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Chukai Y, Furukawa N, Kosegawa O, Bai L, Sugano E, Fukuda T, Tomita H, Ozaki T. Mitochondrial calpain-1 truncates ATP synthase beta subunit. Biochem Biophys Res Commun 2025; 765:151829. [PMID: 40262470 DOI: 10.1016/j.bbrc.2025.151829] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2025] [Accepted: 04/15/2025] [Indexed: 04/24/2025]
Abstract
Calpains cleave proteins in a calcium concentration-dependent manner, modulating their intracellular functions. Calpain-1, a member of the calpain family, is localized in the cytosol and mitochondria. Mitochondrial calpain-1 induces mitochondrial dysfunction and apoptosis by cleaving its substrate. Thus, identifying the substrate of calpain-1 is essential to understand its function. However, little is known about the substrates of mitochondrial calpain-1. To address this issue, we screened mitochondrial proteins using bioinformatics approaches and two-dimensional gel electrophoresis. We identified ATP5B as a potential substrate of mitochondrial calpain-1. Calpeptin, a pan-calpain inhibitor, and Tat-μCL, a mitochondrial calpain-1 specific inhibitor, prevented the truncation of ATP5B during in vitro Ca2+ incubation. Using recombinant human calpain-1 and ATP5B proteins, we demonstrated that calpain-1 directly cleaved ATP5B, generating a fragment of ATP5B. Based on the predicted cleavage sites in ATP5B, this cleavage may disrupt its interaction with ATP5A1, leading to mitochondrial dysfunction in ATP production. This study identified ATP5B as a novel substrate of mitochondrial calpain-1. The results provide new insights into mitochondrial dysfunction.
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Affiliation(s)
- Yusaku Chukai
- Laboratory of Cell Biochemistry, Department of Life Sciences, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan; Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan
| | - Nanami Furukawa
- Laboratory of Cell Biochemistry, Department of Life Sciences, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan; Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan
| | - On Kosegawa
- Laboratory of Cell Biochemistry, Department of Life Sciences, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan
| | - Lanlan Bai
- Laboratory of Cell Engineering and Molecular Genetics, Department of Life Sciences, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan
| | - Eriko Sugano
- Laboratory of Visual Neuroscience, Department of Life Sciences, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan
| | - Tomokazu Fukuda
- Laboratory of Cell Engineering and Molecular Genetics, Department of Life Sciences, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan
| | - Hiroshi Tomita
- Laboratory of Visual Neuroscience, Department of Life Sciences, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan
| | - Taku Ozaki
- Laboratory of Cell Biochemistry, Department of Life Sciences, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan; Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan.
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Villarejo-Zori B, Zapata-Muñoz J, Sierra-Filardi E, Ramírez-Pardo I, Montava-Garriga L, Ganley IG, Boya P. Deferiprone protects against photoreceptor degeneration by inhibiting parthanatos. Cell Death Dis 2025; 16:402. [PMID: 40389413 PMCID: PMC12089389 DOI: 10.1038/s41419-025-07686-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2024] [Revised: 12/18/2024] [Accepted: 04/17/2025] [Indexed: 05/21/2025]
Abstract
Photoreceptor degeneration is the hallmark of retinitis pigmentosa. Identifying general mechanisms underlying photoreceptor cell death is key to developing effective, mutation-independent treatments to prevent vision loss. Mitophagy is a protective pathway that prevents age-dependent vision loss and is upregulated by iron chelators such as deferiprone (DFP). Therefore, we aimed to investigate the ability of DFP to protect against retinal degeneration via mitophagy. First, we treated mitophagy reporter mice with MNU, a classic inducer of photoreceptor degeneration. MNU induced retinal degeneration and comprehensively inhibited mitophagy, while also inducing lysosomal basification and lysosomal membrane permeabilization. Although DFP rescued cells and retinal explants from the toxic effects of MNU, this effect was independent of mitophagy. Further investigation revealed that PAR polymers accumulation associated with parthanatos cell death was reduced to similar extents by DFP and the PARP inhibitor olaparib. In conclusion, iron chelation can protect against MNU-induced photoreceptor degeneration in retinal explants via parthanatos inhibition. Olaparib and DFP rescue parthanatos induced cell death after MNU-induced retinal degeneration. High doses of MNU induce lysosomal damage and mitophagy inhibition. In addition, MNU produces DNA damage and increases oxidative stress, resulting in PAR polymer formation and retinal degeneration (orange panel). DFP and Olaparib are able to rescue retinal degeneration downstream of lysosomal damage (green panel). Sub-lethal doses of MNU induce a peak in mitophagy that is BNIP3L-BNIP3 dependent (blue panel).
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Affiliation(s)
- Beatriz Villarejo-Zori
- Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas Margarita Salas, CSIC, Madrid, Spain.
| | - Juan Zapata-Muñoz
- Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas Margarita Salas, CSIC, Madrid, Spain
| | - Elena Sierra-Filardi
- Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas Margarita Salas, CSIC, Madrid, Spain
| | - Ignacio Ramírez-Pardo
- Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas Margarita Salas, CSIC, Madrid, Spain
- Department of Medicine and Life Sciences, Universitat Pompeu Fabra (UPF), CIBERNED, Barcelona, 08003, Spain
| | - Lambert Montava-Garriga
- MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, DD1 5EH, UK
| | - Ian G Ganley
- MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, DD1 5EH, UK
| | - Patricia Boya
- Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas Margarita Salas, CSIC, Madrid, Spain.
- Department of Neuroscience and Movement Science, University of Fribourg, Chemin. du Musée 14, 1700, Fribourg, Switzerland.
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Chukai Y, Sudo T, Fukuda T, Tomita H, Sugano E, Ozaki T. Proteolysis of mitochondrial calpain-13 in cerebral ischemia-reperfusion injury. Biochem Biophys Rep 2024; 39:101768. [PMID: 39050013 PMCID: PMC11267081 DOI: 10.1016/j.bbrep.2024.101768] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2024] [Revised: 06/21/2024] [Accepted: 06/25/2024] [Indexed: 07/27/2024] Open
Abstract
Calpains are calcium-dependent cysteine proteases activated by intracellular Ca2+. Although calpains mainly exist in the cytosol, calpain-13 is present in the mitochondria in mouse brains; however, the enzymatic properties and physiological functions of calpain-13 remain unknown. Hence, in this study, we predicted and evaluated the enzymatic properties of calpain-13. Based on our bioinformatic approaches, calpain-13 possessed a catalytic triad and EF-hand domain, similar to calpain-1, a well-studied calpain. Therefore, we hypothesized that calpain-13 had calpain-1-like enzymatic properties; however, calpain-13 was not proteolyzed in C57BL/6J mouse brains. Subsequently, cerebral ischemia/reperfusion (I/R) injury caused proteolysis of mitochondrial calpain-13. Thus, our study showed that mitochondrial calpain-13 was proteolyzed in the mitochondria of the I/R injured mouse brain. This finding could be valuable in further research elucidating the involvement of calpain-13 in cell survival or death in brain diseases, such as cerebral infarction.
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Affiliation(s)
- Yusaku Chukai
- Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan
| | - Toru Sudo
- Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan
| | - Tomokazu Fukuda
- Laboratory of Cell Engineering and Molecular Genetics, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan
| | - Hiroshi Tomita
- Laboratory of Visual Neuroscience, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan
| | - Eriko Sugano
- Laboratory of Visual Neuroscience, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan
| | - Taku Ozaki
- Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan
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Wan H, Chen H, Liu J, Yang B, Zhang Y, Bai Y, Chen X, Wang J, Liu T, Zhang Y, Hua Q. PARP1 inhibition prevents oxidative stress in age-related hearing loss via PAR-Ca 2+-AIF axis in cochlear strial marginal cells. Free Radic Biol Med 2024; 220:222-235. [PMID: 38735540 DOI: 10.1016/j.freeradbiomed.2024.05.020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2024] [Revised: 05/02/2024] [Accepted: 05/09/2024] [Indexed: 05/14/2024]
Abstract
Studies have highlighted oxidative damage in the inner ear as a critical pathological basis for sensorineural hearing loss, especially the presbycusis. Poly(ADP-ribose) polymerase-1 (PARP1) activation responds to oxidative stress-induced DNA damage with pro-repair and pro-death effects resembling two sides of the same coin. PARP1-related cell death, known as parthanatos, whose underlying mechanisms are attractive research hotspots but remain to be clarified. In this study, we observed that aged rats showed stria vascularis degeneration and oxidative damage, and PARP1-dependent cell death was prominent in age-related cochlear disorganization and dysfunction. Based on oxidative stress model of primary cultured stria marginal cells (MCs), we revealed that upregulated PARP1 and PAR (Poly(ADP-ribose)) polymers are responsible for MCs oxidative death with high mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse, while inhibition of PARP1 ameliorated the adverse outcomes. Importantly, the PARylation of apoptosis-inducing factor (AIF) is essential for its conformational change and translocation, which subsequently causes DNA break and cell death. Concretely, the interaction of PAR and truncated AIF (tAIF) is the mainstream in the parthanatos pathway. We also found that the effects of AIF cleavage and release were achieved through calpain activity and mPTP opening, both of which could be regulated by PARP1 via mediation of mitochondria Ca2+ concentration. In conclusion, the PAR-Ca2+-tAIF signaling pathway in parthanatos contributes to the oxidative stress damage observed in MCs. Targeting PAR-Ca2+-tAIF might be a potential therapeutic strategy for the early intervention of presbycusis and other oxidative stress-associated sensorineural deafness.
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Affiliation(s)
- Huanzhi Wan
- Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei Province, China; Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China; The First Clinical School of Wuhan University, Wuhan, 430060, Hubei Province, China
| | - Huidong Chen
- Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei Province, China; Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China; The First Clinical School of Wuhan University, Wuhan, 430060, Hubei Province, China
| | - Jingchun Liu
- The First Clinical School of Wuhan University, Wuhan, 430060, Hubei Province, China
| | - Bingqian Yang
- Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei Province, China; Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China; The First Clinical School of Wuhan University, Wuhan, 430060, Hubei Province, China
| | - Yunlong Zhang
- Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China; The First Clinical School of Wuhan University, Wuhan, 430060, Hubei Province, China
| | - Yutong Bai
- Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei Province, China; Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China; The First Clinical School of Wuhan University, Wuhan, 430060, Hubei Province, China
| | - Xiaoying Chen
- Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei Province, China; Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China; The First Clinical School of Wuhan University, Wuhan, 430060, Hubei Province, China
| | - Jie Wang
- Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei Province, China; Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China; The First Clinical School of Wuhan University, Wuhan, 430060, Hubei Province, China
| | - Tianyi Liu
- Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei Province, China; Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China.
| | - Yuanyuan Zhang
- Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei Province, China; Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China.
| | - Qingquan Hua
- Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei Province, China; Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University, Wuhan, 430060, Hubei Province, China.
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Chen Q, Li L, Samidurai A, Thompson J, Hu Y, Willard B, Lesnefsky EJ. Acute endoplasmic reticulum stress-induced mitochondria respiratory chain damage: The role of activated calpains. FASEB J 2024; 38:e23404. [PMID: 38197290 PMCID: PMC11032170 DOI: 10.1096/fj.202301158rr] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2023] [Revised: 11/19/2023] [Accepted: 12/19/2023] [Indexed: 01/11/2024]
Abstract
The induction of acute endoplasmic reticulum (ER) stress damages the electron transport chain (ETC) in cardiac mitochondria. Activation of mitochondria-localized calpain 1 (CPN1) and calpain 2 (CPN2) impairs the ETC in pathological conditions, including aging and ischemia-reperfusion in settings where ER stress is increased. We asked if the activation of calpains causes the damage to the ETC during ER stress. Control littermate and CPNS1 (calpain small regulatory subunit 1) deletion mice were used in the current study. CPNS1 is an essential subunit required to maintain CPN1 and CPN2 activities, and deletion of CPNS1 prevents their activation. Tunicamycin (TUNI, 0.4 mg/kg) was used to induce ER stress in C57BL/6 mice. Cardiac mitochondria were isolated after 72 h of TUNI treatment. ER stress was increased in both control littermate and CPNS1 deletion mice with TUNI treatment. The TUNI treatment activated both cytosolic and mitochondrial CPN1 and 2 (CPN1/2) in control but not in CPNS1 deletion mice. TUNI treatment led to decreased oxidative phosphorylation and complex I activity in control but not in CPNS1 deletion mice compared to vehicle. The contents of complex I subunits, including NDUFV2 and ND5, were decreased in control but not in CPNS1 deletion mice. TUNI treatment also led to decreased oxidation through cytochrome oxidase (COX) only in control mice. Proteomic study showed that subunit 2 of COX was decreased in control but not in CPNS1 deletion mice. Our results provide a direct link between activation of CPN1/2 and complex I and COX damage during acute ER stress.
