B occurred in the cytosol.Further LRP130-SLIRP IP and WB validated LRP130-SLIRP could bind to ATG4 B in an amino acid-sensitive manner.Subsequently,we investigated whether the interaction plays a role in autophagy or mitochondrial function.Results of quantitative real time PCR and WB indicated that ATG4 B loss increased the levels of mt DNA-encoded m RNA and proteins.Measurement of glucose consumption,lactate production and ATP production revealed that ATG4 B affected the ATP production through oxidative phosphorylation pathway.Then by RNA interference,IF and WB,we found that knockdown of LRP130 and SLIRP promoted p62 degradation and LC3 puncta,and overexpression reversed the results.Therefore,LRP130-SLIRP is a negative regulator for autophagy.LRP130 mutants with different domains deletion were expressed in cells,and found that the deletion of the ENTH(Epsin N-terminal homology domain)domain made autophagy insensitive to amino acid deprivation.These results indicate that LRP130 may play a role in amino acid deprivation-induced autophagy dependent on the ENTH domain.