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1
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Roles of the pro-apoptotic factors CaNma111 and CaYbh3 in apoptosis and virulence of Candida albicans. Sci Rep 2022; 12:7574. [PMID: 35534671 PMCID: PMC9085738 DOI: 10.1038/s41598-022-11682-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Accepted: 04/26/2022] [Indexed: 12/25/2022] Open
Abstract
Candida albicans, a commensal and opportunistic pathogen, undergoes apoptosis in response to various stimuli, including hydrogen peroxide, acetic acid, and antifungal agents. Apoptotic processes are highly conserved among mammals, plants, and fungi, but little is known about the apoptosis-regulating factors in C. albicans. In this study, C. albicans homologs of the putative apoptosis factors were identified by database screening followed by overexpression analysis. CaNma111, a homolog of the pro-apoptotic mammalian HtrA2/Omi, and CaYbh3, a homolog of BH3-only protein, yielded increased apoptotic phenotypes upon overexpression. We showed that CaNma111 and CaYbh3 functions as pro-apoptotic regulators by examining intracellular ROS accumulation, DNA end breaks (TUNEL assay), and cell survival in Canma111/Canma111 and Caybh3/Caybh3 deletion strains. We found that the protein level of CaBir1, an inhibitor-of-apoptosis (IAP) protein, was down-regulated by CaNma111. Interestingly, the Canma111/Canma111 and Caybh3/Caybh3 deletion strains showed hyperfilamentation phenotypes and increased virulence in a mouse infection model. Together, our results suggest that CaNma111 and CaYbh3 play key regulatory roles in the apoptosis and virulence of C. albicans.
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2
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Witkop EM, Proestou DA, Gomez-Chiarri M. The expanded inhibitor of apoptosis gene family in oysters possesses novel domain architectures and may play diverse roles in apoptosis following immune challenge. BMC Genomics 2022; 23:201. [PMID: 35279090 PMCID: PMC8917759 DOI: 10.1186/s12864-021-08233-6] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Accepted: 12/06/2021] [Indexed: 11/23/2022] Open
Abstract
Background Apoptosis plays important roles in a variety of functions, including immunity and response to environmental stress. The Inhibitor of Apoptosis (IAP) gene family of apoptosis regulators is expanded in molluscs, including eastern, Crassostrea virginica, and Pacific, Crassostrea gigas, oysters. The functional importance of IAP expansion in apoptosis and immunity in oysters remains unknown. Results Phylogenetic analysis of IAP genes in 10 molluscs identified lineage specific gene expansion in bivalve species. Greater IAP gene family expansion was observed in C. virginica than C. gigas (69 vs. 40), resulting mainly from tandem duplications. Functional domain analysis of oyster IAP proteins revealed 3 novel Baculoviral IAP Repeat (BIR) domain types and 14 domain architecture types across gene clusters, 4 of which are not present in model organisms. Phylogenetic analysis of bivalve IAPs suggests a complex history of domain loss and gain. Most IAP genes in oysters (76% of C. virginica and 82% of C. gigas), representing all domain architecture types, were expressed in response to immune challenge (Ostreid Herpesvirus OsHV-1, bacterial probionts Phaeobacter inhibens and Bacillus pumilus, several Vibrio spp., pathogenic Aliiroseovarius crassostreae, and protozoan parasite Perkinsus marinus). Patterns of IAP and apoptosis-related differential gene expression differed between the two oyster species, where C. virginica, in general, differentially expressed a unique set of IAP genes in each challenge, while C. gigas differentially expressed an overlapping set of IAP genes across challenges. Apoptosis gene expression patterns clustered mainly by resistance/susceptibility of the oyster host to immune challenge. Weighted Gene Correlation Network Analysis (WGCNA) revealed unique combinations of transcripts for 1 to 12 IAP domain architecture types, including novel types, were significantly co-expressed in response to immune challenge with transcripts in apoptosis-related pathways. Conclusions Unprecedented diversity characterized by novel BIR domains and protein domain architectures was observed in oyster IAPs. Complex patterns of gene expression of novel and conserved IAPs in response to a variety of ecologically-relevant immune challenges, combined with evidence of direct co-expression of IAP genes with apoptosis-related transcripts, suggests IAP expansion facilitates complex and nuanced regulation of apoptosis and other immune responses in oysters. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-021-08233-6.
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3
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Buhlman LM, Krishna G, Jones TB, Thomas TC. Drosophila as a model to explore secondary injury cascades after traumatic brain injury. Biomed Pharmacother 2021; 142:112079. [PMID: 34463269 PMCID: PMC8458259 DOI: 10.1016/j.biopha.2021.112079] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 08/11/2021] [Accepted: 08/17/2021] [Indexed: 12/14/2022] Open
Abstract
Drosophilae are emerging as a valuable model to study traumatic brain injury (TBI)-induced secondary injury cascades that drive persisting neuroinflammation and neurodegenerative pathology that imposes significant risk for long-term neurological deficits. As in mammals, TBI in Drosophila triggers axonal injury, metabolic crisis, oxidative stress, and a robust innate immune response. Subsequent neurodegeneration stresses quality control systems and perpetuates an environment for neuroprotection, regeneration, and delayed cell death via highly conserved cell signaling pathways. Fly injury models continue to be developed and validated for both whole-body and head-specific injury to isolate, evaluate, and modulate these parallel pathways. In conjunction with powerful genetic tools, the ability for longitudinal evaluation, and associated neurological deficits that can be tested with established behavioral tasks, Drosophilae are an attractive model to explore secondary injury cascades and therapeutic intervention after TBI. Here, we review similarities and differences between mammalian and fly pathophysiology and highlight strategies for their use in translational neurotrauma research.
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Affiliation(s)
- Lori M Buhlman
- Biomedical Sciences Program, Midwestern University, Glendale, AZ, USA.
| | - Gokul Krishna
- Department of Child Health, University of Arizona College of Medicine - Phoenix, Phoenix, AZ, USA; Barrow Neurological Institute at Phoenix Children's Hospital, Phoenix, AZ, USA
| | - T Bucky Jones
- Department of Anatomy, Midwestern University, Glendale, AZ, USA
| | - Theresa Currier Thomas
- Department of Child Health, University of Arizona College of Medicine - Phoenix, Phoenix, AZ, USA; Barrow Neurological Institute at Phoenix Children's Hospital, Phoenix, AZ, USA; Phoenix VA Health Care System, Phoenix, AZ, USA.
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4
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Hounsell C, Fan Y. The Duality of Caspases in Cancer, as Told through the Fly. Int J Mol Sci 2021; 22:8927. [PMID: 34445633 PMCID: PMC8396359 DOI: 10.3390/ijms22168927] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Revised: 08/15/2021] [Accepted: 08/17/2021] [Indexed: 12/12/2022] Open
Abstract
Caspases, a family of cysteine-aspartic proteases, have an established role as critical components in the activation and initiation of apoptosis. Alongside this a variety of non-apoptotic caspase functions in proliferation, differentiation, cellular plasticity and cell migration have been reported. The activity level and context are important factors in determining caspase function. As a consequence of their critical role in apoptosis and beyond, caspases are uniquely situated to have pathological roles, including in cancer. Altered caspase function is a common trait in a variety of cancers, with apoptotic evasion defined as a "hallmark of cancer". However, the role that caspases play in cancer is much more complex, acting both to prevent and to promote tumourigenesis. This review focuses on the major findings in Drosophila on the dual role of caspases in tumourigenesis. This has major implications for cancer treatments, including chemotherapy and radiotherapy, with the activation of apoptosis being the end goal. However, such treatments may inadvertently have adverse effects on promoting tumour progression and acerbating the cancer. A comprehensive understanding of the dual role of caspases will aid in the development of successful cancer therapeutic approaches.