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Affiliation(s)
- Qun Chen
- Department of Internal Medicine, Division of Cardiology, Pauley Heart Center, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Ling Li
- Proteomics Core, Cleveland Clinic, Cleveland, Ohio, USA
| | - Arun Samidurai
- Department of Internal Medicine, Division of Cardiology, Pauley Heart Center, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Jeremy Thompson
- Department of Internal Medicine, Division of Cardiology, Pauley Heart Center, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Ying Hu
- Department of Internal Medicine, Division of Cardiology, Pauley Heart Center, Virginia Commonwealth University, Richmond, Virginia, USA
| | | | - Edward J. Lesnefsky
- Department of Internal Medicine, Division of Cardiology, Pauley Heart Center, Virginia Commonwealth University, Richmond, Virginia, USA
- Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, USA
- Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia, USA
- Richmond Department of Veterans Affairs Medical Center, Richmond, Virginia, USA
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Chukai Y, Ito G, Miki Y, Wakabayashi K, Itoh K, Sugano E, Tomita H, Fukuda T, Ozaki T. Role of calpain-5 in cerebral ischemia and reperfusion injury. Biochim Biophys Acta Gen Subj 2024; 1868:130506. [PMID: 37949151 DOI: 10.1016/j.bbagen.2023.130506] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Revised: 10/27/2023] [Accepted: 10/30/2023] [Indexed: 11/12/2023]
Abstract
BACKGROUND Ischemia and reperfusion (I/R) injury exacerbate the prognosis of ischemic diseases. The cause of this exacerbation is partly a mitochondrial cell death pathway. Mitochondrial calpain-5 is proteolyzed/autolyzed under endoplasmic reticulum stress, resulting in inflammatory caspase-4 activation. However, the role of calpain-5 in I/R injury remains unclear. We hypothesized that calpain-5 is involved in ischemic brain disease. METHODS Mitochondria from C57BL/6J mice were extracted via centrifugation with/without proteinase K treatment. The expression and proteolysis/autolysis of calpain-5 were determined using western blotting. The mouse and human brains with I/R injury were analyzed using hematoxylin and eosin staining and immunohistochemistry. HT22 cells were treated with tunicamycin and CAPN5 siRNA. RESULTS Calpain-5 was expressed in the mitochondria of mouse tissues. Mitochondrial calpain-5 in mouse brains was responsive to calcium earlier than cytosolic calpain-5 in vitro calcium assays and in vivo bilateral common carotid artery occlusion model mice. Immunohistochemistry revealed that neurons were positive for calpain-5 in the normal brains of mice and humans. The expression of calpain-5 was increased in reactive astrocytes at human infarction sites. The knockdown of calpain-5 suppressed of cleaved caspase-11. CONCLUSIONS The neurons of human and mouse brains express calpain-5, which is proteolyzed/autolyzed in the mitochondria in the early stage of I/R injury and upregulated in reactive astrocytes in the end-stage. GENERAL SIGNIFICANCE Our results provide a comprehensive understanding of the mechanisms underlying I/R injury. Targeting the expression or activity of mitochondrial calpain-5 may suppress the inflammation during I/R injuries such as cerebrovascular diseases.
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Affiliation(s)
- Yusaku Chukai
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Iwate, Japan
| | - Ginga Ito
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Iwate, Japan
| | - Yasuo Miki
- Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, Aomori, Japan
| | - Koichi Wakabayashi
- Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, Aomori, Japan
| | - Ken Itoh
- Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, Aomori, Japan
| | - Eriko Sugano
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Iwate, Japan
| | - Hiroshi Tomita
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Iwate, Japan
| | - Tomokazu Fukuda
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Iwate, Japan
| | - Taku Ozaki
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, Iwate, Japan.
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Dong X, Zang C, Sun Y, Zhang S, Liu C, Qian J. Hydroxyapatite nanoparticles induced calcium overload-initiated cancer cell-specific apoptosis through inhibition of PMCA and activation of calpain. J Mater Chem B 2023; 11:7609-7622. [PMID: 37403708 DOI: 10.1039/d3tb00542a] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/06/2023]
Abstract
Hydroxyapatite nanoparticles (HAPNs) have been reported to specifically induce apoptosis and sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in cancer cells. However, it remains unclear whether calcium overload, the abnormal intracellular accumulation of Ca2+, is the intrinsic cause of cell apoptosis, how HAPNs specifically evoke calcium overload in cancer cells, and which potential pathways were involved in apoptosis initiation in response to calcium overload. In this study, using various cancer and normal cells, we observed a positive correlation between the degree of increased [Ca2+]i and the specific toxicity of HAPNs. Moreover, chelating intracellular Ca2+ with BAPTA-AM inhibited HAPN-induced calcium overload and apoptosis, thus demonstrating that calcium overload was the main cause of HAPN-induced cytotoxicity in cancer cells. Notably, the dissolution of particles outside the cells did not affect cell viability or [Ca2+]i. In contrast, internalized HAPNs dissolved more readily in cancer cells than in normal cells and inhibited the activity of plasma membrane calcium-ATPase solely in cancer cells to prevent extrusion of excessive Ca2+, hence leading to calcium overload in tumor cells. Upon exposure to HAPNs, the Ca2+-sensitive cysteine protease calpain was activated and then cleaved the BH3-only protein Bid. Consequently, cytochrome c was released, and caspase-9 and -3 were activated, leading to mitochondrial apoptosis. However, these effects were alleviated by the calpain inhibitor calpeptin, confirming the involvement of calpain in HANP-induced apoptosis. Therefore, our results demonstrated that calcium overload induced by HAPNs caused cancer cell-specific apoptosis by inhibiting PMCA and activating calpain in tumor cells and thus may contribute to a more comprehensive understanding of biological effects of this nanomaterial and facilitate the development of calcium overload cancer therapy.
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Affiliation(s)
- Xiulin Dong
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China.
| | - Chunyu Zang
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China.
| | - Yi Sun
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China.
| | - Shuiquan Zhang
- Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237, P. R. China
| | - Changsheng Liu
- Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237, P. R. China
| | - Jiangchao Qian
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China.
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Liu GY, Xie WL, Wang YT, Chen L, Xu ZZ, Lv Y, Wu QP. Calpain: the regulatory point of myocardial ischemia-reperfusion injury. Front Cardiovasc Med 2023; 10:1194402. [PMID: 37456811 PMCID: PMC10346867 DOI: 10.3389/fcvm.2023.1194402] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Accepted: 06/13/2023] [Indexed: 07/18/2023] Open
Abstract
Calpain is a conserved cysteine protease readily expressed in several mammalian tissues, which is usually activated by Ca2+ and with maximum activity at neutral pH. The activity of calpain is tightly regulated because its aberrant activation will nonspecifically cleave various proteins in cells. Abnormally elevation of Ca2+ promotes the abnormal activation of calpain during myocardial ischemia-reperfusion, resulting in myocardial injury and cardiac dysfunction. In this paper, we mainly reviewed the effects of calpain in various programmed cell death (such as apoptosis, mitochondrial-mediated necrosis, autophagy-dependent cell death, and parthanatos) in myocardial ischemia-reperfusion. In addition, we also discussed the abnormal activation of calpain during myocardial ischemia-reperfusion, the effect of calpain on myocardial repair, and the possible future research directions of calpain.
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Affiliation(s)
- Guo-Yang Liu
- Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Institute of Anesthesia and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Key Laboratory of Anesthesiology and Resuscitation (Huazhong University of Science and Technology), Ministry of Education, Wuhan, China
| | - Wan-Li Xie
- Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Institute of Anesthesia and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Key Laboratory of Anesthesiology and Resuscitation (Huazhong University of Science and Technology), Ministry of Education, Wuhan, China
| | - Yan-Ting Wang
- Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Institute of Anesthesia and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Key Laboratory of Anesthesiology and Resuscitation (Huazhong University of Science and Technology), Ministry of Education, Wuhan, China
| | - Lu Chen
- Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Institute of Anesthesia and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Key Laboratory of Anesthesiology and Resuscitation (Huazhong University of Science and Technology), Ministry of Education, Wuhan, China
| | - Zhen-Zhen Xu
- Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Institute of Anesthesia and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Key Laboratory of Anesthesiology and Resuscitation (Huazhong University of Science and Technology), Ministry of Education, Wuhan, China
| | - Yong Lv
- Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Institute of Anesthesia and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Key Laboratory of Anesthesiology and Resuscitation (Huazhong University of Science and Technology), Ministry of Education, Wuhan, China
| | - Qing-Ping Wu
- Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Institute of Anesthesia and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Key Laboratory of Anesthesiology and Resuscitation (Huazhong University of Science and Technology), Ministry of Education, Wuhan, China
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Ito G, Tatara Y, Itoh K, Yamada M, Yamashita T, Sakamoto K, Nozaki T, Ishida K, Wake Y, Kaneko T, Fukuda T, Sugano E, Tomita H, Ozaki T. Novel dicarbonyl metabolic pathway via mitochondrial ES1 possessing glyoxalase III activity. BBA ADVANCES 2023; 3:100092. [PMID: 37250100 PMCID: PMC10209487 DOI: 10.1016/j.bbadva.2023.100092] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/31/2023] Open
Abstract
Glycation, caused by reactive dicarbonyls, plays a role in various diseases by forming advanced glycation end products. In live cells, reactive dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) are produced during cell metabolism, and these should be removed consistently. However, the dicarbonyl metabolic system in the mitochondria remains unclear. It has been speculated that the mammalian mitochondrial protein ES1 is a homolog of bacterial elbB possessing glyoxalase III (GLO3) activity. Therefore, in this study, to investigate ES1 functions and GLO3 activity, we generated ES1-knockout (KO) mice and recombinant mouse ES1 protein and investigated the biochemical and histological analyses. In the mitochondrial fraction obtained from ES1-KO mouse brains, the GO metabolism and cytochrome c oxidase activity were significantly lower than those in the mitochondrial fraction obtained from wildtype (WT) mouse brains. However, the morphological features of the mitochondria did not change noticeably in the ES1-KO mouse brains compared with those in the WT mouse brains. The mitochondrial proteome analysis showed that the MGO degradation III pathway and oxidative phosphorylation-related proteins were increased. These should be the response to the reduced GO metabolism caused by ES1 deletion to compensate for the dicarbonyl metabolism and damaged cytochrome c oxidase by elevated GO. Recombinant mouse ES1 protein exhibited catalytic activity of converting GO to glycolic acid. These results indicate that ES1 possesses GLO3 activity and modulates the metabolism of GO in the mitochondria. To our knowledge, this is the first study to show a novel metabolic pathway for reactive dicarbonyls in mitochondria.
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Affiliation(s)
- Ginga Ito
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Yota Tatara
- Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifuchou, Hirosaki, Aomori 036-8562, Japan
| | - Ken Itoh
- Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifuchou, Hirosaki, Aomori 036-8562, Japan
| | - Miwa Yamada
- Department of Biological Chemistry, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan
| | - Tetsuro Yamashita
- Department of Biological Chemistry, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan
| | - Kimitoshi Sakamoto
- Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori 036-8561, Japan
| | - Takayuki Nozaki
- Technical Support Center for Life Science Research, Iwate Medical University, 1-1-1 Idaidori, Yahaba, Iwate 028-3694, Japan
| | - Kinji Ishida
- Technical Support Center for Life Science Research, Iwate Medical University, 1-1-1 Idaidori, Yahaba, Iwate 028-3694, Japan
| | - Yui Wake
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Takehito Kaneko
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Tomokazu Fukuda
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Eriko Sugano
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Hiroshi Tomita
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Taku Ozaki
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
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10
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Gao T, Huang J, Yin H, Huang J, Xie J, Zhou T, Fan W, Yang X, Gao G, Li Z. Inhibition of extranodal NK/T-cell lymphoma by Chiauranib through an AIF-dependent pathway and its synergy with L-asparaginase. Cell Death Dis 2023; 14:316. [PMID: 37160920 PMCID: PMC10169864 DOI: 10.1038/s41419-023-05833-w] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 03/28/2023] [Accepted: 04/24/2023] [Indexed: 05/11/2023]
Abstract
Extranodal NK/T-cell lymphoma (NKTL) is a rare and aggressive form of extranodal lymphoma with a poor prognosis. Currently, there are very limited treatment options for patients with advanced-stage disease or those with relapsed/recurrent disease. Here we show that Chiauranib, an orally small molecule inhibitor of select serine-threonine kinases (aurora B, VEGFRs, PDGFR, CSF1R, c-Kit), inhibited NKTL cell proliferation, induced cell cycle arrest, as well as suppressed the microvessel density in vitro and in vivo similar as in other types of cancer cells. Surprisingly, Chiauranib unfolded a new effect to induce apoptosis of NKTL cells by triggering AIF-dependent apoptosis other than the traditional cyt-c/caspase mitochondrial apoptosis pathway. The knockdown of AIF in vitro and in vivo dramatically blocked the efficacy of Chiauranib on NKTL. Mechanistically, the release of AIF from mitochondria is due to the upregulation of VDAC1 by the AKT-GSK3β pathway and activation of calcium-dependent m-calpain, which promotes the cleavage of VDAC1 and therefore permits the release of AIF. Notably, the low expression of Bax in both NKTL cells and patient tissues restrained the cyt-c release. It resulted in the inhibition of cyt-c/caspase mitochondrial pathway, suggesting that drugs targeting this traditional pathway may not be effective in NKTL. Furthermore, we found that L-asparaginase triggered CD95 (Fas/Apo-1)-caspase 8-caspase 3 apoptotic pathway in NKTL cells, and combination of Chiauranib and L-asparaginase exhibited a synergistic effect, suggesting a feasibility to combine these two drugs for effective treatment of NKTL. This study demonstrates Chiauranib's positive efficacy toward NKTL through the activation of the AIF-dependent apoptosis pathway for the first time. The novel and multi-targets of Chiauranib and the synergistic effect with L-asparaginase may provide a promising therapy for NKTL patients.
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Affiliation(s)
- Tianxiao Gao
- Department of Nuclear Medicine, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, 510060, P.R. China
- Department of Medical Oncology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, 510060, P.R. China
| | - Jieye Huang
- Department of Biochemistry, Zhongshan School of Medicine, SunYat-sen University, Guangzhou, China
| | - Haofan Yin
- Department of Biochemistry, Zhongshan School of Medicine, SunYat-sen University, Guangzhou, China
| | - Jiajia Huang
- Department of Medical Oncology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, 510060, P.R. China
| | - Jinye Xie
- Department of Biochemistry, Zhongshan School of Medicine, SunYat-sen University, Guangzhou, China
| | - Ti Zhou
- Department of Biochemistry, Zhongshan School of Medicine, SunYat-sen University, Guangzhou, China
- Guangdong Engineering & Technology Research Center for Gene Manipulation and Biomacromolecular Products, Sun Yat-sen University, Guangzhou, China
| | - Wei Fan
- Department of Nuclear Medicine, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, 510060, P.R. China.
| | - Xia Yang
- Department of Biochemistry, Zhongshan School of Medicine, SunYat-sen University, Guangzhou, China.
- Guangdong Provincial Key Laboratory of Brain Function and Disease, Sun Yat-sen University, Guangzhou, China.
| | - Guoquan Gao
- Department of Biochemistry, Zhongshan School of Medicine, SunYat-sen University, Guangzhou, China.
- Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education, Guangzhou, China.
| | - Zhiming Li
- Department of Medical Oncology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, 510060, P.R. China.
