Abstract
We have reported that chymotrypsin B (CtrB) is not just a digestive enzyme but is also stored in lysosomes. Herein, we demonstrated a broad distribution of CtrB and explored the involvement of CtrB in apoptosis. Exposure of RH-35 cells to H2O2 or palmitate induced the redistribution of lysosomal CtrB into the cytoplasm as a result of lysosomal membrane permeabilization (LMP). Suppression of CtrB significantly blocked apoptosis, while overexpression of CtrB sensitized apoptosis markedly. CtrB could cleave Bid under neutral conditions. In RH-35 cells with Bid silenced, apoptosis induced by CtrB protein was attenuated, suggesting that CtrB mediates apoptosis of RH-35 cells mainly through processing Bid. Our data also suggest that LMP occurs earlier than mitochondrial outer membrane permeabilization; Bid activation initiated by caspase-8 might be reinforced by CtrB in consequence of LMP, which causes a positive feedback loop leading to the accumulation of tBid, and results in lysosome- and mitochondrion-dependent apoptosis.
Similar content being viewed by others
References
Ferri KF, Kroemer G (2001) Organelle-specific initiation of cell death pathways. Nat Cell Biol 3:E255–E263
Guicciardi ME, Leist M, Gores GJ (2004) Lysosomes in cell death. Oncogene 23:2881–2890
Feldstein AE, Werneburg NW, Li Z, Bronk SF, Gores GJ (2006) Bax inhibition protects against free fatty acid-induced lysosomal permeabilization. Am J Physiol Gastrointest Liver Physiol 290:G1339–G1346
Feldstein AE, Werneburg NW, Canbay A, Guicciardi ME, Bronk SF, Rydzewski R, Burgart LJ, Gores GJ (2004) Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway. Hepatology 40:185–194
Li Z, Berk M, McIntyre TM, Gores GJ, Feldstein AE (2008) The lysosomal-mitochondrial axis in free fatty acid-induced hepatic lipotoxicity. Hepatology 47:1495–1503
Cirman T, Oresic K, Mazovec GD, Turk V, Reed JC, Myers RM, Salvesen GS, Turk B (2004) Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem 279:3578–3587
Droga-Mazovec G, Bojic L, Petelin A, Ivanova S, Romih R, Repnik U, Salvesen GS, Stoka V, Turk V, Turk B (2008) Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues. J Biol Chem 283:19140–19150
Turk B, Turk V (2009) Lysosomes as “suicide bags” in cell death: myth or reality? J Biol Chem 284:21783–21787
Grabarek J, Dragan M, Lee BW, Johnson GL, Darzynkiewicz Z (2002) Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor. Int J Oncol 20:225–233
Stenson-Cox C, FitzGerald U, Samali A (2003) In the cut and thrust of apoptosis, serine proteases come of age. Biochem Pharmacol 66:1469–1474
Autefage H, Albinet V, Garcia V, Berges H, Nicolau ML, Therville N, Altie MF, Caillaud C, Levade T, Andrieu-Abadie N (2009) Lysosomal serine protease CLN2 regulates tumor necrosis factor-alpha-mediated apoptosis in a bid-dependent manner. J Biol Chem 284:11507–11516
Yan L, Miao Q, Sun Y, Yang F (2003) tBid forms a pore in the liposome membrane. FEBS Lett 555:545–550
Zhai D, Miao Q, Xin X, Yang F (2001) Leakage and aggregation of phospholipid vesicles induced by the BH3-only Bcl-2 family member, BID. Eur J Biochem 268:48–55
Miao Q, Sun Y, Wei T, Zhao X, Zhao K, Yan L, Zhang X, Shu H, Yang F (2008) Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway. J Biol Chem 283:8218–8228
Shi L, Wu L, Wang S, Fan Z (2009) Granzyme F induces a novel death pathway characterized by Bid-independent cytochrome c release without caspase activation. Cell Death Differ 16:1694–1706
Arthur PG, Niu X, Rigby P, Steer JH, Jeffrey GP (2008) Oxidative stress causes a decline in lysosomal integrity during hypothermic incubation of rat hepatocytes. Free Radic Biol Med 44:24–33
Doulias PT, Kotoglou P, Tenopoulou M, Keramisanou D, Tzavaras T, Brunk U, Galaris D, Angelidis C (2007) Involvement of heat shock protein-70 in the mechanism of hydrogen peroxide-induced DNA damage: the role of lysosomes and iron. Free Radic Biol Med 42:567–577
Zhang Z, Wei T, Hou J, Li G, Yu S, Xin W (2003) Iron-induced oxidative damage and apoptosis in cerebellar granule cells: attenuation by tetramethylpyrazine and ferulic acid. Eur J Pharmacol 467:41–47
Werneburg N, Guicciardi ME, Yin XM, Gores GJ (2004) TNF-alpha-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent. Am J Physiol Gastrointest Liver Physiol 287:G436–G443
Blomgran R, Zheng L, Stendahl O (2007) Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization. J Leukoc Biol 81:1213–1223
Castino R, Bellio N, Nicotra G, Follo C, Trincheri NF, Isidoro C (2007) Cathepsin D-Bax death pathway in oxidative stressed neuroblastoma cells. Free Radic Biol Med 42:1305–1316
Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, Kaufmann SH, Gores GJ (2000) Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 106:1127–1137
Reiners JJ Jr, Caruso JA, Mathieu P, Chelladurai B, Yin XM, Kessel D (2002) Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves Bid cleavage. Cell Death Differ 9:934–944
Boya P, Gonzalez-Polo RA, Poncet D, Andreau K, Vieira HL, Roumier T, Perfettini JL, Kroemer G (2003) Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine. Oncogene 22:3927–3936
Werneburg NW, Guicciardi ME, Bronk SF, Kaufmann SH, Gores GJ (2007) Tumor necrosis factor-related apoptosis-inducing ligand activates a lysosomal pathway of apoptosis that is regulated by Bcl-2 proteins. J Biol Chem 282:28960–28970
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (Grants 30900246 and 30770492), the National Basic Research Program of China (Grants 2006CB705600, 2006CB911001 and 2010CB833700), and the K. C. Wong Education Foundation, Hong Kong. We would like to thank Dr. L. Gráf (Eötvös Lorárd University, Hungary) for kindly providing plasmid encoding rat chymotrypsin B, and Prof. R. S. Chen (Institute of Biophysics, CAS) and Prof. Frank Dowd (Creighton University, USA) for valuable suggestions and discussions. The authors are also indebted to W. M. Zhong, W. Li, H. J. Zhou and P. T. Jiang for their technical assistance.
Author information
Authors and Affiliations
Corresponding authors
Additional information
K. Zhao and X. Zhao contributed equally to this work.
Rights and permissions
About this article
Cite this article
Zhao, K., Zhao, X., Tu, Y. et al. Lysosomal chymotrypsin B potentiates apoptosis via cleavage of Bid. Cell. Mol. Life Sci. 67, 2665–2678 (2010). https://doi.org/10.1007/s00018-010-0356-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00018-010-0356-0