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Affiliation(s)
| | - Yun Fan
- School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK;
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5
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Qu C, Sun J, Xu Q, Lv X, Yang W, Wang F, Wang Y, Yi Q, Jia Z, Wang L, Song L. An inhibitor of apoptosis protein (EsIAP1) from Chinese mitten crab Eriocheir sinensis regulates apoptosis through inhibiting the activity of EsCaspase-3/7-1. Sci Rep 2019; 9:20421. [PMID: 31892728 PMCID: PMC6938513 DOI: 10.1038/s41598-019-56971-1] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2019] [Accepted: 12/19/2019] [Indexed: 01/13/2023] Open
Abstract
Inhibitor of apoptosis proteins (IAPs) maintain the balance between cell proliferation and cell death by inhibiting caspase activities and mediating immune responses. In the present study, a homolog of IAP (designated as EsIAP1) was identified from Chinese mitten crab Eriocheir sinensis. EsIAP1 consisted of 451 amino acids containing two baculoviral IAP repeat (BIR) domains with the conserved Cx2 Cx6 Wx3 Dx5 Hx6 C motifs. EsIAP1 mRNA was expressed in various tissues and its expression level in hemocytes increased significantly (p < 0.01) at 12–48 h after lipopolysaccharide stimulation. In the hemocytes, EsIAP1 protein was mainly distributed in the cytoplasm. The hydrolytic activity of recombinant EsCaspase-3/7-1 against the substrate Ac-DEVD-pNA decreased after incubation with rEsIAP1. Moreover, rEsIAP1 could directly combine with rEsCaspase-3/7-1 in vitro. After EsIAP1 was interfered by dsRNA, the mRNA expression and the hydrolytic activity of EsCaspase-3/7-1 increased significantly, which was 2.26-fold (p < 0.05) and 1.71-fold (p < 0.05) compared to that in the dsGFP group, respectively. These results collectively demonstrated that EsIAP1 might play an important role in apoptosis pathway by regulating the activity of EsCaspase-3/7-1 in E. sinensis.
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Affiliation(s)
- Chen Qu
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China
| | - Jiejie Sun
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China
| | - Qingsong Xu
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China.,Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China
| | - Xiaojing Lv
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China.,Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China.,Liaoning Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, Dalian, 116023, China.,Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China
| | - Wen Yang
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China
| | - Feifei Wang
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China
| | - Ying Wang
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China
| | - Qilin Yi
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China.,Liaoning Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, Dalian, 116023, China.,Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China
| | - Zhihao Jia
- Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China
| | - Lingling Wang
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China.,Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China.,Liaoning Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, Dalian, 116023, China.,Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China
| | - Linsheng Song
- Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China. .,Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China. .,Liaoning Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, Dalian, 116023, China. .,Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China.
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6
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Rosner A, Kravchenko O, Rinkevich B. IAP genes partake weighty roles in the astogeny and whole body regeneration in the colonial urochordate Botryllus schlosseri. Dev Biol 2018; 448:320-341. [PMID: 30385275 DOI: 10.1016/j.ydbio.2018.10.015] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2018] [Revised: 09/29/2018] [Accepted: 10/17/2018] [Indexed: 12/12/2022]
Abstract
Inhibitors of Apoptosis Protein (IAP) genes participate in processes like apoptosis, proliferation, innate immunity, inflammation, cell motility, differentiation and in malignancies. Here we reveal 25 IAP genes in the tunicate Botryllus schlosseri's genome and their functions in two developmental biology phenomena, a new mode of whole body regeneration (WBR) induced by budectomy, and blastogenesis, the four-staged cycles of botryllid ascidian astogeny. IAP genes that were specifically upregulated during these developmental phenomena were identified, and protein expression patterns of one of these genes, IAP28, were followed. Most of the IAP genes upregulation recorded at blastogenetic stages C/D was in concert with the upregulation at 100 μM H2O2 apoptotic-induced treatment and in parallel to expressions of AIF1, Bax, Mcl1, caspase 2 and two orthologues of caspase 7. Wnt agonist altered the takeover duration along with reduced IAP expressions, and displacement of IAP28+ phagocytes. WBR was initiated solely at blastogenetic stage D, where zooidal absorption was attenuated and regeneration centers were formed either from remains of partially absorbed zooids or from deformed ampullae. Subsequently, bud-bearing zooids developed, in concert with a massive IAP28-dependent phagocytic wave that eliminated the old zooids, then proceeded with the establishment of morphologically normal-looking colonies. IAP4, IAP14 and IAP28 were also involved in WBR, in conjunction with the expression of the pro-survival PI3K-Akt pathway. IAPs function deregulation by Smac mimetics resulted in severe morphological damages, attenuation in bud growth and differentiation, and in destabilization of colonial coordination. Longtime knockdown of IAP functions prior to the budectomy, resulted in colonial death.
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Affiliation(s)
- Amalia Rosner
- Israel Oceanographic&Limnological Research Institute, Tel Shikmona, P.O.B. 8030, Haifa 31080, Israel.
| | - Olha Kravchenko
- Israel Oceanographic&Limnological Research Institute, Tel Shikmona, P.O.B. 8030, Haifa 31080, Israel; National University of Life and Environmental Sciences of Ukraine, Heroiv Oborony, Str 17, building 2, of 45, Kyiv 03041, Ukraine
| | - Baruch Rinkevich
- Israel Oceanographic&Limnological Research Institute, Tel Shikmona, P.O.B. 8030, Haifa 31080, Israel
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7
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Liu J, Giri BR, Chen Y, Luo R, Xia T, Grevelding CG, Cheng G. Schistosoma japonicum IAP and Teg20 safeguard tegumental integrity by inhibiting cellular apoptosis. PLoS Negl Trop Dis 2018; 12:e0006654. [PMID: 30044778 PMCID: PMC6078320 DOI: 10.1371/journal.pntd.0006654] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2018] [Revised: 08/06/2018] [Accepted: 06/29/2018] [Indexed: 11/18/2022] Open
Abstract
Schistosomes are causative agents of human schistosomiasis, which is endemic in tropical and subtropical areas of the world. Adult schistosomes can survive in their final hosts for several decades, and they have evolved various strategies to overcome the host immune response. Consequently, understanding the mechanisms that regulate parasitic cell survival will open avenues for developing novel strategies against schistosomiasis. Our previous study suggested that an inhibitor of apoptosis protein in Schistosoma japonicum (SjIAP) may play important roles in parasitic survival and development. Here, we demonstrated that SjIAP can negatively regulate cellular apoptosis in S. japonicum by suppressing caspase activity. Immunohistochemistry analysis indicated that SjIAP ubiquitously expressed within the worm body including the tegument. Silencing of SjIAP expression via small interfering RNA led to destruction of the tegument integrity in schistosomes. We further used co-immunoprecipitation to identify interaction partners of SjIAP and revealed the tegument protein SjTeg-20 as a putative interacting partner of SjIAP. The interaction between SjIAP and SjTeg-20 was confirmed by a yeast two-hybrid (Y2H) assay. Moreover, results of a TUNEL assay, RNA interference, scanning and transmission electron microscopy, caspase assays, transcript profiling, and protein localization of both interacting molecules provided first evidence for an essential role of SjIAP and SjTeg-20 to maintain the structural integrity of the tegument by negatively regulating apoptosis. Taken together, our findings suggest that the cooperative activities of SjIAP and SjTeg-20 belong to the strategic inventory of S. japonicum ensuring survival in the hostile environment within the vasculature of the final host. Schistosomiasis is a worldwide public health concern particularly in developing countries. The causative agents, schistosomes, can survive within the vascular system of their final hosts for several decades despite facing the host’s immune response. Therefore, elucidating the mechanism of cell survival will contribute to the understanding of host-parasite interaction and may lead to the identification of suitable targets for developing novel strategies against schistosomiasis. Inhibitor of apoptosis proteins are highly conserved proteins functioning as endogenous inhibitors of apoptotic cell death. Here, we demonstrated that an inhibitor of apoptosis protein of Schistosoma japonicum (SjIAP) governs the integrity of the tegument of schistosomes by inhibiting cellular apoptosis of the parasite. Further studies revealed that SjTeg-20, an S. japonicum tegumental protein, cooperates with SjIAP to inhibit apoptosis in schistosomes. Our findings provide new insights into the role of SjIAP and SjTeg-20 in maintaining the integrity of the worm tegument by negatively regulating apoptosis.
