title: DRAM1 regulates autophagy flux through lysosomes.
abstract: We have previously reported that the mitochondria inhibitor 3-nitropropionic acid (3-NP), induces the expression of DNA damage-regulated autophagy modulator1 (DRAM1) and activation of autophagy in rat striatum. Although the role of DRAM1 in autophagy has been previously characterized, the detailed mechanism by which DRAM1 regulates autophagy activity has not been fully understood. The present study investigated the role of DRAM1 in regulating autophagy flux. In A549 cells expressing wilt-type TP53, 3-NP increased the protein levels of DRAM1 and LC3-II, whereas decreased the levels of SQSTM1 (sequestosome 1). The increase in LC3-II and decrease in SQSTM1 were blocked by the autophagy inhibitor 3-methyl-adenine. Lack of TP53 or knock-down of TP53 in cells impaired the induction of DRAM1. Knock-down of DRAM1 with siRNA significantly reduced 3-NP-induced upregulation of LC3-II and downregulation of SQSTM1, indicating DRAM1 contributes to autophagy activation. Knock-down of DRAM1 robustly decreased rate of disappearance of induced autophagosomes, increased RFP-LC3 fluorescence dots and decreased the decline of LC3-II after withdraw of rapamycin, indicating DRAM1 promotes autophagy flux. DRAM1 siRNA inhibited lysosomal V-ATPase and acidification of lysosomes. As a result, DRAM1 siRNA reduced activation of lysosomal cathepsin D. Similar to DRAM1 siRNA, lysosomal inhibitors E64d and chloroquine also inhibited clearance of autophagosomes and activation of lysosomal cathapsin D after 3-NP treatment. These data suggest that DRAM1 plays important roles in autophagy activation induced by mitochondria dysfunction. DRAM1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes and clearance of autophagosomes.
authors.affiliation: Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science, Suzhou, China.
authors.forename: Xing-Ding
authors.initials: XD
authors.lastname: Zhang
authors.forename: Lin
authors.initials: L
authors.lastname: Qi
authors.forename: Jun-Chao
authors.initials: JC
authors.lastname: Wu
authors.forename: Zheng-Hong
authors.initials: ZH
authors.lastname: Qin
mesh_terms: D000136:Acid-Base Equilibrium; D001343:Autophagy; D002402:Cathepsin D / Q000378:metabolism; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000502:physiology*; D009574:Nitro Compounds / Q000494:pharmacology; D010588:Phagosomes / Q000378:metabolism; D011422:Propionates / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: C512709:DRAM1 protein, human; D008565:Membrane Proteins; D009574:Nitro Compounds; D011422:Propionates; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C533922:CTSD protein, human; D002402:Cathepsin D; C015392:3-nitropropionic acid