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11
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Chukai Y, Ito G, Konno M, Sakata Y, Ozaki T. Mitochondrial calpain-5 truncates caspase-4 during endoplasmic reticulum stress. Biochem Biophys Res Commun 2022; 608:156-162. [DOI: 10.1016/j.bbrc.2022.03.156] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2022] [Revised: 03/29/2022] [Accepted: 03/30/2022] [Indexed: 02/06/2023]
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12
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Funajima E, Ito G, Ishiyama E, Ishida K, Ozaki T. Mitochondrial localization of calpain-13 in mouse brain. Biochem Biophys Res Commun 2022; 609:149-155. [DOI: 10.1016/j.bbrc.2022.04.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Accepted: 04/01/2022] [Indexed: 12/27/2022]
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13
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Oikawa T, Fukuda T, Yamashita T, Tomita H, Ozaki T. Lentiviral expression of calpain-1 C2-like domain peptide prevents glutamate-induced cell death in mouse hippocampal neuronal HT22 cells. In Vitro Cell Dev Biol Anim 2022; 58:289-294. [PMID: 35469046 DOI: 10.1007/s11626-022-00683-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Accepted: 03/30/2022] [Indexed: 11/05/2022]
Abstract
Glutamate neurotoxicity is involved in neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Excess glutamate causes caspase-independent programmed cell death via oxidative stress and calcium influx. Our previous study showed that calpain-1 localizes to both the cytoplasm and mitochondria, where apoptosis-inducing factor (AIF) is cleaved by calpain-1 and translocates to the nucleus to induce DNA fragmentation. The autoinhibitory region of calpain-1 conjugated with the cell-penetrating peptide HIV1-Tat (namely Tat-μCL) specifically prevents the activity of mitochondrial calpain-1 and attenuates neuronal cell death in animal models of retinitis pigmentosa, as well as glutamate-induced cell death in mouse hippocampal HT22 cells. In the present study, we constructed a lentiviral vector expressing the Tat-μCL peptide and evaluated its protective effect against glutamate-induced cell death in HT22 cells. Lentiviral transduction with Tat-μCL significantly suppressed glutamate-induced nuclear translocation of AIF and DNA fragmentation. The findings of the present study suggest that the stable expression of Tat-μCL may be a potential gene therapy modality for neurodegenerative diseases.
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Affiliation(s)
- Takenori Oikawa
- Department of Biological Chemistry, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate, 020-8550, Japan
| | - Tomokazu Fukuda
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate, 020-8551, Japan
| | - Tetsuro Yamashita
- Department of Biological Chemistry, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate, 020-8550, Japan
| | - Hiroshi Tomita
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate, 020-8551, Japan
| | - Taku Ozaki
- Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate, 020-8551, Japan.
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14
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Te Boekhorst V, Jiang L, Mählen M, Meerlo M, Dunkel G, Durst FC, Yang Y, Levine H, Burgering BMT, Friedl P. Calpain-2 regulates hypoxia/HIF-induced plasticity toward amoeboid cancer cell migration and metastasis. Curr Biol 2022; 32:412-427.e8. [PMID: 34883047 PMCID: PMC10439789 DOI: 10.1016/j.cub.2021.11.040] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2019] [Revised: 07/05/2021] [Accepted: 11/16/2021] [Indexed: 12/29/2022]
Abstract
Hypoxia, through hypoxia inducible factor (HIF), drives cancer cell invasion and metastatic progression in various cancer types. In epithelial cancer, hypoxia induces the transition to amoeboid cancer cell dissemination, yet the molecular mechanisms, relevance for metastasis, and effective intervention to combat hypoxia-induced amoeboid reprogramming remain unclear. Here, we identify calpain-2 as a key regulator and anti-metastasis target of hypoxia-induced transition from collective to amoeboid dissemination of breast and head and neck (HN) carcinoma cells. Hypoxia-induced amoeboid dissemination occurred through low extracellular matrix (ECM)-adhesive, predominantly bleb-based amoeboid movement, which was maintained by a low-oxidative and -glycolytic energy metabolism ("eco-mode"). Hypoxia induced calpain-2-mediated amoeboid conversion by deactivating β1 integrins through enzymatic cleavage of the focal adhesion adaptor protein talin-1. Consequently, targeted downregulation or pharmacological inhibition of calpain-2 restored talin-1 integrity and β1 integrin engagement and reverted amoeboid to elongated phenotypes under hypoxia. Calpain-2 activity was required for hypoxia-induced amoeboid conversion in the orthotopic mouse dermis and upregulated in invasive HN tumor xenografts in vivo, and attenuation of calpain activity prevented hypoxia-induced metastasis to the lungs. This identifies the calpain-2/talin-1/β1 integrin axis as a druggable mechanosignaling program that conserves energy yet enables metastatic dissemination that can be reverted by interfering with calpain activity.
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Affiliation(s)
- Veronika Te Boekhorst
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Cell Biology, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands
| | - Liying Jiang
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Marius Mählen
- Department of Cell Biology, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands
| | - Maaike Meerlo
- Department of Molecular Cancer Research, Center for Molecular Medicine, UMC Utrecht, the Netherlands; Oncode Institute, 3521 AL Utrecht, the Netherlands
| | - Gina Dunkel
- Department of Cell Biology, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands
| | - Franziska C Durst
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Yanjun Yang
- Center for Theoretical Biological Physics, Department of Applied Physics, Rice University, Houston, TX 77005, USA; Department of Physics, Northeastern University, Boston, MA 02115, USA
| | - Herbert Levine
- Center for Theoretical Biological Physics, Department of Applied Physics, Rice University, Houston, TX 77005, USA; Department of Physics, Northeastern University, Boston, MA 02115, USA
| | - Boudewijn M T Burgering
- Department of Molecular Cancer Research, Center for Molecular Medicine, UMC Utrecht, the Netherlands; Oncode Institute, 3521 AL Utrecht, the Netherlands; Cancer Genomics Center, 3584 CG Utrecht, the Netherlands
| | - Peter Friedl
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Cell Biology, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands; Cancer Genomics Center, 3584 CG Utrecht, the Netherlands.
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15
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Fares M, Oerther S, Hultenby K, Gubrianska D, Zhao Y, Abedi-Valugerdi M, Hassan M. COL-3-Induced Molecular and Ultrastructural Alterations in K562 Cells. J Pers Med 2022; 12:jpm12010042. [PMID: 35055357 PMCID: PMC8778770 DOI: 10.3390/jpm12010042] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Revised: 12/28/2021] [Accepted: 12/30/2021] [Indexed: 01/05/2023] Open
Abstract
Tetracycline-3 (4-dedimethylamino sancycline, COL-3) is a non-antibiotic tetracycline derivative. COL-3 exerts potent anti-metalloproteinase activity and its antitumor effects have been reported both in vitro and in vivo. In this study, we investigated the mechanisms of COL-3-induced cytotoxicity in a chronic myeloid leukemia cell line, K562, characterized by the BCR-ABL fusion protein. COL-3 induced K562 cell death in a concentration-dependent manner with an IC50 of 10.8 µg/mL and exhibited features of both apoptosis and necrosis. However, flow cytometry analysis revealed that necrotic cells dominated over the early and late apoptotic cells upon treatment with COL-3. Transmission electron microscopy analysis in combination with Western blotting (WB) analysis revealed early mitochondrial swelling accompanied by the early release of cytochrome c and truncated apoptosis inducing factor (tAIF). In addition, ultrastructural changes were detected in the endoplasmic reticulum (ER). COL-3 affected the levels of glucose-regulated protein-94 (GRP94) and resulted in m-calpain activation. DNA double strand breaks as a signature for DNA damage was also confirmed using an antibody against γH2AX. WB analyses did not demonstrate caspase activation, while Bcl-xL protein remained unaffected. In conclusion, COL-3-induced cell death involves DNA damage as well as mitochondrial and ER perturbation with features of paraptosis and programmed necrosis.
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Affiliation(s)
- Mona Fares
- Experimental Cancer Medicine, Division of Biomolecular and Cellular Medicine (BCM), Department of Laboratory Medicine, Novum, Karolinska Institutet, 141 57 Huddinge, Sweden; (M.F.); (S.O.); (D.G.); (Y.Z.); (M.A.-V.)
- Clinical Research Center and Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital, 141 86 Huddinge, Sweden
| | - Sandra Oerther
- Experimental Cancer Medicine, Division of Biomolecular and Cellular Medicine (BCM), Department of Laboratory Medicine, Novum, Karolinska Institutet, 141 57 Huddinge, Sweden; (M.F.); (S.O.); (D.G.); (Y.Z.); (M.A.-V.)
- Clinical Research Center and Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital, 141 86 Huddinge, Sweden
| | - Kjell Hultenby
- Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, 141 57 Huddinge, Sweden;
| | - Danica Gubrianska
- Experimental Cancer Medicine, Division of Biomolecular and Cellular Medicine (BCM), Department of Laboratory Medicine, Novum, Karolinska Institutet, 141 57 Huddinge, Sweden; (M.F.); (S.O.); (D.G.); (Y.Z.); (M.A.-V.)
- Clinical Research Center and Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital, 141 86 Huddinge, Sweden
| | - Ying Zhao
- Experimental Cancer Medicine, Division of Biomolecular and Cellular Medicine (BCM), Department of Laboratory Medicine, Novum, Karolinska Institutet, 141 57 Huddinge, Sweden; (M.F.); (S.O.); (D.G.); (Y.Z.); (M.A.-V.)
- Clinical Research Center and Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital, 141 86 Huddinge, Sweden
| | - Manuchehr Abedi-Valugerdi
- Experimental Cancer Medicine, Division of Biomolecular and Cellular Medicine (BCM), Department of Laboratory Medicine, Novum, Karolinska Institutet, 141 57 Huddinge, Sweden; (M.F.); (S.O.); (D.G.); (Y.Z.); (M.A.-V.)
- Clinical Research Center and Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital, 141 86 Huddinge, Sweden
| | - Moustapha Hassan
- Experimental Cancer Medicine, Division of Biomolecular and Cellular Medicine (BCM), Department of Laboratory Medicine, Novum, Karolinska Institutet, 141 57 Huddinge, Sweden; (M.F.); (S.O.); (D.G.); (Y.Z.); (M.A.-V.)
- Clinical Research Center and Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital, 141 86 Huddinge, Sweden
- Correspondence:
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16
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Keasey MP, Razskazovskiy V, Jia C, Peterknecht ED, Bradshaw PC, Hagg T. PDIA3 inhibits mitochondrial respiratory function in brain endothelial cells and C. elegans through STAT3 signaling and decreases survival after OGD. Cell Commun Signal 2021; 19:119. [PMID: 34922569 PMCID: PMC8684072 DOI: 10.1186/s12964-021-00794-z] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2021] [Accepted: 10/14/2021] [Indexed: 11/20/2022] Open
Abstract
BACKGROUND Protein disulfide isomerase A3 (PDIA3, also named GRP58, ER-60, ERp57) is conserved across species and mediates protein folding in the endoplasmic reticulum. PDIA3 is, reportedly, a chaperone for STAT3. However, the role of PDIA3 in regulating mitochondrial bioenergetics and STAT3 phosphorylation at serine 727 (S727) has not been described. METHODS Mitochondrial respiration was compared in immortalized human cerebral microvascular cells (CMEC) wild type or null for PDIA3 and in whole organism C. Elegans WT or null for pdi-3 (worm homologue). Mitochondrial morphology and cell signaling pathways in PDIA3-/- and WT cells were assessed. PDIA3-/- cells were subjected to oxygen-glucose deprivation (OGD) to determine the effects of PDIA3 on cell survival after injury. RESULTS We show that PDIA3 gene deletion using CRISPR-Cas9 in cultured CMECs leads to an increase in mitochondrial bioenergetic function. In C. elegans, gene deletion or RNAi knockdown of pdi-3 also increased respiratory rates, confirming a conserved role for this gene in regulating mitochondrial bioenergetics. The PDIA3-/- bioenergetic phenotype was reversed by overexpression of WT PDIA3 in cultured PDIA3-/- CMECs. PDIA3-/- and siRNA knockdown caused an increase in phosphorylation of the S727 residue of STAT3, which is known to promote mitochondrial bioenergetic function. Increased respiration in PDIA3-/- CMECs was reversed by a STAT3 inhibitor. In PDIA3-/- CMECs, mitochondrial membrane potential and reactive oxygen species production, but not mitochondrial mass, was increased, suggesting an increased mitochondrial bioenergetic capacity. Finally, PDIA3-/- CMECs were more resistant to oxygen-glucose deprivation, while STAT3 inhibition reduced the protective effect. CONCLUSIONS We have discovered a novel role for PDIA3 in suppressing mitochondrial bioenergetic function by inhibiting STAT3 S727 phosphorylation.
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Affiliation(s)
- Matt. P. Keasey
- Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN 37614 USA
| | - V. Razskazovskiy
- Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN 37614 USA
| | - C. Jia
- Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN 37614 USA
- Sandwell and West, Birmingham Hospitals NHS Trust, Birmingham, UK
| | | | - P. C. Bradshaw
- Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN 37614 USA
| | - T. Hagg
- Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN 37614 USA
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Yan J, Chen Y, Zhu Y, Paquet-Durand F. Programmed Non-Apoptotic Cell Death in Hereditary Retinal Degeneration: Crosstalk between cGMP-Dependent Pathways and PARthanatos? Int J Mol Sci 2021; 22:10567. [PMID: 34638907 PMCID: PMC8508647 DOI: 10.3390/ijms221910567] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Revised: 09/26/2021] [Accepted: 09/27/2021] [Indexed: 12/20/2022] Open
Abstract
Programmed cell death (PCD) is a highly regulated process that results in the orderly destruction of a cell. Many different forms of PCD may be distinguished, including apoptosis, PARthanatos, and cGMP-dependent cell death. Misregulation of PCD mechanisms may be the underlying cause of neurodegenerative diseases of the retina, including hereditary retinal degeneration (RD). RD relates to a group of diseases that affect photoreceptors and that are triggered by gene mutations that are often well known nowadays. Nevertheless, the cellular mechanisms of PCD triggered by disease-causing mutations are still poorly understood, and RD is mostly still untreatable. While investigations into the neurodegenerative mechanisms of RD have focused on apoptosis in the past two decades, recent evidence suggests a predominance of non-apoptotic processes as causative mechanisms. Research into these mechanisms carries the hope that the knowledge created can eventually be used to design targeted treatments to prevent photoreceptor loss. Hence, in this review, we summarize studies on PCD in RD, including on apoptosis, PARthanatos, and cGMP-dependent cell death. Then, we focus on a possible interplay between these mechanisms, covering cGMP-signaling targets, overactivation of poly(ADP-ribose)polymerase (PARP), energy depletion, Ca2+-permeable channels, and Ca2+-dependent proteases. Finally, an outlook is given into how specific features of cGMP-signaling and PARthanatos may be targeted by therapeutic interventions.