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Affiliation(s)
- Juntao Liu
- Department of Animal Schistosomiasis, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, Shanghai, China
| | - Bikash R. Giri
- Department of Animal Schistosomiasis, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, Shanghai, China
| | - Yongjun Chen
- Department of Animal Schistosomiasis, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, Shanghai, China
| | - Rong Luo
- Department of Animal Schistosomiasis, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, Shanghai, China
| | - Tianqi Xia
- Department of Animal Schistosomiasis, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, Shanghai, China
| | | | - Guofeng Cheng
- Department of Animal Schistosomiasis, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, Shanghai, China
- * E-mail: ,
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8
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Singh S, Gupta M, Pandher S, Kaur G, Rathore P, Palli SR. Selection of housekeeping genes and demonstration of RNAi in cotton leafhopper, Amrasca biguttula biguttula (Ishida). PLoS One 2018; 13:e0191116. [PMID: 29329327 PMCID: PMC5766320 DOI: 10.1371/journal.pone.0191116] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2017] [Accepted: 12/28/2017] [Indexed: 01/14/2023] Open
Abstract
Amrasca biguttula biguttula (Ishida) commonly known as cotton leafhopper is a severe pest of cotton and okra. Not much is known on this insect at molecular level due to lack of genomic and transcriptomic data. To prepare for functional genomic studies in this insect, we evaluated 15 common housekeeping genes (Tub, B-Tub, EF alpha, GADPH, UbiCF, RP13, Ubiq, G3PD, VATPase, Actin, 18s, 28s, TATA, ETF, SOD and Cytolytic actin) during different developmental stages and under starvation stress. We selected early (1st and 2nd), late (3rd and 4th) stage nymphs and adults for identification of stable housekeeping genes using geNorm, NormFinder, BestKeeper and RefFinder software. Based on the different algorithms, RP13 and VATPase are identified as the most suitable reference genes for quantification of gene expression by reverse transcriptase quantitative PCR (RT-qPCR). Based on RefFinder which comprehended the results of three algorithms, RP13 in adults, Tubulin (Tub) in late nymphs, 28S in early nymph and UbiCF under starvation stress were identified as the most stable genes. We also developed methods for feeding double-stranded RNA (dsRNA) incorporated in the diet. Feeding dsRNA targeting Snf7, IAP, AQP1, and VATPase caused 56.17-77.12% knockdown of targeted genes compared to control and 16 to 48% mortality of treated insects when compared to control.
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Affiliation(s)
- Satnam Singh
- Punjab Agricultural University, Regional Station, Faridkot, Punjab, India
| | - Mridula Gupta
- Punjab Agricultural University, Regional Station, Faridkot, Punjab, India
| | - Suneet Pandher
- Punjab Agricultural University, Regional Station, Faridkot, Punjab, India
| | - Gurmeet Kaur
- Punjab Agricultural University, Regional Station, Faridkot, Punjab, India
| | - Pankaj Rathore
- Punjab Agricultural University, Regional Station, Faridkot, Punjab, India
| | - Subba Reddy Palli
- Department of Entomology, University of Kentucky, Lexington, KY, United States of America
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9
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Clancy-Thompson E, Ali L, Bruck PT, Exley MA, Blumberg RS, Dranoff G, Dougan M, Dougan SK. IAP Antagonists Enhance Cytokine Production from Mouse and Human iNKT Cells. Cancer Immunol Res 2018; 6:25-35. [PMID: 29187357 PMCID: PMC5754232 DOI: 10.1158/2326-6066.cir-17-0490] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2017] [Revised: 11/18/2017] [Accepted: 11/22/2017] [Indexed: 01/05/2023]
Abstract
Inhibitor of apoptosis protein (IAP) antagonists are in clinical trials for a variety of cancers, and mouse models show synergism between IAP antagonists and anti-PD-1 immunotherapy. Although IAP antagonists affect the intrinsic signaling of tumor cells, their most pronounced effects are on immune cells and the generation of antitumor immunity. Here, we examined the effects of IAP antagonism on T-cell development using mouse fetal thymic organ culture and observed a selective loss of iNKT cells, an effector cell type of potential importance for cancer immunotherapy. Thymic iNKT-cell development probably failed due to increased strength of TCR signal leading to negative selection, given that mature iNKT cells treated with IAP antagonists were not depleted, but had enhanced cytokine production in both mouse and human ex vivo cultures. Consistent with this, mature mouse primary iNKT cells and iNKT hybridomas increased production of effector cytokines in the presence of IAP antagonists. In vivo administration of IAP antagonists and α-GalCer resulted in increased IFNγ and IL-2 production from iNKT cells and decreased tumor burden in a mouse model of melanoma lung metastasis. Human iNKT cells also proliferated and increased IFNγ production dramatically in the presence of IAP antagonists, demonstrating the utility of these compounds in adoptive therapy of iNKT cells. Cancer Immunol Res; 6(1); 25-35. ©2017 AACR.
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Affiliation(s)
- Eleanor Clancy-Thompson
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Lestat Ali
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Patrick T Bruck
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Mark A Exley
- Beth Israel Deaconess Medical Center, Boston, Massachusetts
- Division of Gastroenterology, Brigham and Women's Hospital, Boston, Massachusetts
- Harvard Medical School, Boston, Massachusetts
| | - Richard S Blumberg
- Division of Gastroenterology, Brigham and Women's Hospital, Boston, Massachusetts
- Harvard Medical School, Boston, Massachusetts
| | - Glenn Dranoff
- Harvard Medical School, Boston, Massachusetts
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Michael Dougan
- Harvard Medical School, Boston, Massachusetts.
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
- Division of Gastroenterology, Massachusetts General Hospital, Boston, Massachusetts
| | - Stephanie K Dougan
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts.
- Harvard Medical School, Boston, Massachusetts
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10
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Modulation of apoptotic response by LAR family phosphatases-cIAP1 signaling during urinary tract morphogenesis. Proc Natl Acad Sci U S A 2017; 114:E9016-E9025. [PMID: 29073098 DOI: 10.1073/pnas.1707229114] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The elimination of unwanted cells by apoptosis is necessary for tissue morphogenesis. However, the cellular control of morphogenetic apoptosis is poorly understood, notably the modulation of cell sensitivity to apoptotic stimuli. Ureter maturation, the process by which the ureter is displaced to the bladder wall, represents an exquisite example of morphogenetic apoptosis, requiring the receptor protein tyrosine phosphatases (RPTPs): LAR and RPTPσ. Here we show that LAR-RPTPs act through cellular inhibitor of apoptosis protein 1 (cIAP1) to modulate caspase 3,7-mediated ureter maturation. Pharmacologic or genetic inactivation of cIAP1 reverts the apoptotic deficit of LAR-RPTP-deficient embryos. Moreover, Birc2 (cIAP1) inactivation generates excessive apoptosis leading to vesicoureteral reflux in newborns, which underscores the importance of apoptotic modulation during urinary tract morphogenesis. We finally demonstrate that LAR-RPTP deficiency increases cIAP1 stability during apoptotic cell death. Together these results identify a mode of cIAP1 regulation playing a critical role in the cellular response to apoptotic pathway activation in the embryo.
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11
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PD-L1 and IAPs co-operate to protect tumors from cytotoxic lymphocyte-derived TNF. Cell Death Differ 2017; 24:1705-1716. [PMID: 28665401 DOI: 10.1038/cdd.2017.94] [Citation(s) in RCA: 64] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2017] [Revised: 04/18/2017] [Accepted: 05/08/2017] [Indexed: 12/24/2022] Open
Abstract
Smac-mimetics are emerging as promising anti-cancer agents and are being evaluated in clinical trials for a variety of malignancies. Smac-mimetics can induce TNF production from a subset of tumor cells and simultaneously sensitize them to TNF-induced apoptosis. However, TNF derived from other cellular sources, such as cytotoxic lymphocytes (CLs) within the tumor, may also contribute to the anti-tumor activity of SMs. Here, we show that CD8+ T cells and NK cells potently kill tumor cells in the presence of the SM, birinapant. Enhanced CL killing occurred through TNF secretion upon tumor antigen recognition or NK-activating receptor ligation. Importantly, the perforin/granzyme route to CL-mediated tumor cell killing was dispensable for the efficacy of birinapant, emphasizing the importance of the TNF-mediated apoptosis pathway. Time-lapse microscopy revealed that birinapant sensitized tumor cells to apoptosis as bystanders and to membrane-bound TNF delivered to tumor cells within the immunological synapse. Furthermore, PD-L1 expression on tumor cells suppressed antigen-driven TNF production by CD8+ T cells, which could be antagonized through PD-1 blockade. Importantly, the elevated levels of TNF produced upon PD-1 blockade further enhanced tumor cell killing when combined with birinapant. The combined anti-tumor activity of IAP antagonism and PD-1 blockade occurred independently of perforin-mediated tumor cell death. Taken together, we identify CL-derived TNF as a potent effector of birinapant mediated anti-tumor immunity and opportunity for combination therapy through co-inhibition of immune checkpoints.
