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Title: Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

Abstract

Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution.

Authors:
 [1];  [2];  [3];  [4];  [5];  [6];  [7]
  1. Department of Genetics, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536 (United States)
  2. Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502 (Japan)
  3. Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)
  4. Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871 (Japan)
  5. (Japan)
  6. Department of Neurology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawaharacho, Sakyoku, Kyoto 606-8507 (Japan). E-mail: ryosuket@kuhp.kyoto-u.ac.jp
  7. Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan). E-mail: miura@mol.f.u-tokyo.ac.jp
Publication Date:
OSTI Identifier:
20991347
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 356; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2007.03.079; PII: S0006-291X(07)00573-6; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRONMENTAL POLLUTANT EFFECTS ON LIVING ORGANISMS AND BIOLOGICAL MATERIALS; APOPTOSIS; DROSOPHILA; IN VITRO; IN VIVO; IRRADIATION; MITOCHONDRIA; PROTEINS; SERINE; STIMULI

Citation Formats

Igaki, Tatsushi, Suzuki, Yasuyuki, Tokushige, Naoko, Aonuma, Hiroka, Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Takahashi, Ryosuke, and Miura, Masayuki. Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.03.079.
Igaki, Tatsushi, Suzuki, Yasuyuki, Tokushige, Naoko, Aonuma, Hiroka, Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Takahashi, Ryosuke, & Miura, Masayuki. Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila. United States. doi:10.1016/j.bbrc.2007.03.079.
Igaki, Tatsushi, Suzuki, Yasuyuki, Tokushige, Naoko, Aonuma, Hiroka, Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Takahashi, Ryosuke, and Miura, Masayuki. Fri . "Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila". United States. doi:10.1016/j.bbrc.2007.03.079.
@article{osti_20991347,
title = {Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila},
author = {Igaki, Tatsushi and Suzuki, Yasuyuki and Tokushige, Naoko and Aonuma, Hiroka and Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 and Takahashi, Ryosuke and Miura, Masayuki},
abstractNote = {Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution.},
doi = {10.1016/j.bbrc.2007.03.079},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 356,
place = {United States},
year = {Fri May 18 00:00:00 EDT 2007},
month = {Fri May 18 00:00:00 EDT 2007}
}
  • Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongatedmore » mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.« less
  • No abstract prepared.
  • The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediatedmore » through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.« less
  • The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assaysmore » to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.« less
  • Diclofenac is a widely used nonsteroidal anti-inflammatory drug that has been associated with rare but serious hepatotoxicity. Experimental evidence indicates that diclofenac targets mitochondria and induces the permeability transition (mPT) which leads to apoptotic cell death in hepatocytes. While the downstream effector mechanisms have been well characterized, the more proximal pathways leading to the mPT are not known. The purpose of this study was to explore the role of free cytosolic calcium (Ca{sup 2+} {sub c}) in diclofenac-induced cell injury in immortalized human hepatocytes. We show that exposure to diclofenac caused time- and concentration-dependent cell injury, which was prevented bymore » the specific mPT inhibitor cyclosporin A (CsA, 5 {mu}M). At 8 h, diclofenac caused increases in [Ca{sup 2+}]{sub c} (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca{sup 2+} chelator) inhibited cell injury, indicating that Ca{sup 2+} plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, {delta}{psi}{sub m} (JC-1 fluorescence), even in the presence of CsA or BAPTA, indicating that mitochondrial depolarization was not a consequence of the mPT or elevated [Ca{sup 2+}]{sub c}. The CYP2C9 inhibitor sulphaphenazole (10 {mu}M) protected from diclofenac-induced cell injury and prevented increases in [Ca{sup 2+}]{sub c}, while it had no effect on the dissipation of the {delta}{psi}{sub m}. Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca{sup 2+}]{sub c}. In conclusion, these data demonstrate that diclofenac has direct depolarizing effects on mitochondria which does not lead to cell injury, while CYP2C9-mediated bioactivation causes increases in [Ca{sup 2+}]{sub c}, triggering the mPT and precipitating cell death.« less