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Affiliation(s)
| | | | | | - François Paquet-Durand
- Cell Death Mechanism Group, Institute for Ophthalmic Research, University of Tübingen, Elfriede-Aulhorn-Strasse 7, 72076 Tübingen, Germany; (J.Y.); (Y.C.); (Y.Z.)
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18
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Calpain-1 C2L domain peptide protects mouse hippocampus-derived neuronal HT22 cells against glutamate-induced oxytosis. Biochem Biophys Rep 2021; 27:101101. [PMID: 34430716 PMCID: PMC8374356 DOI: 10.1016/j.bbrep.2021.101101] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2021] [Revised: 08/02/2021] [Accepted: 08/06/2021] [Indexed: 01/13/2023] Open
Abstract
Calpains are Ca2+-dependent cysteine proteases; their aberrant activation is associated with several neurodegenerative diseases. The μ-calpain catalytic subunit, calpain-1, is located in the cytoplasm as well as in the mitochondria. Mitochondrial calpain-1 cleaves apoptosis-inducing factor (AIF), leading to apoptotic cell death. We have previously reported that short peptides of calpain-1 C2-like domain conjugated with cell penetrating peptide HIV-Tat (Tat-μCL) selectively inhibit mitochondrial calpain-1 and effectively prevent neurodegenerative diseases of the eye. In this study, we determined whether mitochondrial calpain-1 mediates oxytosis (oxidative glutamate toxicity) in hippocampal HT22 cells using Tat-μCL and newly generated polyhistidine-conjugated μCL peptide and compared their efficacies in preventing oxytosis. TUNEL assay and single strand DNA staining revealed that both μCL peptides inhibited glutamate-induced oxytosis. Additionally, both the peptides suppressed the mitochondrial AIF translocation into the nucleus. All polyhistidine-μCL peptides (containing 4–16 histidine residues) showed higher cell permeability than Tat-μCL. Notably, tetrahistidine (H4)-μCL exerted the highest cytoprotective activity. Thus, H4-μCL may be a potential peptide drug for calpain-1-mediated neurodegenerative diseases such as Alzheimer's disease.
Mitochondrial calpain-1 mediates glutamate-induced oxytosis in HT22 cells. CPP-μCL inhibits AIF translocation and DNA fragmentation in oxytosis. Polyhistidine conjugation enhances intracellular μCL peptide uptake efficiency.
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Key Words
- AIF, apoptosis-inducing factor
- BBB, blood–brain barrier
- CPP, cell penetrating peptide
- Cell penetrating peptide
- DAPI, 4′,6-diamidino-2-phenylindole
- DMEM, Dulbecco's Modified Eagle Medium
- Hippocampal HT22 cells
- Hn-μCL, polyhistidine-conjugated μCL
- MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
- Mitochondrial calpain-1
- Neurodegeneration
- Oxytosis
- PBS, phosphate-buffered saline
- ssDNA, single stranded DNA
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19
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Feng Y, Huang W, Paul C, Liu X, Sadayappan S, Wang Y, Pauklin S. Mitochondrial nucleoid in cardiac homeostasis: bidirectional signaling of mitochondria and nucleus in cardiac diseases. Basic Res Cardiol 2021; 116:49. [PMID: 34392401 PMCID: PMC8364536 DOI: 10.1007/s00395-021-00889-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Accepted: 07/20/2021] [Indexed: 01/11/2023]
Abstract
Metabolic function and energy production in eukaryotic cells are regulated by mitochondria, which have been recognized as the intracellular 'powerhouses' of eukaryotic cells for their regulation of cellular homeostasis. Mitochondrial function is important not only in normal developmental and physiological processes, but also in a variety of human pathologies, including cardiac diseases. An emerging topic in the field of cardiovascular medicine is the implication of mitochondrial nucleoid for metabolic reprogramming. This review describes the linear/3D architecture of the mitochondrial nucleoid (e.g., highly organized protein-DNA structure of nucleoid) and how it is regulated by a variety of factors, such as noncoding RNA and its associated R-loop, for metabolic reprogramming in cardiac diseases. In addition, we highlight many of the presently unsolved questions regarding cardiac metabolism in terms of bidirectional signaling of mitochondrial nucleoid and 3D chromatin structure in the nucleus. In particular, we explore novel techniques to dissect the 3D structure of mitochondrial nucleoid and propose new insights into the mitochondrial retrograde signaling, and how it regulates the nuclear (3D) chromatin structures in mitochondrial diseases.
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Affiliation(s)
- Yuliang Feng
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Oxford, OX3 7LD, UK
| | - Wei Huang
- Department of Pathology and Laboratory Medicine, Regenerative Medicine Research, University of Cincinnati College of Medicine, 231 Albert Sabin Way, CincinnatiCincinnati, OH, 45267-0529, USA
| | - Christian Paul
- Department of Pathology and Laboratory Medicine, Regenerative Medicine Research, University of Cincinnati College of Medicine, 231 Albert Sabin Way, CincinnatiCincinnati, OH, 45267-0529, USA
| | - Xingguo Liu
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Guangzhou Regenerative Medicine and Health Guangdong Laboratory, CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Sakthivel Sadayappan
- Heart, Lung and Vascular Institute, Division of Cardiovascular Health and Disease, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH, 45267, USA
| | - Yigang Wang
- Department of Pathology and Laboratory Medicine, Regenerative Medicine Research, University of Cincinnati College of Medicine, 231 Albert Sabin Way, CincinnatiCincinnati, OH, 45267-0529, USA.
| | - Siim Pauklin
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Oxford, OX3 7LD, UK.
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20
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Calpain-Mediated Mitochondrial Damage: An Emerging Mechanism Contributing to Cardiac Disease. Cells 2021; 10:cells10082024. [PMID: 34440793 PMCID: PMC8392834 DOI: 10.3390/cells10082024] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Revised: 07/19/2021] [Accepted: 08/06/2021] [Indexed: 12/13/2022] Open
Abstract
Calpains belong to the family of calcium-dependent cysteine proteases expressed ubiquitously in mammals and many other organisms. Activation of calpain is observed in diseased hearts and is implicated in cardiac cell death, hypertrophy, fibrosis, and inflammation. However, the underlying mechanisms remain incompletely understood. Recent studies have revealed that calpains target and impair mitochondria in cardiac disease. The objective of this review is to discuss the role of calpains in mediating mitochondrial damage and the underlying mechanisms, and to evaluate whether targeted inhibition of mitochondrial calpain is a potential strategy in treating cardiac disease. We expect to describe the wealth of new evidence surrounding calpain-mediated mitochondrial damage to facilitate future mechanistic studies and therapy development for cardiac disease.
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21
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Kobayashi-Otsugu M, Orihara K, Nakajima E, Shearer TR, Azuma M. Activation of Cytosolic Calpain, Not Caspase, Is Underlying Mechanism for Hypoxic RGC Damage in Human Retinal Explants. Invest Ophthalmol Vis Sci 2021; 61:13. [PMID: 33156340 PMCID: PMC7671854 DOI: 10.1167/iovs.61.13.13] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Purpose Activation of proteolytic enzymes, calpains and caspases, have been observed in many models of retinal disease. We previously demonstrated calpain activation in monkey retinal explants cultured under hypoxia. However, cellular responses are often species-specific. The purpose of the present study was to determine whether calpains or caspase-3 was involved in retinal ganglion cell (RGC) damage caused by hypoxia/reoxygenation in human retinal explants. The explant model was improved by use of an oxygen-controlled chamber. Methods Human and monkey retinal explants were cultured under hypoxic conditions in an oxygen-controlled chamber and then reoxygenated. Calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting were performed for calpains 1 and 2, calpastatin, α-spectrin, calpain-specific α-spectrin breakdown product at 150 kDa (SBDP150), caspase-3, and apoptosis-inducing factor (AIF). Propidium iodide (PI) staining measured membrane disruption, and TUNEL staining detected DNA fragmentation. Results Activation of calpains in nerve fibers and increases of PI-positive RGCs were observed in retinal explants incubated for 16-hour hypoxia/8-hour reoxygenation. Except for autolysis of calpain 2, SNJ-1945 ameliorated these changes. In longer incubations under 24-hour hypoxia/16-hour reoxygenation, TUNEL-positive cells appeared, although activated caspase-3 and truncated AIF were not observed. DNA fragmentation was inhibited by SNJ-1945. Conclusions An improved human retinal explant model showed that calpains, not caspase-3, were involved in cell damage induced by hypoxia/reoxygenation. This finding could be relevant for patient treatment with a calpain inhibitor if calpain activation is documented in human retinal ischemic diseases.
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Affiliation(s)
- Momoko Kobayashi-Otsugu
- Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Portland, Oregon, United States.,Department of Integrative Biomedical & Diagnostic Sciences, Oregon Health & Science University, Portland, Oregon, United States
| | - Kana Orihara
- Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Portland, Oregon, United States.,Department of Integrative Biomedical & Diagnostic Sciences, Oregon Health & Science University, Portland, Oregon, United States
| | - Emi Nakajima
- Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Portland, Oregon, United States.,Department of Integrative Biomedical & Diagnostic Sciences, Oregon Health & Science University, Portland, Oregon, United States
| | - Thomas R Shearer
- Department of Integrative Biomedical & Diagnostic Sciences, Oregon Health & Science University, Portland, Oregon, United States
| | - Mitsuyoshi Azuma
- Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Portland, Oregon, United States.,Department of Integrative Biomedical & Diagnostic Sciences, Oregon Health & Science University, Portland, Oregon, United States
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22
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Belkheir AM, Reunert J, Elpers C, van den Heuvel L, Rodenburg R, Seelhöfer A, Rust S, Jeibmann A, Frosch M, Marquardt T. Severe Form of ßIV-Spectrin Deficiency With Mitochondrial Dysfunction and Cardiomyopathy-A Case Report. Front Neurol 2021; 12:643805. [PMID: 33986717 PMCID: PMC8110827 DOI: 10.3389/fneur.2021.643805] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Accepted: 03/25/2021] [Indexed: 11/13/2022] Open
Abstract
ßIV-spectrin is a protein of the spectrin family which is involved in the organization of the cytoskeleton structure and is found in high quantity in the axon initial segment and the nodes of Ranvier. Together with ankyrin G, ßIV-spectrin is responsible for the clustering of KCNQ2/3-potassium channels and NaV-sodium channels. Loss or reduction of ßIV-spectrin causes a destabilization of the cytoskeleton and an impairment in the generation of the action potential, which leads to neuronal degeneration. Furthermore, ßIV-spectrin has been described to play an important role in the maintenance of the neuronal polarity and of the diffusion barrier. ßIV-spectrin is also located in the heart where it takes an important part in the structural organization of ion channels and has also been described to participate in cell signaling pathways through binding of transcription factors. We describe two patients with a severe form of ßIV-spectrin deficiency. Whole-exome sequencing revealed the homozygous stop mutation c.6016C>T (p.R2006*) in the SPTBN4 gene. The phenotype of these patients is characterized by profound psychomotor developmental arrest, respiratory insufficiency and deafness. Additionally one of the patients presents with cardiomyopathy, optical nerve atrophy, and mitochondrial dysfunction. This is the first report of a severe form of ßIV-spectrin deficiency with hypertrophic cardiomyopathy and mitochondrial dysfunction.
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Affiliation(s)
- Aziza Miriam Belkheir
- Department of General Paediatrics, Metabolic Diseases, University Children's Hospital Muenster, Münster, Germany
| | - Janine Reunert
- Department of General Paediatrics, Metabolic Diseases, University Children's Hospital Muenster, Münster, Germany
| | - Christiane Elpers
- Department of General Paediatrics, Metabolic Diseases, University Children's Hospital Muenster, Münster, Germany
| | - Lambert van den Heuvel
- Translational Metabolic Laboratory, Department of Paediatrics, Radboud Center for Mitochondrial Medicine, Radboud UMC, Nijmegen, Netherlands
| | - Richard Rodenburg
- Translational Metabolic Laboratory, Department of Paediatrics, Radboud Center for Mitochondrial Medicine, Radboud UMC, Nijmegen, Netherlands
| | - Anja Seelhöfer
- Department of General Paediatrics, Metabolic Diseases, University Children's Hospital Muenster, Münster, Germany
| | - Stephan Rust
- Department of General Paediatrics, Metabolic Diseases, University Children's Hospital Muenster, Münster, Germany
| | - Astrid Jeibmann
- Institute of Neuropathology, University Hospital Muenster, Münster, Germany
| | - Michael Frosch
- Department of Children's Pain Therapy and Paediatric Palliative Care, Faculty of Health-School of Medicine, Witten/Herdecke University, Witten, Germany
| | - Thorsten Marquardt
- Department of General Paediatrics, Metabolic Diseases, University Children's Hospital Muenster, Münster, Germany
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23
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Chukai Y, Iwamoto T, Itoh K, Tomita H, Ozaki T. Characterization of mitochondrial calpain-5. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2021; 1868:118989. [PMID: 33607190 DOI: 10.1016/j.bbamcr.2021.118989] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 07/17/2020] [Revised: 01/21/2021] [Accepted: 02/09/2021] [Indexed: 01/08/2023]
Abstract
Calpain, a Ca2+-dependent cysteine protease, plays a significant role in gene expression, signal transduction, and apoptosis. Mutations in human calpain-5 cause autosomal dominant neovascular inflammatory vitreoretinopathy and the inhibition of calpain-5 activity may constitute an effective therapeutic strategy for this condition. Although calpain-5 is ubiquitously expressed in mammalian tissues and was recently found to be present in the mitochondria as well as in the cytosol, its physiological function and enzymological properties require further elucidation. The objective of the current study was to determine the characteristics of mitochondrial calpain-5 in porcine retinas, human HeLa cells, and C57BL/6J mice using subcellular fractionation. We found that mitochondrial calpain-5 was proteolyzed/autolyzed at low Ca2+ concentrations in mitochondria isolated from porcine retinas and by thapsigargin-induced endoplasmic reticulum (ER) stress in HeLa cells. Further, mitochondrial calpain-5, as opposed to cytosolic calpain-5, was activated during the early stages of ER stress in C57BL/6J mice. These results showed that mitochondrial calpain-5 was activated at low Ca2+ concentrations in vitro and in response to ER stress in vivo. The present study provides new insights into a novel calpain system in the mitochondria that includes stress responses during the early phases of ER stress. Further, activation of mitochondrial calpain-5 by treatment using low-molecular-weight compounds may have therapeutic potential for diseases related to ER stress, including neurodegenerative diseases, metabolic syndromes, diabetes, and cancer.
