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. 2010 Jan;6(1):61-6.
doi: 10.4161/auto.6.1.10326. Epub 2010 Jan 13.

Control of basal autophagy by calpain1 mediated cleavage of ATG5

Hong-Guang Xia  1 , Lihong Zhang, Gang Chen, Tao Zhang, Junli Liu, Mingzhi Jin, Xiuquan Ma, Dawei Ma, Junying Yuan
Affiliations

Control of basal autophagy by calpain1 mediated cleavage of ATG5

Hong-Guang Xia et al. Autophagy. 2010 Jan.

Abstract

Autophagy functions as an important catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes. Although the induction of autophagy by starvation has been extensively studied, we still understand very little about how autophagy is regulated under normal nutritional conditions. Here we describe a study using a small molecule autophagy inducer, fluspirilene, as a tool to explore the mechanism of autophagy induction in normal living cells. We confirm the activity of fluspirilene in inhibiting Ca(2+) flux VSports手机版. Furthermore, we show that reducing intracellular Ca(2+) prevents the cleavage of ATG5, which in turn increases the levels of full-length ATG5 and ATG12-ATG5 conjugate. Using siRNA mediated gene silencing, we demonstrate that inhibiting calpain1 is sufficient to induce autophagy in living cells. We conclude that calpain1 plays an important role in controlling the levels of autophagy in normal living cells by regulating the levels of a key signaling molecule, ATG12-ATG5 conjugate. .

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Figures

Figure 1
Figure 1
Induction of autophagy by fluspirilene. (A) Dose-dependent induction of LC3II by fluspirilene in H4 cells. H4 cells were treated with indicated concentrations of fluspirilene for 4 hours. The cell lysates were harvested and analyzed by western blotting using anti-LC3. Anti-tubulin was used as a loading control. (B) H4 cells were treated with fluspirilene (10 μM), bafilomycin A1 (100 nM), or fluspirilene and bafilomycin A1 together for 4 hours. 0.1% DMSO is used as negative control. The cell lysates were harvested and analyzed by western blotting using anti-LC3 antibody. Anti-tubulin was used as a loading control.
Figure 2
Figure 2
The effects of fluspirilene on the levels of ATG12-ATG5 in H4 cells. (A) H4 cells were treated with 10μM fluspirilene for indicated length of time. The cell lysates were harvested and analyzed by western blotting using anti-ATG12 antibody. Anti-tubulin was used as a loading control. (B) H4 cells were treated with 10μM fluspirilene for indicated length of time. The mRNA levels of ATG5 are analyzed by RT-PCR as described in the methods. (C) H4 cells were treated with 10μM fluspirilene for indicated length of time. The cell lysates were harvested and analyzed by western blotting with anti-ATG5 antibodies. Anti-tubulin was used as a loading control.
Figure 3
Figure 3
The effects of Ca2+ channel inhibitors or agonist on ATG5. (A) Fluspirilene inhibits ATP induced Ca2+ flux. HeLa cells were treated with 0.1% DMSO (above) or 10 μM fluspirilene (below) for 10 min before adding ATP. The Ca2+ flux was measured using dye fura-2. (B) The effects of Ca2+ channel inhibitors on the levels of ATG12-ATG5 and LC3II in H4 cells. H4 cells were treated with indicated compounds (10 μM) for 4 hours. 0.1% DMSO was used as a negative control. The cell lysates were harvested and analyzed by western blotting using anti-ATG12 and anti-LC3. Anti-tubulin was used as a loading control. (C) The effects of Bay K-8644 on the levels of ATG5 and tATG5 in H4 cells. H4 cells were treated with 10 μM Bay K-8644 for indicated length of time. The cell lysates were harvested and analyzed by western blotting using anti-ATG5. Anti-tubulin was used as a loading control.
Figure 4
Figure 4
The effects of inhibiting calpains on the levels of ATG12-ATG5 and LC3II in H4 cells. (A) H4 cells were treated with indicated concentrations of rapamycin (0.25 μM), fluspirilene (10 μM), MDL-28170 (10 μM), MG-101 (10 μM) and Calpeptin (10 μM), respectively, for 4 hours. 0.1% DMSO was used as a negative control. The cell lysates were harvested and analyzed by western blotting using anti-ATG12 and anti-LC3. Anti-tubulin was used as a loading control. (B) H4 cells were treated with 10μM fluspirilene for indicated length of time. The cell lysates were harvested and analyzed by western blotting with anti-MARCKs antibodies. Anti-tubulin was used as a loading control. (C–E) H4 cells were transfected with indicated siRNAs for 72 hrs and no targets siRNA (N. T. siRNA) was used as negative control. The cell lysates were harvested and analyzed by western blotting using anti-calpain1 (C), anti-calpain2 (D), anti-calpain4 (E), anti-ATG12 and anti-LC3 (C–E). Anti-tubulin was used as loading controls. (F) H4 cells were transfected with siRNAs indicated for 72 hrs or treated with MDL-28170 (10 μM) for 4 hrs and the cell lysates were harvested and analyzed by western blotting using anti-Calpain1, anti-Calpain2, anti-Calpain4, anti-ATG12, anti-ATG5 and anti-LC3 antibody. Anti-tubulin was used as a loading control.
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References (VSports手机版)

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