Abstract: Objective To explore the regulatory role of mechanistic target of rapamycin complex 1(mTORC1)on the protein stability of triphenyl peptidase 1(TPP1)through β-TrCP. Methods Interactions between TPP1 and mTORC1 or β-TrCP were investigated via GST Pull-down,coimmunoprecipitation and Western blotting.The cells were cultured under various nutrient stress conditions or treated with rapamycin to inhibit mTORC1 activity.TPP1 protein expression was assessed via Western blotting,while TPP1 transcriptional levels were measured via qPCR.Proteasome or cullin neddylation inhibitors were used to determine whether TPP1 degradation is mediated by the proteasome and Cullin-dependent E3 ubiquitin ligases.Putative β-TrCP recognition phosphodegron motifs in TPP1 were mutated via site-directed mutagenesis,replacing serine(S)residues with alanine(A).Lentivirus-delivered shRNA was employed to silence β-TrCP or Cullin1 gene expression.TPP1 phosphorylation and ubiquitination levels were detected by Western blotting,and the essential role of two serine residues within the phosphodegron in its ubiquitination was functionally validated.In vitro TPP1 activity assays were performed to assess whether phosphodegron mutations affect TPP1 enzymatic activity. Results TPP1 interacted with both mTORC1 and β-TrCP.TPP1 protein levels were negatively correlated with mTORC1 activity,and this regulation was not due to transcriptional changes.Inhibition of the proteasome or knockdown of β-TrCP increased TPP1 protein levels.In vitro kinase and in vivo ubiquitination assays confirmed that TPP1 was phosphorylated by mTORC1 and ubiquitinated by β-TrCP.Mutations in the TPP1 phosphodegron did not affect its enzymatic activity. Conclusion mTORC1 phosphorylates TPP1,promoting its specific binding to β-TrCP,leading to TPP1 ubiquitination and subsequent proteasomal degradation.

Key words: mTORC1, β-TrCP, TPP1, ubiquitin-proteasome system