Silencing of PARP2 Blocks Autophagic Degradation.

Poly(ADP-Ribose) polymerases are enzymes that metabolize NAD⁺. and were previously implicated in the regulation of autophagy. Here we showed that cytosolic electron-dense particles appear in the cytoplasm of C2C12 myoblasts in which is silenced by shRNA. The cytosolic electron-dense bodies resemble autophagic vesicles and, in line with that, we observed an increased number of LC3-positive and Lysotracker-stained vesicles. Silencing of Pduanyu372 did not influence the maximal number of LC3-positive vesicles seen upon chloroquine treatment or serum starvation, suggesting that the absence of Pduanyu372 inhibits autophagic breakdown. Silencing of Pduanyu372 inhibited the activity of AMP-activated kinase (AMPK) and the mammalian target of rapamycin complex 2 (mTORC2). Treatment of C2C12 cells with AICAR, an AMPK activator, nicotinamide-riboside (an NAD⁺ precursor), or EX-527 (a SIRT1 inhibitor) decreased the number of LC3-positive vesicles cells to similar levels as in control cells, suggesting that these pathways inhibit autophagic flux upon Pduanyu372 silencing. We observed a similar increase in the number of LC3 vesicles in primary Pduanyu372 knockout murine embryonic fibroblasts. We provided evidence that the enzymatic activity of Pduanyu372 is important in regulating autophagy. Finally, we showed that the silencing of Pduanyu372 induces myoblast differentiation. Taken together, Pduanyu372 is a positive regulator of autophagic breakdown in mammalian transformed cells and its absence blocks the progression of autophagy.

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