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Affiliation(s)
- Yusaku Chukai
- Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Takeshi Iwamoto
- Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Ken Itoh
- Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifuchou, Hirosaki, Aomori 036-8562, Japan
| | - Hiroshi Tomita
- Laboratory of Visual Neuroscience, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Taku Ozaki
- Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan.
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24
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Herrmann JM, Riemer J. Apoptosis inducing factor and mitochondrial NADH dehydrogenases: redox-controlled gear boxes to switch between mitochondrial biogenesis and cell death. Biol Chem 2020; 402:289-297. [PMID: 32769219 DOI: 10.1515/hsz-2020-0254] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Accepted: 08/03/2020] [Indexed: 02/07/2023]
Abstract
The mitochondrial complex I serves as entry point for NADH into the electron transport chain. In animals, fungi and plants, additional NADH dehydrogenases carry out the same electron transfer reaction, however they do not pump protons. The apoptosis inducing factor (AIF, AIFM1 in humans) is a famous member of this group as it was the first pro-apoptotic protein identified that can induce caspase-independent cell death. Recent studies on AIFM1 and the NADH dehydrogenase Nde1 of baker's yeast revealed two independent and experimentally separable activities of this class of enzymes: On the one hand, these proteins promote the functionality of mitochondrial respiration in different ways: They channel electrons into the respiratory chain and, at least in animals, promote the import of Mia40 (named MIA40 or CHCHD4 in humans) and the assembly of complex I. On the other hand, they can give rise to pro-apoptotic fragments that are released from the mitochondria to trigger cell death. Here we propose that AIFM1 and Nde1 serve as conserved redox switches which measure metabolic conditions on the mitochondrial surface and translate it into a binary life/death decision. This function is conserved among eukaryotic cells and apparently used to purge metabolically compromised cells from populations.
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Affiliation(s)
- Johannes M Herrmann
- Department of Cell Biology, University of Kaiserslautern, Erwin-Schrödinger-Strasse 13, D-67663Kaiserslautern, Germany
| | - Jan Riemer
- Department of Biochemistry, University of Cologne, Zülpicher Str. 47A, D-50674Cologne, Germany
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25
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Tian W, Czopka T, López-Schier H. Systemic loss of Sarm1 protects Schwann cells from chemotoxicity by delaying axon degeneration. Commun Biol 2020; 3:49. [PMID: 32001778 PMCID: PMC6992705 DOI: 10.1038/s42003-020-0776-9] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2019] [Accepted: 01/09/2020] [Indexed: 12/11/2022] Open
Abstract
Protecting the nervous system from chronic effects of physical and chemical stress is a pressing clinical challenge. The obligate pro-degenerative protein Sarm1 is essential for Wallerian axon degeneration. Thus, blocking Sarm1 function is emerging as a promising neuroprotective strategy with therapeutic relevance. Yet, the conditions that will most benefit from inhibiting Sarm1 remain undefined. Here we combine genome engineering, pharmacology and high-resolution intravital videmicroscopy in zebrafish to show that genetic elimination of Sarm1 increases Schwann-cell resistance to toxicity by diverse chemotherapeutic agents after axonal injury. Synthetic degradation of Sarm1-deficient axons reversed this effect, suggesting that glioprotection is a non-autonomous effect of delayed axon degeneration. Moreover, loss of Sarm1 does not affect macrophage recruitment to nerve-wound microenvironment, injury resolution, or neural-circuit repair. These findings anticipate that interventions aimed at inhibiting Sarm1 can counter heightened glial vulnerability to chemical stressors and may be an effective strategy to reduce chronic consequences of neurotrauma.
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Affiliation(s)
- Weili Tian
- Sensory Biology & Organogenesis, Helmholtz Zentrum Munich, Munich, Germany
| | - Tim Czopka
- Institute of Neuronal Cell Biology, Technical University of Munich, Munich, Germany
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26
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Kutluer M, Huang L, Marigo V. Targeting molecular pathways for the treatment of inherited retinal degeneration. Neural Regen Res 2020; 15:1784-1791. [PMID: 32246618 PMCID: PMC7513962 DOI: 10.4103/1673-5374.280303] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Inherited retinal degeneration is a major cause of incurable blindness characterized by loss of retinal photoreceptor cells. Inherited retinal degeneration is characterized by high genetic and phenotypic heterogeneity with several genes mutated in patients affected by these genetic diseases. The high genetic heterogeneity of these diseases hampers the development of effective therapeutic interventions for the cure of a large cohort of patients. Common cell demise mechanisms can be envisioned as targets to treat patients regardless the specific mutation. One of these targets is the increase of intracellular calcium ions, that has been detected in several murine models of inherited retinal degeneration. Recently, neurotrophic factors that favor the efflux of calcium ions to concentrations below toxic levels have been identified as promising molecules that should be evaluated as new treatments for retinal degeneration. Here, we discuss therapeutic options for inherited retinal degeneration and we will focus on neuroprotective approaches, such as the neuroprotective activity of the Pigment epithelium-derived factor. The characterization of specific targets for neuroprotection opens new perspectives together with many questions that require deep analyses to take advantage of this knowledge and develop new therapeutic approaches. We believe that minimizing cell demise by neuroprotection may represent a promising treatment strategy for retinal degeneration.
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Affiliation(s)
- Meltem Kutluer
- Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Li Huang
- Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Valeria Marigo
- Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
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27
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Weber JJ, Clemensson LE, Schiöth HB, Nguyen HP. Olesoxime in neurodegenerative diseases: Scrutinising a promising drug candidate. Biochem Pharmacol 2019; 168:305-318. [PMID: 31283931 DOI: 10.1016/j.bcp.2019.07.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2019] [Accepted: 07/03/2019] [Indexed: 12/14/2022]
Abstract
Over the last years, the experimental compound olesoxime, a mitochondria-targeting cholesterol derivative, has emerged as a promising drug candidate for neurodegenerative diseases. Numerous preclinical studies have successfully proved olesoxime's neuroprotective properties in cell and animal models of clinical conditions such as amyotrophic lateral sclerosis, Huntington disease, Parkinson disease, peripheral neuropathy and spinal muscular atrophy. The beneficial effects were attributed to olesoxime's potential impact on oxidative stress, mitochondrial permeability transition or cholesterol homoeostasis. Although no significant benefits have been demonstrated in patients of amyotrophic lateral sclerosis, and only the first 12 months of a phase II/III clinical trial showed an improvement in motor symptoms of spinal muscular atrophy, this orphan drug may still offer undiscovered potential in the treatment of neurological diseases. In our earlier preclinical studies, we demonstrated that administration of olesoxime in mouse and rat models of Huntington disease improved psychiatric and molecular phenotypes. Aside from stabilising mitochondrial function, the drug reduced the overactivation of calpains, a class of calcium-dependent proteases entangled in neurodegenerative conditions. This observation may be credited to olesoxime's action on calcium dyshomeostasis, a further hallmark in neurodegeneration, and linked to its targets TSPO and VDAC, two proteins of the outer mitochondrial membrane associated with mitochondrial calcium handling. Further research into the mode of action of olesoxime under pathological conditions, including its effect on neuronal calcium homeostasis, may strengthen the untapped potential of olesoxime or other similar compounds as a therapeutic for neurodegenerative diseases.
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Affiliation(s)
- Jonasz Jeremiasz Weber
- Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.
| | | | - Helgi Birgir Schiöth
- Department of Neuroscience, Uppsala University, Uppsala, Sweden; Institute for Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, Moscow, Russia.
| | - Huu Phuc Nguyen
- Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany; Department of Human Genetics, Ruhr-University Bochum, Bochum, Germany.
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28
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Li J, Guo M, Liu Y, Wu G, Miao L, Zhang J, Zuo Z, Li Y. Both GSK-3β/CRMP2 and CDK5/CRMP2 pathways participate in the protection of dexmedetomidine against propofol-induced learning and memory impairment in neonatal rats. Toxicol Sci 2019; 171:193-210. [PMID: 31187143 DOI: 10.1093/toxsci/kfz135] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2018] [Revised: 05/17/2019] [Accepted: 05/29/2019] [Indexed: 11/13/2022] Open
Abstract
Dexmedetomidine has been reported to ameliorate propofol-induced neurotoxicity in neonatal animals. However, the underlying mechanism is still undetermined. Glycogen synthase kinase-3β (GSK-3β), cycline dependent kinase-5 (CDK5) and Rho-kinase (RhoA) pathways play critical roles in neuronal development. The present study is to investigate whether GSK-3β, CDK5 and RhoA pathways are involved in the neuroprotection of dexmedetomidine. Seven-day-old (P7) Sprague-Dawley rats were anesthetized with propofol for 6 h. Dexmedetomidine at various concentrations were administered before propofol exposure. Neuroapoptosis, the neuronal proliferation and the level of neurotransmitter in the hippocampus were evaluated. The effects of GSK-3β inhibitor SB415286, CDK5 inhibitor roscovitine or RhoA inhibitor Y276321 on propofol-induced neurotoxicity were assessed. Propofol induced apoptosis in the hippocampal neurons and astrocytes, inhibited neuronal proliferation in the DG region, down-regulated the level of γ-aminobutyric acid (GABA) and glutamate in the hippocampus, and impaired long-term cognitive function. These harmful effects were reduced by pretreatment with 50 μg·kg-1 dexmedetomidine. Moreover, propofol activated GSK-3β and CDK5 pathways, but not RhoA pathway, by reducing the phosphorylation of GSK-3β (ser 9), increasing the expression of CDK5 activator P25 and increasing the phosphorylation of their target sites on CRMP2 shortly after exposure. These effects were reversed by pretreatment with 50 μg·kg-1 dexmedetomidine. Furthermore, SB415286 and roscovitine, not Y276321, attenuated the propofol-induced neuroapoptosis, brain cell proliferation inhibition, GABA and glutamate downregulation, and learning and memory dysfunction. Our results indicate that dexmedetomidine reduces propofol-induced neurotoxicity and neurocognitive impairment via inhibiting activation of GSK-3β/CRMP2 and CDK5/CRMP2 pathways in the hippocampus of neonatal rats.
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Affiliation(s)
- Junhua Li
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.,Laboratory of RNA and Major Diseases of Brain and Hearts, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - Minyan Guo
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.,Laboratory of RNA and Major Diseases of Brain and Hearts, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - Yafang Liu
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.,Laboratory of RNA and Major Diseases of Brain and Hearts, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - Guiyun Wu
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - Liping Miao
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - Jing Zhang
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - Zhiyi Zuo
- Department of Anesthesiology, University of Virginia Health System, Charlottesville, Virginia, 22908-0710, USA
| | - Yujuan Li
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.,Laboratory of RNA and Major Diseases of Brain and Hearts, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.,Guangdong Province Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
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29
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Randriamboavonjy V, Kyselova A, Fleming I. Redox Regulation of Calpains: Consequences on Vascular Function. Antioxid Redox Signal 2019; 30:1011-1026. [PMID: 30266074 DOI: 10.1089/ars.2018.7607] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
SIGNIFICANCE Calpains (CAPNs) are a family of calcium-activated cysteine proteases. The ubiquitous isoforms CAPN1 and CAPN2 have been involved in the maintenance of vascular integrity, but uncontrolled CAPN activation plays a role in the pathogenesis of vascular diseases. Recent Advances: It is well accepted that chronic and acute overproduction of reactive oxygen species (ROS) is associated with the development of vascular diseases. There is increasing evidence that ROS can also affect the CAPN activity, suggesting CAPN as a potential link between oxidative stress and vascular disease. CRITICAL ISSUES The physiopathological relevance of ROS in regulating the CAPN activity is not fully understood but seems to involve direct effects on CAPNs, redox modifications of CAPN substrates, as well as indirect effect on CAPNs via changes in Ca2+ levels. Finally, CAPNs can also stimulate ROS production; however, data showing in which context ROS are the causes or the consequences of CAPN activation are missing. FUTURE DIRECTIONS Detailed characterization of the molecular mechanisms underlying the regulation of the different members of the CAPN system by specific ROS would help understanding the pathophysiological role of CAPN in the modulation of the vascular function. Moreover, given that CAPNs have been found in different cellular compartments such as mitochondria and nucleus as well as in the extracellular space, identification of new CAPN targets as well as their functional consequences would add new insights in the function of these enigmatic proteases.
