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.

Mechanism of DRAM1's Regulation of Autophagy and Apoptosis

“…The results also support the involvement of DRAM1 in the degradation of autophagosomes [6]. However, Galavotti et al found that DRAM1 was not involved in starvation-and mTORmediated autophagy activation [7].…”

“…Knock-down of DRAM1 resulted in the impairment of lysosomal acidification and the accumulation of LC3-II, indicating reduced autophagy flux [6]. DRAM1 appeared to regulate autophagy in both the early and later stages.…”

“…DRAM1 appeared to regulate autophagy in both the early and later stages. It also increased the formation of and clearance of autophagosomes [6]. The mechanism of DRAM1 is a lysosomal protein, and it may regulate the dynamics of lysosomal membranes to increase V-ATPase activity and facilitate membrane recycling for autophagosomal formation.…”

“…Lysosomes are rich in hydrolytic enzymes and are responsible for the degradation of intracellular materials captured by autophagy [8]. The increase in acidic lysosomes was noticeable, as indicated by a fluorescent dye.Knock-down of DRAM1 resulted in the impairment of lysosomal acidification and the accumulation of LC3-II, indicating reduced autophagy flux [6]. DRAM1 appeared to regulate autophagy in both the early and later stages.…”

Imatinib Triggers Phagolysosome Acidification and Antimicrobial Activity againstMycobacterium bovisBacille Calmette–Guérin in Glucocorticoid-Treated Human Macrophages

“…DRAM1 was found to trigger selective autophagy downstream of TLR signaling promoting mycobacterial phagosome maturation, phagolysosome fusion, and anti-mycobacterial activity (33). Knockdown of DRAM1 inhibits the v-ATPase and acidification of lysosomes (51). Thus, it is tempting to speculate that DRAM1 links IFN-γ induction of autophagy, TCIRG1, and acidification of lysosomes.…”