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12
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Koltai E, Bori Z, Chabert C, Dubouchaud H, Naito H, Machida S, Davies KJ, Murlasits Z, Fry AC, Boldogh I, Radak Z. SIRT1 may play a crucial role in overload-induced hypertrophy of skeletal muscle. J Physiol 2017; 595:3361-3376. [PMID: 28251652 PMCID: PMC5451718 DOI: 10.1113/jp273774] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2016] [Accepted: 02/23/2017] [Indexed: 01/08/2023] Open
Abstract
KEY POINTS Silent mating type information regulation 2 homologue 1 (SIRT1) activity and content increased significantly in overload-induced hypertrophy. SIRT1-mediated signalling through Akt, the endothelial nitric oxide synthase mediated pathway, regulates anabolic process in the hypertrophy of skeletal muscle. The regulation of catabolic signalling via forkhead box O 1 and protein ubiquitination is SIRT1 dependent. Overload-induced changes in microRNA levels regulate SIRT1 and insulin-like growth factor 1 signalling. ABSTRACT Significant skeletal muscle mass guarantees functional wellbeing and is important for high level performance in many sports. Although the molecular mechanism for skeletal muscle hypertrophy has been well studied, it still is not completely understood. In the present study, we used a functional overload model to induce plantaris muscle hypertrophy by surgically removing the soleus and gastrocnemius muscles in rats. Two weeks of muscle ablation resulted in a 40% increase in muscle mass, which was associated with a significant increase in silent mating type information regulation 2 homologue 1 (SIRT1) content and activity (P < 0.001). SIRT1-regulated Akt, endothelial nitric oxide synthase and GLUT4 levels were also induced in hypertrophied muscles, and SIRT1 levels correlated with muscle mass, paired box protein 7 (Pax7), proliferating cell nuclear antigen (PCNA) and nicotinamide phosphoribosyltransferase (Nampt) levels. Alternatively, decreased forkhead box O 1 (FOXO1) and increased K48 polyubiquitination also suggest that SIRT1 could be involved in the catabolic process of hypertrophy. Furthermore, increased levels of K63 and muscle RING finger 2 (MuRF2) protein could also be important enhancers of muscle mass. We report here that the levels of miR1 and miR133a decrease in hypertrophy and negatively correlate with muscle mass, SIRT1 and Nampt levels. Our results reveal a strong correlation between SIRT1 levels and activity, SIRT1-regulated pathways and overload-induced hypertrophy. These findings, along with the well-known regulatory roles that SIRT1 plays in modulating both anabolic and catabolic pathways, allow us to propose the hypothesis that SIRT1 may actually play a crucial causal role in overload-induced hypertrophy of skeletal muscle. This hypothesis will now require rigorous direct and functional testing.
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Affiliation(s)
- Erika Koltai
- Research Institute of Sport Science, University of Physical Education, Budapest, Hungary
| | - Zoltán Bori
- Research Institute of Sport Science, University of Physical Education, Budapest, Hungary
| | - Clovis Chabert
- Université Joseph Fourier, Laboratoire de Bioénergétique Fondamentale et Appliquée, Grenoble Cedex, 0938041, France
| | - Hervé Dubouchaud
- Université Joseph Fourier, Laboratoire de Bioénergétique Fondamentale et Appliquée, Grenoble Cedex, 0938041, France
| | - Hisashi Naito
- Department of Exercise Physiology, Graduate School of Health and Sports Science & Medicine, Juntendo University, Japan
| | - Shuichi Machida
- Department of Exercise Physiology, Graduate School of Health and Sports Science & Medicine, Juntendo University, Japan
| | - Kelvin Ja Davies
- Ethel Percy Andrus Gerontology Centre of the Leonard Davis School of Gerontology; and Division of Molecular & Computational Biology, Department of Biological Sciences, of the Dornsife College of Letters, Arts, and Sciences, the University of Southern California, Los Angeles, CA, 90089-0191, USA
| | | | - Andrew C Fry
- Osness Human Performance Laboratories, Department of Health, Sport, and Exercise Sciences, University of Kansas, Lawrence, KS, 66045, USA
| | - Istvan Boldogh
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX, 77555, USA
| | - Zsolt Radak
- Research Institute of Sport Science, University of Physical Education, Budapest, Hungary.,Institute of Sport Sciences and Physical Education, University of Pecs, Pecs, Hungary
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Boudida Y, Gagaoua M, Becila S, Picard B, Boudjellal A, Herrera-Mendez CH, Sentandreu M, Ouali A. Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions? Crit Rev Food Sci Nutr 2017; 56:957-72. [PMID: 25085261 DOI: 10.1080/10408398.2012.741630] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50-70 kDa), F2 (Mr:40-60 kDa) and F3 (Mr:10-15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.
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Affiliation(s)
- Yasmine Boudida
- a Equipe Maquav, INATAA, Université Frères Mentouri , Constantine , Algeria
| | - Mohammed Gagaoua
- a Equipe Maquav, INATAA, Université Frères Mentouri , Constantine , Algeria
| | - Samira Becila
- a Equipe Maquav, INATAA, Université Frères Mentouri , Constantine , Algeria
| | - Brigitte Picard
- b UMR1213 Herbivores, URH - AMUVI, INRA de Clermont Ferrand Theix, St Genès Champanelle , France
| | | | - Carlos H Herrera-Mendez
- c Agroindustrial Engineering Department, Universidad De Guanajuato, Salvatierra , Guanajuato , Mexico
| | - Miguel Sentandreu
- d Instituto de Agroquímica y Tecnología de Alimentos, CSIC , Burjassot (Valencia ), Spain
| | - Ahmed Ouali
- e UR370, QuaPA, INRA de Clermont Ferrand - Theix, St Genès Champanelle , France
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Sumi H, Inazuka M, Hashimoto K, Ishikawa T, Yoshida S, Yabuki M. T-3256336, a novel and orally available small molecule IAP antagonist, induced tumor cell death via induction of systemic TNF alpha production. Biochem Biophys Res Commun 2016; 479:179-185. [PMID: 27608596 DOI: 10.1016/j.bbrc.2016.09.019] [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] [Received: 08/30/2016] [Accepted: 09/03/2016] [Indexed: 01/12/2023]
Abstract
Inhibitors of apoptosis proteins (IAPs) are a family of antiapoptotic regulators that have attracted attention as potential targets for cancer therapeutics. Although recent studies have revealed that small-molecule IAP antagonists induce tumor selective cell death in an autocrine tumor necrosis factor (TNF)α-dependent manner, the single-agent efficacy of IAP antagonists is restricted to a small subset of cancer cells. In this study, we showed that the single-agent activity of T-3256336 was limited to a few cancer cell lines in vitro, and these cell lines were defined by relatively high levels of TNFα mRNA expression. However, some other cancer cells, including PANC-1 cells, become drastically sensitive to T-3256336 when costimulated with exogenous TNFα. In PANC-1 mouse xenograft models, the administration of T-3256336 increased levels of several cytokines including TNFα and lead to tumor regression as a single agent, which was attenuated by the neutralization of circulating mouse TNFα with an antibody. These results suggest dual roles of IAP antagonists, increase systemic cytokines including TNFα, and sensitization of tumors to IAP antagonist-induced death.