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Affiliation(s)
- Voahanginirina Randriamboavonjy
- 1 Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany.,2 German Center of Cardiovascular Research (DZHK), Partner Site Rhein-Main, Frankfurt am Main, Germany
| | - Anastasia Kyselova
- 1 Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany.,2 German Center of Cardiovascular Research (DZHK), Partner Site Rhein-Main, Frankfurt am Main, Germany
| | - Ingrid Fleming
- 1 Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany.,2 German Center of Cardiovascular Research (DZHK), Partner Site Rhein-Main, Frankfurt am Main, Germany
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30
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Mitochondrial calcium signalling and neurodegenerative diseases. Neuronal Signal 2018; 2:NS20180061. [PMID: 32714593 PMCID: PMC7373239 DOI: 10.1042/ns20180061] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2018] [Revised: 09/06/2018] [Accepted: 09/20/2018] [Indexed: 12/11/2022] Open
Abstract
Calcium is utilised by cells in signalling and in regulating ATP production; it also contributes to cell survival and, when concentrations are unbalanced, triggers pathways for cell death. Mitochondria contribute to calcium buffering, meaning that mitochondrial calcium uptake and release is intimately related to cytosolic calcium concentrations. This review focuses on the proteins contributing to mitochondrial calcium homoeostasis, the roles of the mitochondrial permeability transition pore (MPTP) and mitochondrial calcium-activated proteins, and their relevance in neurodegenerative pathologies. It also covers alterations to calcium homoeostasis in Friedreich ataxia (FA).
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Iwamoto T, Ishiyama E, Ishida K, Yamashita T, Tomita H, Ozaki T. Presence of calpain-5 in mitochondria. Biochem Biophys Res Commun 2018; 504:454-459. [DOI: 10.1016/j.bbrc.2018.08.144] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2018] [Accepted: 08/24/2018] [Indexed: 01/10/2023]
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Plackal Adimuriyil George B, Kumar N, Abrahamse H, Ray SS. Apoptotic efficacy of multifaceted biosynthesized silver nanoparticles on human adenocarcinoma cells. Sci Rep 2018; 8:14368. [PMID: 30254325 PMCID: PMC6156419 DOI: 10.1038/s41598-018-32480-5] [Citation(s) in RCA: 66] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2018] [Accepted: 09/04/2018] [Indexed: 12/31/2022] Open
Abstract
Metallic nanoparticles (NPs) especially silver (Ag) NPs have shown immense potential in medical applications due to their distinctive physio-chemical and biological properties. This article reports the conjugation of Ag NPs with Rubus fairholmianus extract. The modification of Ag NPs was confirmed using various physico-chemical characterization techniques. The cytotoxic effect of Rubus-conjugated Ag NPs (RAgNPs) was studied by LDH assay and proliferation by ATP assay. The apoptotic inducing ability of the NPs were investigated by Annexin V/PI staining, caspase 3/7 analysis, cytochrome c release, intracellular ROS analysis, Hoechst staining and mitochondrial membrane potential analysis using flow cytometry. The expression of apoptotic proteins caspase 3, Bax and P53 were analyzed using ELISA and caspase 3, Bax using western blotting. Cells treated with 10 µg/mL RAgNPs showed an increased number of cell death by microscopic analysis compared to untreated control cells. The RAgNPs induced a statistically significant dose-dependent decrease in proliferation (p < 0.001 for 5 and 10 µg/mL) and increased cytotoxicity in MCF-7 cells. A 1.83 fold increase in cytotoxicity was observed in cells treated with 10 µg/mL (p < 0.05) compared to the untreated cells. Nuclear damage and intracellular ROS production were observed upon treatment with all tested concentrations of RAgNPs and the highest concentrations (5 and 10 µg/mL) showed significant (p < 0.05, p < 0.01) expression of caspase 3, Bax and P53 proteins. The data strongly suggest that RAgNPs induces cell death in MCF-7 cells through the mitochondrial-mediated intrinsic apoptosis pathway. The present investigation supports the potential of RAgNPs in anticancer drug development.
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Affiliation(s)
| | - Neeraj Kumar
- Department of Applied Chemistry, University of Johannesburg, Doornfontein, 2028, South Africa.,DST/CSIR National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, Pretoria, 0001, South Africa
| | - Heidi Abrahamse
- Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, Doornfontein, 2028, South Africa
| | - Suprakas Sinha Ray
- Department of Applied Chemistry, University of Johannesburg, Doornfontein, 2028, South Africa.,DST/CSIR National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, Pretoria, 0001, South Africa
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Akimoto M, Maruyama R, Kawabata Y, Tajima Y, Takenaga K. Antidiabetic adiponectin receptor agonist AdipoRon suppresses tumour growth of pancreatic cancer by inducing RIPK1/ERK-dependent necroptosis. Cell Death Dis 2018; 9:804. [PMID: 30038429 PMCID: PMC6056513 DOI: 10.1038/s41419-018-0851-z] [Citation(s) in RCA: 68] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2018] [Revised: 05/21/2018] [Accepted: 07/04/2018] [Indexed: 12/20/2022]
Abstract
The association between lower circulating adiponectin (APN) levels and the development of pancreatic cancer has been reported. However, the effect of APN on the growth and survival of pancreatic cancer cells remains elusive. Here, we investigate the effects of the anti-diabetic APN receptor (AdipoR) agonist AdipoRon and APN on human pancreatic cancer cells. We found that AdipoRon, but not APN, induces MIAPaCa-2 cell death, mainly through necroptosis. Mechanistically, although both AdipoRon and APN activate AMPK and p38 MAPK in an AdipoR-dependent manner that elicits survival signals, only AdipoRon induces rapid mitochondrial dysfunction through mitochondrial Ca2+ overload, followed by superoxide production via RIPK1 and ERK1/2 activation. Oral administration of AdipoRon suppresses MIAPaCa-2 tumour growth without severe adverse effects and kills cancer cells isolated from patients with pancreatic cancer. Thus, AdipoRon could be a therapeutic agent against pancreatic cancer as well as diabetes.
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Affiliation(s)
- Miho Akimoto
- Department of Life Science, Shimane University Faculty of Medicine, 89-1 Ennya, Izumo, Shimane, 693-8501, Japan.,Department of Biochemistry, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan
| | - Riruke Maruyama
- Department of Pathology, Shimane University Faculty of Medicine, 89-1 Ennya, Izumo, Shimane, 693-8501, Japan
| | - Yasunari Kawabata
- Department of Digestive and General Surgery, Shimane University Faculty of Medicine, 89-1 Ennya, Izumo, Shimane, 693-8501, Japan
| | - Yoshitsugu Tajima
- Department of Digestive and General Surgery, Shimane University Faculty of Medicine, 89-1 Ennya, Izumo, Shimane, 693-8501, Japan
| | - Keizo Takenaga
- Department of Life Science, Shimane University Faculty of Medicine, 89-1 Ennya, Izumo, Shimane, 693-8501, Japan. .,Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute, 666-2 Nitona, Chiba, 260-8717, Japan.
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Protection of Luteolin-7-O-glucoside against apoptosis induced by hypoxia/reoxygenation through the MAPK pathways in H9c2 cells. Mol Med Rep 2018; 17:7156-7162. [PMID: 29568918 PMCID: PMC5928668 DOI: 10.3892/mmr.2018.8774] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Accepted: 09/27/2017] [Indexed: 01/09/2023] Open
Abstract
Myocardial hypertrophy is often associated with myocardial infarction. Luteolin-7-O-glucoside (LUTG) has the prosperity of preventing cardiomyocyte injury. The current study aimed to explore the potential protective effect of LUTG and its relevant mechanisms in the heart. To establish the cardiac hypertrophy model in vitro, Angiotensin II (Ang II) was used to stimuli H9c2 cells in this study. The CCK-8 assay showed that LUTG pretreatment improved cell viability of cardiomyocytes co-treated with Ang II and ischemia/reperfusion. LUTG decreased the reactive oxygen species levels. Furthermore, it was demonstrated LUTG could reduce the release amount of lactate dehydrogenase and recover the catalase activity according to the flow cytometry analysis, and activity detection, respectively in Ang II-H/R-treated H9c2 cells. In addition, the flow cytometry analysis showed that the pretreatment of LUTG mitigated cell apoptosis induced by hypoxia/reoxygenation in the cardiac hypertrophy model. Meanwhile, reverse transcription-quantitative polymerase chain reaction and western blot assays showed that the apoptosis-related genes, including poly (ADP-ribose) polymerase, Fas, Fasl and Caspase-3 were downregulated at the transcriptional and translational levels. Notably, the protien expression of phosphorylated (p)-extracellular signal-regulated kinas (ERK) 1/2, p-janus kinase and p-P38 were reduced, while the expression of p-ERK5 was elevated in the LUTG pretreatment groups compared with the hypoxia/reoxygenation treatment group. Based on these results, it was suggested that the anti-apoptosis effect of LUTG may be associated with regulating the activation of mitogen-activated protein kinases signaling pathways.
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Pahima H, Reina S, Tadmor N, Dadon-Klein D, Shteinfer-Kuzmine A, Mazure NM, De Pinto V, Shoshan-Barmatz V. Hypoxic-induced truncation of voltage-dependent anion channel 1 is mediated by both asparagine endopeptidase and calpain 1 activities. Oncotarget 2018; 9:12825-12841. [PMID: 29560113 PMCID: PMC5849177 DOI: 10.18632/oncotarget.24377] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2017] [Accepted: 01/25/2018] [Indexed: 01/04/2023] Open
Abstract
The voltage-dependent anion channel 1 (VDAC1), an outer mitochondria membrane (OMM)
protein, serves as a mitochondrial gatekeeper, mediating the transport of
nucleotides, Ca2+ and other metabolites across the OMM. VDAC1 also
plays a central role in mitochondria-mediated apoptosis by facilitating the release
of apoptotic proteins and by association with both pro- and anti-apoptotic proteins.
Tumor cells, which are constantly exposed to hypoxic conditions, affect the cell via
the transcription factor hypoxia-inducible factor (HIF) that induces transcriptional
activity. In cultured cells and in lung cancer patients, hypoxia induces VDAC1
truncation at the C-terminus (VDAC1-ΔC). However, the molecular mechanisms
involved in VDAC1-ΔC formation are unknown. Here, we show that hypoxia-induced
VDAC1-ΔC formation is inhibited by the Ca2+ chelator
BAPTA-AM, by calpain inhibitor-1, by inhibitor of the asparagine endopeptidase (AEP)
and by si-RNA targeting HIF1-α or Ca2+-activated protease
calpain-1 expression but not that of calpain-2. Finally, VDAC1-ΔC expressed in
bacteria and reconstituted into a planar lipid bilayer exhibited decreased channel
conductance relative to the full-length protein, yet retained voltage-dependent
conductance. These findings suggest that hypoxia, acting via HIF-1α
expression, leads to VDAC1 cleavage involving the activation of calpain 1 and
AEP.
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Affiliation(s)
- Hadas Pahima
- Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
| | - Simona Reina
- Department of Biomedicine and Biotechnology, University of Catania and National Institute for Biomembranes and Biosystems, Section of Catania, Catania 95125, Italy.,Department of Biological, Geological and Environmental Sciences, University of Catania, Catania 95125, Italy
| | - Noa Tadmor
- Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
| | - Daniella Dadon-Klein
- Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
| | - Anna Shteinfer-Kuzmine
- Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
| | - Nathalie M Mazure
- Institute for Research on Cancer and Aging of Nice, University of Nice Sophia-Antipolis, Centre Antoine Lacassagne, Nice 06189, France.,Present address: INSERM U1065, C3M, Nice 06204, France
| | - Vito De Pinto
- Department of Biomedicine and Biotechnology, University of Catania and National Institute for Biomembranes and Biosystems, Section of Catania, Catania 95125, Italy
| | - Varda Shoshan-Barmatz
- Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
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Han X, Liu C, Zhang K, Guo M, Shen Z, Liu Y, Zuo Z, Cao M, Li Y. Calpain and JNK pathways participate in isoflurane - induced nucleus translocation of apoptosis-inducing factor in the brain of neonatal rats. Toxicol Lett 2017; 285:60-73. [PMID: 29289695 DOI: 10.1016/j.toxlet.2017.12.022] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2017] [Revised: 12/11/2017] [Accepted: 12/27/2017] [Indexed: 01/26/2023]
Abstract
Recent studies have demonstrated that volatile anesthetic causes caspase-dependent neuroapoptosis and persistent cognitive deficits in young animals. Apoptosis-inducing factor (AIF) can trigger apoptosis by caspase-independent pathway. Whether isoflurane induces neuroapoptosis by activation of AIF and its possible mechanism are underdetermined. Rats at postnatal day 7 were exposed to 1.1% isoflurane for 4 h and the expression of AIF, cytochrome c, caspase-3, μ-calpain, m-calpain, Bcl-2 and Bax in the mitochondrial, cytosolic, and nuclear fraction, as well as the number of both AIF and TUNEL positive neurons in the cortices of rats were measured. Moreover, the effects of calpain inhibitor MDL-28170 or JNK inhibitor SP600125 on isoflurane-induced AIF release, caspase activation and cognitive deficits were assessed. We found isoflurane activated CytC-caspase-3 dependent apoptosis pathway mainly in the early phase (0-6 h after exposure). Moreover, isoflurane activated mitochondrial μ-calpain, induced AIF truncation during early phase and activated m-calpain, induced AIF release from the mitochondria to cytosol and translocation into the nucleus in the late phase (6-24 h after exposure). MDL-28170 attenuated the isoflurane-induced mitochondrial AIF truncation, release and nuclear translocation, but did not change the expression of cleaved-caspase-3 and mitochondrial Bax and Bcl-2 proteins. SP600125 attenuated isoflurane-induced neuroapoptosis by inhibiting both AIF and caspase-3 pathways and reduced cognitive impairment in neonatal rats. This is the first study to provide the evidence that isoflurane induced AIF-dependent neuroapoptosis by activation of mitochondrial μ-calpain and m-calpain in neonatal rats. JNK inhibition reversed isoflurane-induced neuroapoptosis and subsequent long-term neurocognitive impairment, acting via inhibiting activation of both AIF and caspase-3 pathways.
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Affiliation(s)
- Xue Han
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China; Laboratory of RNA and Major Diseases of Brain and Heart, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China
| | - Chuiliang Liu
- Department of Anesthesiology, ChanCheng Center Hospital, Guangdong Medical College, Foshan, 528030, PR China
| | - Kun Zhang
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China; Laboratory of RNA and Major Diseases of Brain and Heart, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China
| | - Mingyan Guo
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China
| | - Zhiwen Shen
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China
| | - Yafang Liu
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China
| | - Zhiyi Zuo
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China; Laboratory of RNA and Major Diseases of Brain and Heart, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China; Department of Anesthesiology, University of Virginia Health System, Charlottesville, VA, 22908-0710, USA
| | - Minghui Cao
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China; Laboratory of RNA and Major Diseases of Brain and Heart, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China.
| | - Yujuan Li
- Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China; Laboratory of RNA and Major Diseases of Brain and Heart, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, PR China; Guangdong Province Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, China.
