Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.

Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.

Mechanistic study of TRPM2-Ca(2+)-CAMK2-BECN1 signaling in oxidative stress-induced autophagy inhibition.

Reactive oxygen species (ROS) have been commonly accepted as inducers of autophagy, and autophagy in turn is activated to relieve oxidative stress. Yet, whether and how oxidative stress, generated in various human pathologies, regulates autophagy remains unknown. Here, we mechanistically studied the role of TRPM2 (transient receptor potential cation channel subfamily M member 2)-mediated Ca(2+) influx in oxidative stress-mediated autophagy regulation. On the one hand, we demonstrated that oxidative stress triggered TRPM2-dependent Ca(2+) influx to inhibit the induction of early autophagy, which renders cells more susceptible to death. On the other hand, oxidative stress induced autophagy (and not cell death) in the absence of the TRPM2-mediated Ca(2+) influx. Moreover, in response to oxidative stress, TRPM2-mediated Ca(2+) influx activated CAMK2 (calcium/calmodulin dependent protein kinase II) at levels of both phosphorylation and oxidation, and the activated CAMK2 subsequently phosphorylated BECN1/Beclin 1 on Ser295. Ser295 phosphorylation of BECN1 in turn decreased the association between BECN1 and PIK3C3/VPS34, but induced binding between BECN1 and BCL2. Clinically, acetaminophen (APAP) overdose is the most common cause of acute liver failure worldwide. We demonstrated that APAP overdose also activated ROS-TRPM2-CAMK2-BECN1 signaling to suppress autophagy, thereby causing primary hepatocytes to be more vulnerable to death. Inhibiting the TRPM2-Ca(2+)-CAMK2 cascade significantly mitigated APAP-induced liver injury. In summary, our data clearly demonstrate that oxidative stress activates the TRPM2-Ca(2+)-CAMK2 cascade to phosphorylate BECN1 resulting in autophagy inhibition.

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A.

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca(2+) content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.