Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary.
Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary.
Cells. 2020 Feb 7;9(2):380. doi: 10.3390/cells9020380.
Poly(ADP-Ribose) polymerases (PARPs) are enzymes that metabolize NAD. PARP1 and PARP10 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 PARP2 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 PARP2 did not influence the maximal number of LC3-positive vesicles seen upon chloroquine treatment or serum starvation, suggesting that the absence of PARP2 inhibits autophagic breakdown. Silencing of PARP2 inhibited the activity of AMP-activated kinase (AMPK) and the mammalian target of rapamycin complex 2 (mTORC2). Treatment of PARP2-silenced 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 (scPARP2) cells, suggesting that these pathways inhibit autophagic flux upon PARP2 silencing. We observed a similar increase in the number of LC3 vesicles in primary PARP2 knockout murine embryonic fibroblasts. We provided evidence that the enzymatic activity of PARP2 is important in regulating autophagy. Finally, we showed that the silencing of PARP2 induces myoblast differentiation. Taken together, PARP2 is a positive regulator of autophagic breakdown in mammalian transformed cells and its absence blocks the progression of autophagy.
聚（ADP-核糖）聚合酶（PARPs）是代谢 NAD 的酶。PARP1 和 PARP10 先前被牵连到自噬的调节中。在这里，我们表明，在 PARP2 通过 shRNA 沉默的 C2C12 成肌细胞的细胞质中出现细胞质电子致密颗粒。细胞质电子致密体类似于自噬小泡，并且我们观察到 LC3 阳性和溶酶体追踪染色小泡的数量增加。PARP2 的沉默不影响氯喹处理或血清饥饿时观察到的最大数量的 LC3 阳性小泡，这表明 PARP2 的缺失抑制自噬降解。PARP2 的沉默抑制 AMP 激活的蛋白激酶（AMPK）和雷帕霉素靶蛋白复合物 2（mTORC2）的活性。用 AMPK 激活剂 AICAR、烟酰胺核糖（NAD 前体）或 EX-527（SIRT1 抑制剂）处理 PARP2 沉默的 C2C12 细胞，将 LC3 阳性小泡细胞的数量减少到与对照（scPARP2）细胞相似的水平，这表明这些途径抑制 PARP2 沉默时的自噬通量。我们在原代 PARP2 敲除鼠胚胎成纤维细胞中观察到 LC3 小泡数量的类似增加。我们提供的证据表明，PARP2 的酶活性对于调节自噬很重要。最后，我们表明 PARP2 的沉默诱导成肌细胞分化。总之，PARP2 是哺乳动物转化细胞中自噬降解的正调节剂，其缺失阻止自噬的进展。
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