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Affiliation(s)
- Hiroyuki Sumi
- Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 26-1, Muraoka-higashi, 2-Chome, Fujisawa, Kanagawa 251-8555, Japan.
| | - Masakazu Inazuka
- Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 26-1, Muraoka-higashi, 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Kentaro Hashimoto
- Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 26-1, Muraoka-higashi, 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Tomoyasu Ishikawa
- Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 26-1, Muraoka-higashi, 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Sei Yoshida
- Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 26-1, Muraoka-higashi, 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Masato Yabuki
- Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 26-1, Muraoka-higashi, 2-Chome, Fujisawa, Kanagawa 251-8555, Japan.
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Xu J, Jiang S, Li Y, Li M, Cheng Q, Zhao D, Yang B, Jia Z, Wang L, Song L. Caspase-3 serves as an intracellular immune receptor specific for lipopolysaccharide in oyster Crassostrea gigas. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2016; 61:1-12. [PMID: 26993662 DOI: 10.1016/j.dci.2016.03.015] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/02/2016] [Revised: 03/12/2016] [Accepted: 03/13/2016] [Indexed: 06/05/2023]
Abstract
Apoptosis is a form of programmed cell death process controlled by a family of cysteine proteases called caspases, which plays a crucial role in the immune system homeostasis. The apoptosis and the detailed regulation mechanism have been well studied in vertebrate, but the information in lower animals, especially invertebrates, is still very limited. In the present study, Caspase-3 in the Pacific oyster Crassostrea gigas (designated CgCaspase-3) was enriched by lipopolysaccharide (LPS) affinity chromatography and further identified by MALDI-TOF/TOF-mass spectrometry. The binding activity of CgCaspase-3 to LPS was confirmed by enzyme-linked immunosorbent assay, and surface plasmon resonance analysis revealed its high binding specificity and moderate binding affinity (KD = 1.08 × 10(-6) M) to LPS. The recombinant CgCaspase-3 exhibited high proteolytic activity to substrate Ac-DEVD-pNA and relatively weak activity to substrate Ac-DMQD-pNA and Ac-VDQQD-pNA. The binding of CgCaspase-3 to LPS significantly inhibited its proteolytic activity toward AC-DEVD-pNA in vitro. The over-expression of CgCaspase-3 leaded to the phosphatidylserine exposure on the external plasma membrane and the cleavage of poly (ADP-ribose) polymerase, which reduced cell viability, and finally induced cell apoptosis. But the cell apoptosis mediated by CgCaspase-3 in vivo was significantly inhibited by the treatment of LPS. These results collectively indicated that CgCaspase-3 could serve as an intracellular LPS receptor, and the interaction of LPS with CgCaspase-3 specifically inhibited the cell apoptosis induced by CgCaspase-3.
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Affiliation(s)
- Jiachao Xu
- Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shuai Jiang
- Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
| | - Yiqun Li
- Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Meijia Li
- Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Qi Cheng
- Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Dalian Polytechnic University, Dalian 116034, China
| | - Depeng Zhao
- Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Dalian Polytechnic University, Dalian 116034, China
| | - Bin Yang
- Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Zhihao Jia
- Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Lingling Wang
- Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
| | - Linsheng Song
- Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, China.
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16
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Williams AA, Mehler VJ, Mueller C, Vonhoff F, White R, Duch C. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations. PLoS One 2016; 11:e0159632. [PMID: 27442528 PMCID: PMC4956225 DOI: 10.1371/journal.pone.0159632] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2016] [Accepted: 07/05/2016] [Indexed: 11/22/2022] Open
Abstract
Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.
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Affiliation(s)
- Alison A. Williams
- School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America
- Institute of Zoology- Neurobiology, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Vera J. Mehler
- Institute of Zoology- Neurobiology, Johannes Gutenberg University Mainz, Mainz, Germany
| | | | - Fernando Vonhoff
- Molecular, Cellular, and Developmental Biology Department, Yale University, New Haven, Connecticut, United States of America
| | - Robin White
- Institute of Physiology, University Medical Center, Mainz, Germany
| | - Carsten Duch
- Institute of Zoology- Neurobiology, Johannes Gutenberg University Mainz, Mainz, Germany
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17
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Unsain N, Barker PA. New Views on the Misconstrued: Executioner Caspases and Their Diverse Non-apoptotic Roles. Neuron 2016; 88:461-74. [PMID: 26539888 DOI: 10.1016/j.neuron.2015.08.029] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Initially characterized for their roles in apoptosis, executioner caspases have emerged as important regulators of an array of cellular activities. This is especially true in the nervous system, where sublethal caspase activity has been implicated in axonal pathfinding and branching, axonal degeneration, dendrite pruning, regeneration, long-term depression, and metaplasticity. Here we examine the roles of sublethal executioner caspase activity in nervous system development and maintenance, consider the mechanisms that locally activate and restrain these potential killers, and discuss how their activity be subverted in neurodegenerative disease.
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Affiliation(s)
- Nicolas Unsain
- Laboratorio de Neurobiología, Instituto de Investigación Médica Mercedes y Martín Ferreyra, Instituto Nacional de Investigación Médica Córdoba-Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Córdoba, Friuli 2434, Córdoba (5016), Argentina
| | - Philip A Barker
- Irving K. Barber School of Arts and Sciences, University of British Columbia, Kelowna, BC V1V 1V7, Canada.
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Baculovirus Inhibitor-of-Apoptosis Op-IAP3 Blocks Apoptosis by Interaction with and Stabilization of a Host Insect Cellular IAP. J Virol 2015; 90:533-44. [PMID: 26491164 DOI: 10.1128/jvi.02320-15] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2015] [Accepted: 10/14/2015] [Indexed: 12/18/2022] Open
Abstract
UNLABELLED Baculovirus-encoded inhibitor of apoptosis (IAP) proteins likely evolved from their host cell IAP homologs, which function as critical regulators of cell death. Despite their striking relatedness to cellular IAPs, including the conservation of two baculovirus IAP repeat (BIR) domains and a C-terminal RING, viral IAPs use an unresolved mechanism to suppress apoptosis in insects. To define this mechanism, we investigated Op-IAP3, the prototypical IAP from baculovirus OpMNPV. We found that Op-IAP3 forms a stable complex with SfIAP, the native, short-lived IAP of host insect Spodoptera frugiperda. Long-lived Op-IAP3 prevented virus-induced SfIAP degradation, which normally causes caspase activation and apoptosis. In uninfected cells, Op-IAP3 also increased SfIAP steady-state levels and extended SfIAP's half-life. Conversely, SfIAP stabilization was lost or reversed in the presence of mutated Op-IAP3 that was engineered for reduced stability. Thus, Op-IAP3 stabilizes SfIAP and preserves its antiapoptotic function. In contrast to SfIAP, Op-IAP3 failed to bind or inhibit native Spodoptera caspases. Furthermore, BIR mutations that abrogate binding of well-conserved IAP antagonists did not affect Op-IAP3's capacity to prevent virus-induced apoptosis. Remarkably, Op-IAP3 also failed to prevent apoptosis when endogenous SfIAP was ablated by RNA silencing. Thus, Op-IAP3 requires SfIAP as a cofactor. Our findings suggest a new model wherein Op-IAP3 interacts directly with SfIAP to maintain its intracellular level, thereby suppressing virus-induced apoptosis indirectly. Consistent with this model, Op-IAP3 has evolved an intrinsic stability that may serve to repress signal-induced turnover and autoubiquitination when bound to its targeted cellular IAP. IMPORTANCE The IAPs were first discovered in baculoviruses because of their potency for preventing apoptosis. However, the antiapoptotic mechanism of viral IAPs in host insects has been elusive. We show here that the prototypical viral IAP, Op-IAP3, blocks apoptosis indirectly by associating with unstable, autoubiquitinating host IAP in such a way that cellular IAP levels and antiapoptotic activities are maintained. This mechanism explains Op-IAP3's requirement for native cellular IAP as a cofactor and the dispensability of caspase inhibition. Viral IAP-mediated preservation of the host IAP homolog capitalizes on normal IAP-IAP interactions and is likely the result of viral IAP evolution in which degron-mediated destabilization and ubiquitination potential have been reduced. This mechanism illustrates another novel means by which DNA viruses incorporate host death regulators that are modified for resistance to host regulatory controls for the purpose of suppressing host cell apoptosis and acquiring replication advantages.