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Dutt V, Kaur N, Gupta P, Gujar R, Grewal A, Gupta S, Dua A, Mittal A. Efficient and modified protocol for zymography to detect muscle specific calpain activity. BIOCATALYSIS AND AGRICULTURAL BIOTECHNOLOGY 2017. [DOI: 10.1016/j.bcab.2017.02.011] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Hurst S, Hoek J, Sheu SS. Mitochondrial Ca 2+ and regulation of the permeability transition pore. J Bioenerg Biomembr 2017; 49:27-47. [PMID: 27497945 PMCID: PMC5393273 DOI: 10.1007/s10863-016-9672-x] [Citation(s) in RCA: 160] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2016] [Accepted: 07/31/2016] [Indexed: 02/06/2023]
Abstract
The mitochondrial permeability transition pore was originally described in the 1970's as a Ca2+ activated pore and has since been attributed to the pathogenesis of many diseases. Here we evaluate how each of the current models of the pore complex fit to what is known about how Ca2+ regulates the pore, and any insight that provides into the molecular identity of the pore complex. We also discuss the central role of Ca2+ in modulating the pore's open probability by directly regulating processes, such as ATP/ADP balance through the tricarboxylic acid cycle, electron transport chain, and mitochondrial membrane potential. We review how Ca2+ influences second messengers such as reactive oxygen/nitrogen species production and polyphosphate formation. We discuss the evidence for how Ca2+ regulates post-translational modification of cyclophilin D including phosphorylation by glycogen synthase kinase 3 beta, deacetylation by sirtuins, and oxidation/ nitrosylation of key residues. Lastly we introduce a novel view into how Ca2+ activated proteolysis through calpains in the mitochondria may be a driver of sustained pore opening during pathologies such as ischemia reperfusion injury.
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Affiliation(s)
- Stephen Hurst
- Center for Translational Medicine, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, 1020 Locust Street, Suite 543D, Philadelphia, PA, 19107, USA
| | - Jan Hoek
- Mitocare Center for Mitochondria Research, Department of Pathology Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, 19107, USA
| | - Shey-Shing Sheu
- Center for Translational Medicine, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, 1020 Locust Street, Suite 543D, Philadelphia, PA, 19107, USA.
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Ozaki T, Yamashita T, Tomita H, Sugano E, Ishiguro SI. The protection of rat retinal ganglion cells from ischemia/reperfusion injury by the inhibitory peptide of mitochondrial μ-calpain. Biochem Biophys Res Commun 2016; 478:1700-5. [PMID: 27596965 DOI: 10.1016/j.bbrc.2016.09.006] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2016] [Accepted: 09/01/2016] [Indexed: 11/16/2022]
Abstract
Intracellular Ca(2+)-dependent cysteine proteases such as calpains have been suggested as critical factors in retinal ganglion cell (RGC) death. However, it is unknown whether mitochondrial calpains are involved in RGC death. The purpose of the present study was to determine whether the inhibition of mitochondrial μ-calpain activity protects against RGC death during ischemia/reperfusion (I/R) injury. This study used a well-established rat model of experimental acute glaucoma involving I/R injury. A specific peptide inhibitor of mitochondrial μ-calpain, Tat-μCL, was topically applied to rats via eye drops three times a day for 5 days after I/R. RGC death was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The truncation of apoptosis-inducing factor (AIF) was determined by western blot analyses. Retinal morphology was determined after staining with hematoxyline and eosin. In addition, the number of Fluoro Gold-labeled RGCs in flat-mounted retinas was used to determine the percentage of surviving RGCs after I/R injury. After 1 day of I/R, RGC death was observed in the ganglion cell layer. Treatment with Tat-μCL eye drops significantly prevented the death of RGCs and the truncation of AIF. After 5 days of I/R, RGC death decreased by approximately 40%. However, Tat-μCL significantly inhibited the decrease in the retinal sections and flat-mounted retinas. The results suggested that mitochondrial μ-calpain is associated with RGC death during I/R injury via truncation of AIF. In addition, the inhibition of mitochondrial μ-calpain activity by Tat-μCL had a neuroprotective effect against I/R-induced RGC death.
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Affiliation(s)
- Taku Ozaki
- Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, Morioka, Japan; Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan.
| | - Tetsuro Yamashita
- Department of Biological Chemistry, Faculty of Agriculture, Iwate University, Morioka, Japan
| | - Hiroshi Tomita
- Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, Morioka, Japan
| | - Eriko Sugano
- Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, Morioka, Japan
| | - Sei-Ichi Ishiguro
- Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan; Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan
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40
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Györffy BA, Gulyássy P, Gellén B, Völgyi K, Madarasi D, Kis V, Ozohanics O, Papp I, Kovács P, Lubec G, Dobolyi Á, Kardos J, Drahos L, Juhász G, Kékesi KA. Widespread alterations in the synaptic proteome of the adolescent cerebral cortex following prenatal immune activation in rats. Brain Behav Immun 2016; 56:289-309. [PMID: 27058163 DOI: 10.1016/j.bbi.2016.04.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2016] [Revised: 03/23/2016] [Accepted: 04/04/2016] [Indexed: 01/08/2023] Open
Abstract
An increasing number of studies have revealed associations between pre- and perinatal immune activation and the development of schizophrenia and autism spectrum disorders (ASDs). Accordingly, neuroimmune crosstalk has a considerably large impact on brain development during early ontogenesis. While a plethora of heterogeneous abnormalities have already been described in established maternal immune activation (MIA) rodent and primate animal models, which highly correlate to those found in human diseases, the underlying molecular background remains obscure. In the current study, we describe the long-term effects of MIA on the neocortical pre- and postsynaptic proteome of adolescent rat offspring in detail. Molecular differences were revealed in sub-synaptic fractions, which were first thoroughly characterized using independent methods. The widespread proteomic examination of cortical samples from offspring exposed to maternal lipopolysaccharide administration at embryonic day 13.5 was conducted via combinations of different gel-based proteomic techniques and tandem mass spectrometry. Our experimentally validated proteomic data revealed more pre- than postsynaptic protein level changes in the offspring. The results propose the relevance of altered synaptic vesicle recycling, cytoskeletal structure and energy metabolism in the presynaptic region in addition to alterations in vesicle trafficking, the cytoskeleton and signal transduction in the postsynaptic compartment in MIA offspring. Differing levels of the prominent signaling regulator molecule calcium/calmodulin-dependent protein kinase II in the postsynapse was validated and identified specifically in the prefrontal cortex. Finally, several potential common molecular regulators of these altered proteins, which are already known to be implicated in schizophrenia and ASD, were identified and assessed. In summary, unexpectedly widespread changes in the synaptic molecular machinery in MIA rats were demonstrated which might underlie the pathological cortical functions that are characteristic of schizophrenia and ASD.
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Affiliation(s)
- Balázs A Györffy
- Laboratory of Proteomics, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary; MTA-ELTE NAP B Neuroimmunology Research Group, Department of Biochemistry, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary
| | - Péter Gulyássy
- MTA-TTK NAP B MS Neuroproteomics Group, Hungarian Academy of Sciences, Budapest H-1117, Hungary; Department of Pediatrics, Medical University of Vienna, Vienna A-1090, Austria
| | - Barbara Gellén
- Laboratory of Proteomics, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary; MTA-ELTE NAP B Laboratory of Molecular and Systems Neurobiology, Institute of Biology, Hungarian Academy of Sciences and Eötvös Loránd University, Budapest H-1117, Hungary
| | - Katalin Völgyi
- Laboratory of Proteomics, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary; MTA-ELTE NAP B Laboratory of Molecular and Systems Neurobiology, Institute of Biology, Hungarian Academy of Sciences and Eötvös Loránd University, Budapest H-1117, Hungary
| | - Dóra Madarasi
- Laboratory of Proteomics, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary
| | - Viktor Kis
- Department of Anatomy, Cell and Developmental Biology, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary
| | - Olivér Ozohanics
- MTA-TTK NAP B MS Neuroproteomics Group, Hungarian Academy of Sciences, Budapest H-1117, Hungary
| | | | | | - Gert Lubec
- Department of Pediatrics, Medical University of Vienna, Vienna A-1090, Austria
| | - Árpád Dobolyi
- MTA-ELTE NAP B Laboratory of Molecular and Systems Neurobiology, Institute of Biology, Hungarian Academy of Sciences and Eötvös Loránd University, Budapest H-1117, Hungary
| | - József Kardos
- MTA-ELTE NAP B Neuroimmunology Research Group, Department of Biochemistry, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary
| | - László Drahos
- MTA-TTK NAP B MS Neuroproteomics Group, Hungarian Academy of Sciences, Budapest H-1117, Hungary
| | - Gábor Juhász
- Laboratory of Proteomics, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary; MTA-TTK NAP B MS Neuroproteomics Group, Hungarian Academy of Sciences, Budapest H-1117, Hungary
| | - Katalin A Kékesi
- Laboratory of Proteomics, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary; Department of Physiology and Neurobiology, Institute of Biology, Eötvös Loránd University, Budapest H-1117, Hungary.
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Thompson J, Hu Y, Lesnefsky EJ, Chen Q. Activation of mitochondrial calpain and increased cardiac injury: beyond AIF release. Am J Physiol Heart Circ Physiol 2016; 310:H376-84. [PMID: 26637561 PMCID: PMC4796621 DOI: 10.1152/ajpheart.00748.2015] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2015] [Accepted: 11/23/2015] [Indexed: 12/14/2022]
Abstract
Calpain 1 (CPN1) is a ubiquitous cysteine protease that exists in both cytosol and cardiac mitochondria. Mitochondrial CPN1 (mit-CPN1) is located in the intermembrane space and matrix. Activation of mit-CPN1 within the intermembrane space increases cardiac injury by releasing apoptosis-inducing factor from mitochondria during ischemia-reperfusion (IR). We asked if activation of mit-CPN1 is involved in mitochondrial injury during IR. MDL-28170 (MDL) was used to inhibit CPN1 in buffer-perfused hearts following 25-min ischemia and 30-min reperfusion. MDL treatment decreased the release of lactate dehydrogenase into coronary effluent compared with untreated hearts, indicating that inhibition of CPN1 decreases cardiac injury. MDL also prevented the cleavage of spectrin (a substrate of CPN1) in cytosol during IR, supporting that MDL treatment decreased cytosolic calpain activation. In addition, MDL markedly improved calcium retention capacity compared with untreated heart, suggesting that MDL treatment decreases mitochondrial permeability transition pore opening. In addition, we found that IR led to decreased complex I activity, whereas inhibition of mit-CPN1 using MDL protected complex I. Pyruvate dehydrogenase content was decreased following IR. However, pyruvate dehydrogenase content was preserved in MDL-treated mitochondria. Taken together, MDL treatment decreased cardiac injury during IR by inhibiting both cytosolic and mit-CPN1. Activation of mit-CPN1 increases cardiac injury during IR by sensitizing mitochondrial permeability transition pore opening and impairing mitochondrial metabolism through damage of complex I.
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Affiliation(s)
- Jeremy Thompson
- Division of Cardiology, Department of Medicine, Virginia Commonwealth University, Richmond, Virginia
| | - Ying Hu
- Division of Cardiology, Department of Medicine, Virginia Commonwealth University, Richmond, Virginia
| | - Edward J Lesnefsky
- Division of Cardiology, Department of Medicine, Virginia Commonwealth University, Richmond, Virginia; Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia; Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia; and McGuire Veterans Affairs Medical Center, Richmond, Virginia
| | - Qun Chen
- Division of Cardiology, Department of Medicine, Virginia Commonwealth University, Richmond, Virginia;
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Totzeck M, Hendgen-Cotta U, Rassaf T. Concepts of hypoxic NO signaling in remote ischemic preconditioning. World J Cardiol 2015; 7:645-651. [PMID: 26516418 PMCID: PMC4620075 DOI: 10.4330/wjc.v7.i10.645] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2015] [Revised: 08/17/2015] [Accepted: 09/08/2015] [Indexed: 02/06/2023] Open
Abstract
Acute coronary syndromes remain a leading single cause of death worldwide. Therapeutic strategies to treat cardiomyocyte threatening ischemia/reperfusion injury are urgently needed. Remote ischemic preconditioning (rIPC) applied by brief ischemic episodes to heart-distant organs has been tested in several clinical studies, and the major body of evidence points to beneficial effects of rIPC for patients. The underlying signaling, however, remains incompletely understood. This relates particularly to the mechanism by which the protective signal is transferred from the remote site to the target organ. Many pathways have been forwarded but none can explain the protective effects completely. In light of recent experimental studies, we here outline the current knowledge relating to the generation of the protective signal in the remote organ, the signal transfer to the target organ and the transduction of the transferred signal into cardioprotection. The majority of studies favors a humoral factor that activates cardiomyocyte downstream signaling - receptor-dependent and independently. Cellular targets include deleterious calcium (Ca2+) signaling, reactive oxygen species, mitochondrial function and structure, and cellular apoptosis and necrosis. Following an outline of the existing evidence, we will furthermore characterize the existing knowledge and discuss future perspectives with particular emphasis on the interaction between the recently discovered hypoxic nitrite-nitric oxide signaling in rIPC. This refers to the protective role of nitrite, which can be activated endogenously using rIPC and which then contributes to cardioprotection by rIPC.
