DRAM1 regulates autophagy flux through lysosomes

PLoS One. 2013 May 17;8(5):e63245. doi: 10.1371/journal.pone.0063245. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid-Base Equilibrium
  • Autophagy*
  • Cathepsin D / metabolism
  • Enzyme Activation
  • Gene Knockdown Techniques
  • HeLa Cells
  • Humans
  • Lysosomes / metabolism*
  • Membrane Proteins / physiology*
  • Nitro Compounds / pharmacology
  • Phagosomes / metabolism
  • Propionates / pharmacology
  • RNA, Small Interfering / genetics
  • Transcriptional Activation / drug effects
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • DRAM1 protein, human
  • Membrane Proteins
  • Nitro Compounds
  • Propionates
  • RNA, Small Interfering
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • CTSD protein, human
  • Cathepsin D
  • 3-nitropropionic acid

Grant support

This work was partially supported by the National Natural Science Foundation of China (No 30930035), by the National Basic Science Key Project (973 project, CB510003), by the Priority Academic Program development of Jiangsu Higher Education Institutes, and by Graduate Training Innovation Project of Jiangsu Province (CX09B_042Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.