title: The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins.
abstract: The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.
authors: a Department of Biochemistry and Molecular Biology , Seoul National University College of Medicine , Seoul , Korea.
authors: Yanxialei
authors: Y
authors: Jiang
authors: a Department of Biochemistry and Molecular Biology , Seoul National University College of Medicine , Seoul , Korea.|b Department of Biomedical Sciences , Seoul National University Graduate School , Seoul , Korea.
authors: Jeeyoung
authors: J
authors: Lee
authors: a Department of Biochemistry and Molecular Biology , Seoul National University College of Medicine , Seoul , Korea.|b Department of Biomedical Sciences , Seoul National University Graduate School , Seoul , Korea.
authors: Jung Hoon
authors: JH
authors: Lee
authors: d Department of Applied Chemistry , College of Applied Sciences, Kyung Hee University , Yongin , Korea.
authors: Joon Won
authors: JW
authors: Lee
authors: a Department of Biochemistry and Molecular Biology , Seoul National University College of Medicine , Seoul , Korea.|b Department of Biomedical Sciences , Seoul National University Graduate School , Seoul , Korea.
authors: Ji Hyeon
authors: JH
authors: Kim
authors: a Department of Biochemistry and Molecular Biology , Seoul National University College of Medicine , Seoul , Korea.|b Department of Biomedical Sciences , Seoul National University Graduate School , Seoul , Korea.
authors: Won Hoon
authors: WH
authors: Choi
authors: b Department of Biomedical Sciences , Seoul National University Graduate School , Seoul , Korea.
authors: Young Dong
authors: YD
authors: Yoo
authors: c World Class Institute, Korea Research Institute of Bioscience and Biotechnology , Ochang, Cheongwon , Korea.
authors: Hyunjoo
authors: H
authors: Cha-Molstad
authors: c World Class Institute, Korea Research Institute of Bioscience and Biotechnology , Ochang, Cheongwon , Korea.
authors: Bo Yeon
authors: BY
authors: Kim
authors: b Department of Biomedical Sciences , Seoul National University Graduate School , Seoul , Korea.
authors: Yong Tae
authors: YT
authors: Kwon
authors: d Department of Applied Chemistry , College of Applied Sciences, Kyung Hee University , Yongin , Korea.
authors: Sue Ah
authors: SA
authors: Noh
authors: d Department of Applied Chemistry , College of Applied Sciences, Kyung Hee University , Yongin , Korea.
authors: Kwang Pyo
authors: KP
authors: Kim
authors: a Department of Biochemistry and Molecular Biology , Seoul National University College of Medicine , Seoul , Korea.|b Department of Biomedical Sciences , Seoul National University Graduate School , Seoul , Korea.
authors: Min Jae
authors: MJ
authors: Lee
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism*; D000071182:Autophagosomes / Q000187:drug effects / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D000071058:Huntingtin Protein / Q000378:metabolism; D007553:Isotope Labeling; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects; D051379:Mice; D008954:Models, Biological; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D066329:Protein Aggregates / Q000187:drug effects; D011506:Proteins / Q000633:toxicity*; D059748:Proteolysis / Q000187:drug effects; D040901:Proteomics; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology; D010133:p-Chloroamphetamine / Q000494:pharmacology; D016875:tau Proteins / Q000378:metabolism
chemical_list: D015415:Biomarkers; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D000071058:Huntingtin Protein; D066329:Protein Aggregates; D011506:Proteins; D054852:Small Molecule Libraries; D016875:tau Proteins; D010133:p-Chloroamphetamine; D001120:Arginine; D046988:Proteasome Endopeptidase Complex
keywords: ATE1; HSPA5; N-end rule pathway; autophagy; neurodegenerative disease; para-chloroamphetamine; ubiquitin-proteasome system