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Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray. Virusdisease 2015; 26:9-18. [PMID: 26436116 DOI: 10.1007/s13337-014-0243-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2014] [Accepted: 12/29/2014] [Indexed: 12/30/2022] Open
Abstract
White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.
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20
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Insect inhibitor-of-apoptosis (IAP) proteins are negatively regulated by signal-induced N-terminal degrons absent within viral IAP proteins. J Virol 2015; 89:4481-93. [PMID: 25653450 DOI: 10.1128/jvi.03659-14] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
UNLABELLED Inhibitor-of-apoptosis (IAP) proteins are key regulators of the innate antiviral response by virtue of their capacity to respond to signals affecting cell survival. In insects, wherein the host IAP provides a primary restriction to apoptosis, diverse viruses trigger rapid IAP depletion that initiates caspase-mediated apoptosis, thereby limiting virus multiplication. We report here that the N-terminal leader of two insect IAPs, Spodoptera frugiperda SfIAP and Drosophila melanogaster DIAP1, contain distinct instability motifs that regulate IAP turnover and apoptotic consequences. Functioning as a protein degron, the cellular IAP leader dramatically shortened the life span of a long-lived viral IAP (Op-IAP3) when fused to its N terminus. The SfIAP degron contains mitogen-activated kinase (MAPK)-like regulatory sites, responsible for MAPK inhibitor-sensitive phosphorylation of SfIAP. Hyperphosphorylation correlated with increased SfIAP turnover independent of the E3 ubiquitin-ligase activity of the SfIAP RING, which also regulated IAP stability. Together, our findings suggest that the SfIAP phospho-degron responds rapidly to a signal-activated kinase cascade, which regulates SfIAP levels and thus apoptosis. The N-terminal leader of dipteran DIAP1 also conferred virus-induced IAP depletion by a caspase-independent mechanism. DIAP1 instability mapped to previously unrecognized motifs that are not found in lepidopteran IAPs. Thus, the leaders of cellular IAPs from diverse insects carry unique signal-responsive degrons that control IAP turnover. Rapid response pathways that trigger IAP degradation and initiate apoptosis independent of canonical prodeath gene (Reaper-Grim-Hid) expression may provide important innate immune advantages. Furthermore, the elimination of these response motifs within viral IAPs, including those of baculoviruses, explains their unusual stability and their potent antiapoptotic activity. IMPORTANCE Apoptosis is an effective means by which a host controls virus infection. In insects, inhibitor-of-apoptosis (IAP) proteins act as regulatory sentinels by responding to cellular signals that determine the fate of infected cells. We discovered that lepidopteran (moth and butterfly) IAPs, which are degraded upon baculovirus infection, are controlled by a conserved phosphorylation-sensitive degron within the IAP N-terminal leader. The degron likely responds to virus-induced kinase-specific signals for degradation through SKP1/Cullin/F-box complex-mediated ubiquitination. Such signal-induced destruction of cellular IAPs is distinct from degradation caused by well-known IAP antagonists, which act to expel IAP-bound caspases. The major implication of this study is that insects have multiple signal-responsive mechanisms by which the sentinel IAPs are actively degraded to initiate host apoptosis. Such diversity of pathways likely provides insects with rapid and efficient strategies for pathogen control. Furthermore, the absence of analogous degrons in virus-encoded IAPs explains their relative stability and antiapoptotic potency.
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21
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Cellular inhibitors of apoptosis (cIAP) 1 and 2 are increased in placenta from obese pregnant women. Placenta 2014; 35:831-8. [DOI: 10.1016/j.placenta.2014.07.011] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/19/2014] [Revised: 07/18/2014] [Accepted: 07/23/2014] [Indexed: 12/12/2022]
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22
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Takeda AN, Oberoi-Khanuja TK, Glatz G, Schulenburg K, Scholz RP, Carpy A, Macek B, Remenyi A, Rajalingam K. Ubiquitin-dependent regulation of MEKK2/3-MEK5-ERK5 signaling module by XIAP and cIAP1. EMBO J 2014; 33:1784-801. [PMID: 24975362 PMCID: PMC4195761 DOI: 10.15252/embj.201487808] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2014] [Revised: 05/18/2014] [Accepted: 05/26/2014] [Indexed: 11/09/2022] Open
Abstract
Mitogen-activated protein kinases (MAPKs) are highly conserved protein kinase modules, and they control fundamental cellular processes. While the activation of MAPKs has been well studied, little is known on the mechanisms driving their inactivation. Here we uncover a role for ubiquitination in the inactivation of a MAPK module. Extracellular-signal-regulated kinase 5 (ERK5) is a unique, conserved member of the MAPK family and is activated in response to various stimuli through a three-tier cascade constituting MEK5 and MEKK2/3. We reveal an unexpected role for Inhibitors of Apoptosis Proteins (IAPs) in the inactivation of ERK5 pathway in a bimodal manner involving direct interaction and ubiquitination. XIAP directly interacts with MEKK2/3 and competes with PB1 domain-mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3 which directly impede MEK5-ERK5 interaction in a trimeric complex leading to ERK5 inactivation. Consistently, loss of XIAP or cIAP1 by various strategies leads to hyperactivation of ERK5 in normal and tumorigenic cells. Loss of XIAP promotes differentiation of human primary skeletal myoblasts to myocytes in a MEKK2/3-ERK5-dependent manner. Our results reveal a novel, obligatory role for IAPs and ubiquitination in the physical and functional disassembly of ERK5-MAPK module and human muscle cell differentiation.
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Affiliation(s)
- Armelle-Natsuo Takeda
- Cell Death Signaling Group, Institute of Biochemistry II Goethe University Medical School, Frankfurt, Germany
| | - Tripat Kaur Oberoi-Khanuja
- Cell Death Signaling Group, Institute of Biochemistry II Goethe University Medical School, Frankfurt, Germany
| | - Gabor Glatz
- Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary Institute of Enzymology, Research Centre for Natural Sciences Hungarian Academy of Sciences, Budapest, Hungary
| | - Katharina Schulenburg
- Cell Death Signaling Group, Institute of Biochemistry II Goethe University Medical School, Frankfurt, Germany
| | - Rolf-Peter Scholz
- Cell Death Signaling Group, Institute of Biochemistry II Goethe University Medical School, Frankfurt, Germany
| | - Alejandro Carpy
- Proteome Center Tuebingen, Interfaculty Institute for Cell Biology University of Tuebingen, Tuebingen, Germany
| | - Boris Macek
- Proteome Center Tuebingen, Interfaculty Institute for Cell Biology University of Tuebingen, Tuebingen, Germany
| | - Attila Remenyi
- Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary Institute of Enzymology, Research Centre for Natural Sciences Hungarian Academy of Sciences, Budapest, Hungary
| | - Krishnaraj Rajalingam
- Cell Death Signaling Group, Institute of Biochemistry II Goethe University Medical School, Frankfurt, Germany
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Maycotte P, Thorburn A. Targeting autophagy in breast cancer. World J Clin Oncol 2014; 5:224-240. [PMID: 25114840 PMCID: PMC4127596 DOI: 10.5306/wjco.v5.i3.224] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/07/2014] [Revised: 04/02/2014] [Accepted: 05/29/2014] [Indexed: 02/06/2023] Open
Abstract
Macroautophagy (referred to as autophagy here) is an intracellular degradation pathway enhanced in response to a variety of stresses and in response to nutrient deprivation. This process provides the cell with nutrients and energy by degrading aggregated and damaged proteins as well as compromised organelles. Since autophagy has been linked to diverse diseases including cancer, it has recently become a very interesting target in breast cancer treatment. Indeed, current clinical trials are trying to use chloroquine or hydroxychloroquine, alone or in combination with other drugs to inhibit autophagy during breast cancer therapy since chemotherapy and radiation, regimens that are used to treat breast cancer, are known to induce autophagy in cancer cells. Importantly, in breast cancer, autophagy has been involved in the development of resistance to chemotherapy and to anti-estrogens. Moreover, a close relationship has recently been described between autophagy and the HER2 receptor. Here, we discuss some of the recent findings relating autophagy and cancer with a particular focus on breast cancer therapy.
