Abstract
SummaryAutophagy, a major degradation process for long-lived and aggregate-prone proteins, affects various human processes, such as development, immunity, cancer, and neurodegeneration. Several autophagy regulators have been identified in recent years. Here we show that nitric oxide (NO), a potent cellular messenger, inhibits autophagosome synthesis via a number of mechanisms. NO impairs autophagy by inhibiting the activity of S-nitrosylation substrates, JNK1 and IKKβ. Inhibition of JNK1 by NO reduces Bcl-2 phosphorylation and increases the Bcl-2–Beclin 1 interaction, thereby disrupting hVps34/Beclin 1 complex formation. Additionally, NO inhibits IKKβ and reduces AMPK phosphorylation, leading to mTORC1 activation via TSC2. Overexpression of nNOS, iNOS, or eNOS impairs autophagosome formation primarily via the JNK1–Bcl-2 pathway. Conversely, NOS inhibition enhances the clearance of autophagic substrates and reduces neurodegeneration in models of Huntington's disease. Our data suggest that nitrosative stress-mediated protein aggregation in neurodegenerative diseases may be, in part, due to autophagy inhibition.
Highlights
Macroautophagy is an intracellular bulk degradation process involved in the clearance of long-lived proteins and organelles, which affects various physiological and pathological processes, including development, immunity, longevity, cancer, and neurodegenerative diseases (Mizushima et al, 2008)
nitric oxide (NO) donors decreased the number of autolysosomes in mRFP-GFP-light chain 3 (LC3) HeLa cells cultured in full medium (FM) or when starved with Hank’s buffered salt solution (HBSS), compared with untreated cells (Figures 1B–1E), suggesting that NO inhibited autophagic flux under basal and starvation conditions
We found that NO donors increased the phosphorylation of S6 kinase 1 (S6K) and S6 in Tsc2+/+, but not in Tsc2À/À, MEFs (Figure 4A), suggesting that NO-induced activation of mTOR complex 1 (mTORC1) was TSC2-dependent, by acting at, or above, TSC2
Summary
Macroautophagy ( referred to as ‘‘autophagy’’) is an intracellular bulk degradation process involved in the clearance of long-lived proteins and organelles, which affects various physiological and pathological processes, including development, immunity, longevity, cancer, and neurodegenerative diseases (Mizushima et al, 2008). Autophagy initiates with the formation of isolation membranes called phagophores, which elongate and engulf a portion of the cytoplasm to form mature autophagosomes. Autophagosomes fuse with lysosomes to form autolysosomes, in which acidic lysosomal hydrolases degrade the engulfed contents. Autophagy is a highly conserved process; genetic analyses in yeast have identified several ATG (AuTophaGy related) genes, many of which have mammalian orthologs. The only known mammalian protein that associates with autophagosome membranes throughout their lifespan is microtubule-associated protein 1 (MAP1) light chain 3 (LC3), the mammalian ortholog of yeast Atg. LC3-I conjugates with phosphatidylethanolamine to form the autophagosome-associated LC3-II. LC3-II levels (relative to actin/ tubulin loading controls) correlate with autophagosome numbers (Kabeya et al, 2000)
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