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Chen Q, Lesnefsky EJ. Heart mitochondria and calpain 1: Location, function, and targets. Biochim Biophys Acta Mol Basis Dis 2015; 1852:2372-8. [PMID: 26259540 DOI: 10.1016/j.bbadis.2015.08.004] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2015] [Revised: 07/17/2015] [Accepted: 08/06/2015] [Indexed: 12/22/2022]
Abstract
Calpain 1 is an ubiquitous Ca(2+)-dependent cysteine protease. Although calpain 1 has been found in cardiac mitochondria, the exact location within mitochondrial compartments and its function remain unclear. The aim of the current review is to discuss the localization of calpain 1 in different mitochondrial compartments in relationship to its function, especially in pathophysiological conditions. Briefly, mitochondrial calpain 1 (mit-CPN1) is located within the intermembrane space and mitochondrial matrix. Activation of the mit-CPN1 within intermembrane space cleaves apoptosis inducing factor (AIF), whereas the activated mit-CPN1 within matrix cleaves complex I subunits and metabolic enzymes. Inhibition of the mit-CPN1 could be a potential strategy to decrease cardiac injury during ischemia-reperfusion.
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Affiliation(s)
- Qun Chen
- Department of Medicine (Division of Cardiology, Pauley Heart Center), Virginia Commonwealth University, Richmond, VA 23298, United States.
| | - Edward J Lesnefsky
- Department of Medicine (Division of Cardiology, Pauley Heart Center), Virginia Commonwealth University, Richmond, VA 23298, United States; Department of Biochemistry, Virginia Commonwealth University, Richmond, VA 23298, United States; Department of Physiology, Virginia Commonwealth University, Richmond, VA 23298, United States; McGuire VA Medical Center, Richmond, VA 23249, United States
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Shintani-Ishida K, Yoshida KI. Mitochondrial m-calpain opens the mitochondrial permeability transition pore in ischemia-reperfusion. Int J Cardiol 2015; 197:26-32. [PMID: 26113472 DOI: 10.1016/j.ijcard.2015.06.010] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Revised: 05/14/2015] [Accepted: 06/12/2015] [Indexed: 12/14/2022]
Abstract
BACKGROUND/OBJECTIVES Opening of the mitochondrial permeability transition pore (mPTP) is involved in ischemia-reperfusion injury. Isoforms of Ca(2+)-activated cysteine proteases, calpains, are implicated in the development of myocardial infarction in ischemia-reperfusion. Growing evidence has revealed the presence of calpains in the mitochondria. We aimed to characterize mitochondrial calpains in the rat heart and to investigate the roles of calpains in mPTP opening after ischemia-reperfusion. METHODS AND RESULTS Western blotting analysis showed the expression of μ-calpain, m-calpain and calpain 10 in mitochondria isolated from male Sprague-Dawley rats, but casein zymography detected only m-calpain activity. Subcellular fractionation of mitochondria demonstrated the distribution of m-calpain to the matrix fraction. Addition of >500μM of Ca(2+) to isolated mitochondria induced mitochondrial swelling, reflecting mPTP opening, and calpain activation. Ca(2+)-induced mitochondrial swelling was inhibited partially by the calpain inhibitor calpeptin. These results support a partial contribution of calpain in the opening of the mPTP. The addition of Ca(2+) to the mitochondria induced inactivation of complex I of the electron transport chain, and cleavage of the ND6 complex I subunit, which were inhibited by calpeptin. Mitochondria isolated from rat hearts that underwent 30min of coronary occlusion followed by 30min of reperfusion showed activation of mitochondrial calpains, ND6 cleavage, complex I inactivation, and mPTP opening, which were inhibited by pretreatment with calpain inhibitor 1. CONCLUSIONS We demonstrated for the first time the presence of mitochondrial matrix m-calpain, and its contribution to complex I inactivation and mPTP opening after postischemic reperfusion in the rat heart.
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Affiliation(s)
- Kaori Shintani-Ishida
- Department of Forensic Medicine, Graduate School of Medicine, the University of Tokyo, Japan.
| | - Ken-Ichi Yoshida
- Department of Forensic Medicine, Graduate School of Medicine, the University of Tokyo, Japan
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Ziegler C, Finke J, Grüllich C. Features of cell death, mitochondrial activation and caspase dependence of rabbit anti-T-lymphocyte globulin signaling in lymphoblastic Jurkat cells are distinct from classical apoptosis signaling of CD95. Leuk Lymphoma 2015; 57:177-82. [PMID: 25927246 DOI: 10.3109/10428194.2015.1044449] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Rabbit anti-T-lymphocyte-globulin (ATG) is used for immunosuppression in organ and stem cell transplantation. The aim of this study was to investigate ATG-induced cell death compared to CD95-signaling of apoptosis. We measured features of cell death at the cell membrane, mitochondria, nuclei and caspase-3 cleavage. We used the following inhibitors: the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (O-Me)-fluoromethyl ketone (zVAD-fmk), the serine protease inhibitors 3,4 dichloroisocoumarin (DCI) and N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and the reducing agent N-acetycysteine (NAC). ATG-induced cellular changes were rapid, included mitochondrial membrane permeability (MMP) induction and annexin V/propidium iodide (PI) positivity but little caspase-3 activation and nuclear morphology changes. MMP was not sensitive to caspase inhibition, serine protease inhibition with DCI moderately reduced MMP. These findings were in contrast to CD95-signaling. Interestingly, TLCK massively augmented CD95-induced MMP which could be abrogated by NAC. In conclusion, ATG-signaling differs in features and kinetics from CD95-induced apoptosis with caspase-independent mechanisms involved in MMP.
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Affiliation(s)
- Christian Ziegler
- a Department of Medical Oncology , National Center for Tumor Diseases, Heidelberg University Hospital , Heidelberg , Germany.,b Department of Hematology and Oncology , Albert Ludwigs-University Medical Center Freiburg , Freiburg , Germany.,c Department of Hematology and Oncology , Charité Medical Center Berlin , Campus Virchow Klinik, Berlin, Germany
| | - Jürgen Finke
- b Department of Hematology and Oncology , Albert Ludwigs-University Medical Center Freiburg , Freiburg , Germany
| | - Carsten Grüllich
- a Department of Medical Oncology , National Center for Tumor Diseases, Heidelberg University Hospital , Heidelberg , Germany.,b Department of Hematology and Oncology , Albert Ludwigs-University Medical Center Freiburg , Freiburg , Germany
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46
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Ding ZJ, Chen X, Tang XX, Wang X, Song YL, Chen XD, Wang J, Wang RF, Mi WJ, Chen FQ, Qiu JH. Apoptosis-inducing factor and calpain upregulation in glutamate-induced injury of rat spiral ganglion neurons. Mol Med Rep 2015; 12:1685-92. [PMID: 25891494 PMCID: PMC4464299 DOI: 10.3892/mmr.2015.3626] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2014] [Accepted: 02/27/2015] [Indexed: 01/08/2023] Open
Abstract
Spiral ganglion neuron (SGN) damage and apoptosis can lead to noise-induced hearing loss, age-associated hearing loss and, in certain cases, auditory neuropathy. The apoptosis-inducing factor (AIF)-associated pathway may be important in this process. The present study aimed to investigate the expression levels of AIF and calpain in damaged SGNs. Glutamate (Glu) perfusion and cell culture in different concentrations of Glu were performed to damage the SGNs of Sprague-Dawley (SD) rats, with saline water used as a control Different concentrations (5, 10, 20 and 40 mM) of Glu were injected into the cochlear tympanic canal of 18 SD rats, and 10, 20 and 40 mM Glu were added to SGN cultures. Auditory brainstem responses (ABR) were measured prior to and 2 days following the injection of Glu. Immunofluorescent staining was used to detect the SGN damage and the expression levels of AIF and calpain in vivo and in in vitro. Transmission electron microscopy (TEM) was used to measure cell apoptosis and reverse transcription-quantitative polymerase chain reaction was used to analyse the gene expression levels of AIF and calpain in the damaged SGNs. The TEM identified mitochondrial vacuolisation, swelling of the SGN and hetero-chromatin formation. Injection of Glu reduced the number of SGNs and induced apoptosis. AIF was observed to translocate into the nuclei of the SGNs in the 20 and 40 mM Glu groups, and the expression levels of AIF and calpain were markedly upregulated in the modiolus of the Glu-damaged SGNs. The upregulation of AIF and calpain may be important in the process of SGN damage and apoptosis.
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Affiliation(s)
- Zhong-Jia Ding
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Xin Chen
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Xiao-Xu Tang
- Outpatient Department, Logistics Academy, Beijing 100858, P.R. China
| | - Xi Wang
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Yong-Li Song
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Xiao-Dong Chen
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Jian Wang
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Ren-Feng Wang
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Wen-Juan Mi
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Fu-Quan Chen
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Jian-Hua Qiu
- Department of Otolaryngology‑Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
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Moreira AC, Branco AF, Sampaio SF, Cunha-Oliveira T, Martins TR, Holy J, Oliveira PJ, Sardão VA. Mitochondrial apoptosis-inducing factor is involved in doxorubicin-induced toxicity on H9c2 cardiomyoblasts. Biochim Biophys Acta Mol Basis Dis 2014; 1842:2468-78. [DOI: 10.1016/j.bbadis.2014.09.015] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2013] [Revised: 09/19/2014] [Accepted: 09/26/2014] [Indexed: 01/22/2023]
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Neuhof C, Neuhof H. Calpain system and its involvement in myocardial ischemia and reperfusion injury. World J Cardiol 2014; 6:638-652. [PMID: 25068024 PMCID: PMC4110612 DOI: 10.4330/wjc.v6.i7.638] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2013] [Revised: 01/26/2014] [Accepted: 05/29/2014] [Indexed: 02/06/2023] Open
Abstract
Calpains are ubiquitous non-lysosomal Ca2+-dependent cysteine proteases also present in myocardial cytosol and mitochondria. Numerous experimental studies reveal an essential role of the calpain system in myocardial injury during ischemia, reperfusion and postischemic structural remodelling. The increasing Ca2+-content and Ca2+-overload in myocardial cytosol and mitochondria during ischemia and reperfusion causes an activation of calpains. Upon activation they are able to injure the contractile apparatus and impair the energy production by cleaving structural and functional proteins of myocytes and mitochondria. Besides their causal involvement in acute myocardial dysfunction they are also involved in structural remodelling after myocardial infarction by the generation and release of proapoptotic factors from mitochondria. Calpain inhibition can prevent or attenuate myocardial injury during ischemia, reperfusion, and in later stages of myocardial infarction.
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Singh R, Brewer MK, Mashburn CB, Lou D, Bondada V, Graham B, Geddes JW. Calpain 5 is highly expressed in the central nervous system (CNS), carries dual nuclear localization signals, and is associated with nuclear promyelocytic leukemia protein bodies. J Biol Chem 2014; 289:19383-94. [PMID: 24838245 PMCID: PMC4094050 DOI: 10.1074/jbc.m114.575159] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2014] [Revised: 05/14/2014] [Indexed: 12/20/2022] Open
Abstract
Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF hand motif characteristic of classical calpains but retains catalytic and Ca(2+) binding domains, and it contains a unique C-terminal domain. TRA-3, an ortholog of CAPN5, has been shown to be involved in necrotic cell death in Caenorhabditis elegans. CAPN5 is expressed throughout the CNS, but its expression relative to other calpains and subcellular distribution has not been investigated previously. Based on relative mRNA levels, Capn5 is the second most highly expressed calpain in the rat CNS, with Capn2 mRNA being the most abundant. Unlike classical calpains, CAPN5 is a non-cytosolic protein localized to the nucleus and extra-nuclear locations. CAPN5 possesses two nuclear localization signals (NLS): an N-terminal monopartite NLS and a unique bipartite NLS closer to the C terminus. The C-terminal NLS contains a SUMO-interacting motif that contributes to nuclear localization, and mutation or deletion of both NLS renders CAPN5 exclusively cytosolic. Dual NLS motifs are common among transcription factors. Interestingly, CAPN5 is found in punctate domains associated with promyelocytic leukemia (PML) protein within the nucleus. PML nuclear bodies are implicated in transcriptional regulation, cell differentiation, cellular response to stress, viral defense, apoptosis, and cell senescence as well as protein sequestration, modification, and degradation. The roles of nuclear CAPN5 remain to be determined.
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Affiliation(s)
- Ranjana Singh
- From the Spinal Cord and Brain Injury Research Center and the Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky 40536
| | | | | | - Dingyuan Lou
- From the Spinal Cord and Brain Injury Research Center and
| | - Vimala Bondada
- From the Spinal Cord and Brain Injury Research Center and
| | | | - James W Geddes
- From the Spinal Cord and Brain Injury Research Center and the Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky 40536
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Inhibition of calpain reduces oxidative stress and attenuates endothelial dysfunction in diabetes. Cardiovasc Diabetol 2014; 13:88. [PMID: 24886224 PMCID: PMC4045988 DOI: 10.1186/1475-2840-13-88] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2014] [Accepted: 04/25/2014] [Indexed: 12/25/2022] Open
Abstract
Aims The present study was to investigate the role of calpain in reactive oxygen species (ROS) production in endothelial cells and endothelium-dependent vascular dysfunction under experimental conditions of diabetes. Methods and results Exposure to high glucose activated calpain, induced apoptosis and reduced nitric oxide (NO) production without changing eNOS protein expression, its phosphorylation and dimers formation in primary human umbilical vein endothelial cells (HUVECs). These effects of high glucose correlated with intracellular ROS production and mitochondrial superoxide generation. Selectively scavenging mitochondrial superoxide increased NO production in high glucose-stimulated HUVECs. Inhibition of calpain using over-expression of calpastatin or pharmacological calpain inhibitor prevented high glucose-induced ROS production, mitochondrial superoxide generation and apoptosis, which were concurrent with an elevation of NO production in HUVECs. In mouse models of streptozotocin-induced type-1 diabetes and OVE26 type-1 diabetic mice, calpain activation correlated with an increase in ROS production and peroxynitrite formation in aortas. Transgenic over-expression of calpastatin reduced ROS production and peroxynitrite formation in diabetic mice. In parallel, diabetes-induced reduction of endothelium-dependent relaxation in aortic ring was reversed by over-expression of calpastatin in mouse models of diabetes. However, the protective effect of calpastatin on endothelium-dependent relaxation was abrogated by eNOS deletion in diabetic mice. Conclusions This study suggests that calpain may play a role in vascular endothelial cell ROS production and endothelium-dependent dysfunction in diabetes. Thus, calpain may be an important therapeutic target to overcome diabetes-induced vascular dysfunction.
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