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Lappas M. Cellular Inhibitors of Apoptosis Proteins cIAP1 and cIAP2 are Increased after Labour in Foetal Membranes and Myometrium and are Essential for TNF-α-Induced Expression of Pro-Labour Mediators. Am J Reprod Immunol 2014; 73:313-29. [DOI: 10.1111/aji.12295] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2014] [Accepted: 06/30/2014] [Indexed: 11/30/2022] Open
Affiliation(s)
- Martha Lappas
- Mercy Perinatal Research Centre; Mercy Hospital for Women; Heidelberg VIC Australia
- Obstetrics, Nutrition and Endocrinology Group; Department of Obstetrics and Gynaecology; University of Melbourne; Melbourne VIC Australia
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25
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Fan X, Huang Q, Ye X, Lin Y, Chen Y, Lin X, Qu J. Drosophila USP5 controls the activation of apoptosis and the Jun N-terminal kinase pathway during eye development. PLoS One 2014; 9:e92250. [PMID: 24643212 PMCID: PMC3958489 DOI: 10.1371/journal.pone.0092250] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2013] [Accepted: 02/20/2014] [Indexed: 11/18/2022] Open
Abstract
The Jun N-terminal kinase pathway plays an important role in inducing programmed cell death (apoptosis) and is activated in a variety of contexts. The deubiquitinating enzymes (DUBs) are proteases regulating the protein stability by ubiquitin-proteasome system. Here, for the first time, we report the phenotypes observed during eye development that are induced by deleting Drosophila USP5 gene, which encodes one of the USP subfamily of DUBs. usp5 mutants displayed defects in photoreceptor differentiation. Using genetic epistasis analysis and molecular markers, we show that most of these phenotypes are caused by the activation of apoptosis and JNK pathway. These data may provide a mechanistic model for understanding the mammalian usp5 gene.
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Affiliation(s)
- Xiaolan Fan
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Qinzhu Huang
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Xiaolei Ye
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yi Lin
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yuting Chen
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Xinhua Lin
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Jia Qu
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
- * E-mail:
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26
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Guo S, Messmer-Blust AF, Wu J, Song X, Philbrick MJ, Shie JL, Rana JS, Li J. Role of A20 in cIAP-2 protection against tumor necrosis factor α (TNF-α)-mediated apoptosis in endothelial cells. Int J Mol Sci 2014; 15:3816-33. [PMID: 24595242 PMCID: PMC3975369 DOI: 10.3390/ijms15033816] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2013] [Revised: 01/30/2014] [Accepted: 02/06/2014] [Indexed: 12/13/2022] Open
Abstract
Tumor necrosis factor α (TNF-α) influences endothelial cell viability by altering the regulatory molecules involved in induction or suppression of apoptosis. However, the underlying mechanisms are still not completely understood. In this study, we demonstrated that A20 (also known as TNFAIP3, tumor necrosis factor α-induced protein 3, and an anti-apoptotic protein) regulates the inhibitor of apoptosis protein-2 (cIAP-2) expression upon TNF-α induction in endothelial cells. Inhibition of A20 expression by its siRNA resulted in attenuating expression of TNF-α-induced cIAP-2, yet not cIAP-1 or XIAP. A20-induced cIAP-2 expression can be blocked by the inhibition of phosphatidyl inositol-3 kinase (PI3-K), but not nuclear factor (NF)-κB, while concomitantly increasing the number of endothelial apoptotic cells and caspase 3 activation. Moreover, TNF-α-mediated induction of apoptosis was enhanced by A20 inhibition, which could be rescued by cIAP-2. Taken together, these results identify A20 as a cytoprotective factor involved in cIAP-2 inhibitory pathway of TNF-α-induced apoptosis. This is consistent with the idea that endothelial cell viability is dependent on interactions between inducers and suppressors of apoptosis, susceptible to modulation by TNF-α.
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Affiliation(s)
- Shuzhen Guo
- School of Preclinical Medicine, Beijing University of Chinese Medicine, Beijing 100029, China.
| | - Angela F Messmer-Blust
- CardioVascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
| | - Jiaping Wu
- CardioVascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
| | - Xiaoxiao Song
- CardioVascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
| | - Melissa J Philbrick
- CardioVascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
| | - Jue-Lon Shie
- CardioVascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
| | - Jamal S Rana
- CardioVascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
| | - Jian Li
- CardioVascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
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27
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Dekkers MPJ, Nikoletopoulou V, Barde YA. Cell biology in neuroscience: Death of developing neurons: new insights and implications for connectivity. ACTA ACUST UNITED AC 2014; 203:385-93. [PMID: 24217616 PMCID: PMC3824005 DOI: 10.1083/jcb.201306136] [Citation(s) in RCA: 135] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
The concept that target tissues determine the survival of neurons has inspired much of the thinking on neuronal development in vertebrates, not least because it is supported by decades of research on nerve growth factor (NGF) in the peripheral nervous system (PNS). Recent discoveries now help to understand why only some developing neurons selectively depend on NGF. They also indicate that the survival of most neurons in the central nervous system (CNS) is not simply regulated by single growth factors like in the PNS. Additionally, components of the cell death machinery have begun to be recognized as regulators of selective axonal degeneration and synaptic function, thus playing a critical role in wiring up the nervous system.
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28
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Wang H, Zhang X, Wang L, Zheng G, Du L, Yang Y, Dong Z, Liu Y, Qu A, Wang C. Investigation of cell free BIRC5 mRNA as a serum diagnostic and prognostic biomarker for colorectal cancer. J Surg Oncol 2013; 109:574-9. [PMID: 24338523 DOI: 10.1002/jso.23526] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2013] [Accepted: 11/17/2013] [Indexed: 12/18/2022]
Affiliation(s)
- Haiyan Wang
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
| | - Xin Zhang
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
| | - Lili Wang
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
| | - Guixi Zheng
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
| | - Lutao Du
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
| | - Yongmei Yang
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
| | - Zhaogang Dong
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
| | - Yimin Liu
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
| | - Ailin Qu
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
| | - Chuanxin Wang
- Department of Clinical Laboratory; Qilu Hospital, Shandong University; Jinan China
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29
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Interactions between herpesvirus entry mediator (TNFRSF14) and latency-associated transcript during herpes simplex virus 1 latency. J Virol 2013; 88:1961-71. [PMID: 24307582 DOI: 10.1128/jvi.02467-13] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Herpesvirus entry mediator (HVEM) is one of several cell surface proteins herpes simplex virus (HSV) uses for attachment/entry. HVEM regulates cellular immune responses and can also increase cell survival. Interestingly, latency-associated transcript (LAT), the only viral gene consistently expressed during neuronal latency, enhances latency and reactivation by promoting cell survival and by helping the virus evade the host immune response. However, the mechanisms of these LAT activities are not well understood. We show here for the first time that one mechanism by which LAT enhances latency and reactivation appears to be by upregulating HVEM expression. HSV-1 latency/reactivation was significantly reduced in Hvem(-/-) mice, indicating that HVEM plays a significant role in HSV-1 latency/reactivation. Furthermore, LAT upregulated HVEM expression during latency in vivo and also when expressed in vitro in the absence of other viral factors. This study suggests a mechanism whereby LAT upregulates HVEM expression potentially through binding of two LAT small noncoding RNAs to the HVEM promoter and that the increased HVEM then leads to downregulation of immune responses in the latent microenvironment and increased survival of latently infected cells. Thus, one of the mechanisms by which LAT enhances latency/reactivation appears to be through increasing expression of HVEM.
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30
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Ortega-Arellano HF, Jimenez-Del-Rio M, Velez-Pardo C. Dmp53, basket and drICE gene knockdown and polyphenol gallic acid increase life span and locomotor activity in a Drosophila Parkinson's disease model. Genet Mol Biol 2013; 36:608-15. [PMID: 24385865 PMCID: PMC3873193 DOI: 10.1590/s1415-47572013000400020] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2013] [Accepted: 10/08/2013] [Indexed: 12/22/2022] Open
Abstract
Understanding the mechanism(s) by which dopaminergic (DAergic) neurons are eroded in Parkinson's disease (PD) is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH)-Gal4/UAS-X RNAi Drosophila melanogaster system, we report that Dmp53, basket and drICE gene knockdown in dopaminergic neurons prolong life span (p < 0.05; log-rank test) and locomotor activity (p < 0.05; χ(2) test) in D. melanogaster lines chronically exposed to (1 mM) paraquat (PQ, oxidative stress (OS) generator) compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA) significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s) involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. We propose that a therapy with antioxidants and selectively "switching off" death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition.
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Affiliation(s)
- Hector Flavio Ortega-Arellano
- Neuroscience Research Group, Medical Research Institute, Faculty of Medicine, University of Antioquia, Medellin, Colombia
| | - Marlene Jimenez-Del-Rio
- Neuroscience Research Group, Medical Research Institute, Faculty of Medicine, University of Antioquia, Medellin, Colombia
| | - Carlos Velez-Pardo
- Neuroscience Research Group, Medical Research Institute, Faculty of Medicine, University of Antioquia, Medellin, Colombia
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31
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Abstract
Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination–dependent inactivation of Rac1 signaling.
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Affiliation(s)
- Tripat Kaur Oberoi-Khanuja
- Emmy Noether Group of the DFG, Institute of Biochemistry II, Goethe University Medical School; Frankfurt, Germany
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32
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Saladi SV, Wong PG, Trivedi AR, Marathe HG, Keenen B, Aras S, Liew ZQ, Setaluri V, de la Serna IL. BRG1 promotes survival of UV-irradiated melanoma cells by cooperating with MITF to activate the melanoma inhibitor of apoptosis gene. Pigment Cell Melanoma Res 2013; 26:377-91. [PMID: 23480510 PMCID: PMC3633630 DOI: 10.1111/pcmr.12088] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2012] [Accepted: 03/02/2013] [Indexed: 01/19/2023]
Abstract
Microphthalmia-associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells. MITF regulates expression of antiapoptotic genes and promotes lineage-specific survival in response to ultraviolet (UV) radiation and to chemotherapeutics. SWI/SNF chromatin-remodeling enzymes interact with MITF to regulate MITF target gene expression. We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV-induced death. BRG1 prevents apoptosis in UV-irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML-IAP). Down-regulation of ML-IAP compromises BRG1-mediated survival of melanoma cells in response to UV radiation. BRG1 regulates ML-IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML-IAP promoter. The alternative catalytic subunit, BRM, and the BRG1-associated factor, BAF180, were found to be dispensable for elevated expression of ML-IAP in melanoma cells. Thus, we illuminate a lineage-specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an antiapoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the prosurvival function of MITF.
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Affiliation(s)
- Srinivas V Saladi
- Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, OH, USA
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33
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Park YH, Seo SY, Lee E, Ku JH, Kim HH, Kwak C. Simvastatin induces apoptosis in castrate resistant prostate cancer cells by deregulating nuclear factor-κB pathway. J Urol 2013; 189:1547-1552. [PMID: 23085058 DOI: 10.1016/j.juro.2012.10.030] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/11/2012] [Indexed: 11/24/2022]
Abstract
PURPOSE In castrate resistant prostate cancer cells we investigated the cytotoxic effect of simvastatin and the mechanism involved. MATERIALS AND METHODS After treating PC3 and DU-145 cells with simvastatin, cell viability and apoptosis were determined using tetrazolium salt based colorimetric assay and annexin-V-fluorescein isothiocyanate/propidium iodide double staining assay, respectively. To determine whether simvastatin affects the nuclear factor-κB pathway, we assessed IκBα and phosphorylated IκBα expression, and p65 and phosphorylated p65 subcellular localization by Western blot analysis. Also, changes in nuclear factor-κB transcriptional activity were assessed using a luciferase reporter assay. RESULTS After treating PC3 and DU-145 cells with 0, 20 or 40 μM simvastatin for 24, 48 or 72 hours, the proportion of viable cells decreased and the proportion of apoptotic cells increased in a dose and time dependent manner. Western blot analysis showed that simvastatin inhibited IκBα phosphorylation and degradation. It also demonstrated that simvastatin increased p65 protein levels in cytoplasmic fractions and decreased phosphorylated p65 protein levels in nuclear fractions but did not change p65 protein levels in cytoplasm. Luciferase reporter assay showed that simvastatin dose dependently reduced nuclear factor-κB activity. Reverse transcriptase-polymerase chain reaction and Western blot revealed that simvastatin inhibited nuclear factor-κB regulated cIAP-1 and 2, cFLIP-S and XIAP expression in dose and time dependent fashion. CONCLUSIONS Simvastatin inhibited castrate resistant prostate cancer cell growth by inducing apoptosis. These effects were probably mediated by the inhibition of IκBα phosphorylation and nuclear translocation of p50/p65 dimer in the nuclear factor-κB pathway.
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34
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Yacobi-Sharon K, Namdar Y, Arama E. Alternative germ cell death pathway in Drosophila involves HtrA2/Omi, lysosomes, and a caspase-9 counterpart. Dev Cell 2013; 25:29-42. [PMID: 23523076 DOI: 10.1016/j.devcel.2013.02.002] [Citation(s) in RCA: 83] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2012] [Revised: 01/03/2013] [Accepted: 02/04/2013] [Indexed: 12/15/2022]
Abstract
In both flies and mammals, almost one-third of the newly emerging male germ cells are spontaneously eliminated before entering meiosis. Here, we show that in Drosophila, germ cell death (GCD) involves the initiator caspase Dronc independently of the apoptosome and the main executioner caspases. Electron microscopy of dying germ cells revealed mixed morphologies of apoptosis and necrosis. We further show that the lysosomes and their catabolic enzymes, but not macroautophagy, are involved in the execution of GCD. We then identified, in a screen, the Parkinson's disease-associated mitochondrial protease, HtrA2/Omi, as an important mediator of GCD, acting mainly through its catalytic activity rather than by antagonizing inhibitor of apoptosis proteins. Concomitantly, other mitochondrial-associated factors were also implicated in GCD, including Pink1 (but not Parkin), the Bcl-2-related proteins, and endonuclease G, which establish the mitochondria as central mediators of GCD. These findings uncover an alternative developmental cell death pathway in metazoans.
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Affiliation(s)
- Keren Yacobi-Sharon
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
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35
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Rudrapatna VA, Bangi E, Cagan RL. Caspase signalling in the absence of apoptosis drives Jnk-dependent invasion. EMBO Rep 2013; 14:172-7. [PMID: 23306653 DOI: 10.1038/embor.2012.217] [Citation(s) in RCA: 64] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2012] [Revised: 12/04/2012] [Accepted: 12/12/2012] [Indexed: 11/09/2022] Open
Abstract
Tumours evolve several mechanisms to evade apoptosis, yet many resected carcinomas show significantly elevated caspase activity. Moreover, caspase activity is positively correlated with tumour aggression and adverse patient outcome. These observations indicate that caspases might have a functional role in promoting tumour invasion and metastasis. Using a Drosophila model of invasion, we show that precise effector caspase activity drives cell invasion without initiating apoptosis. Affected cells express the matrix metalloprotinase Mmp1 and invade by activating Jnk. Our results link Jnk and effector caspase signalling during the invasive process and suggest that tumours under apoptotic stresses from treatment, immune surveillance or intrinsic signals might be induced further along the metastatic cascade.
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Affiliation(s)
- Vivek A Rudrapatna
- Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029, USA
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36
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Ikeda M, Yamada H, Hamajima R, Kobayashi M. Baculovirus genes modulating intracellular innate antiviral immunity of lepidopteran insect cells. Virology 2013; 435:1-13. [DOI: 10.1016/j.virol.2012.10.016] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2012] [Revised: 10/08/2012] [Accepted: 10/08/2012] [Indexed: 12/11/2022]
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37
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Kearney CJ, Sheridan C, Cullen SP, Tynan GA, Logue SE, Afonina IS, Vucic D, Lavelle EC, Martin SJ. Inhibitor of apoptosis proteins (IAPs) and their antagonists regulate spontaneous and tumor necrosis factor (TNF)-induced proinflammatory cytokine and chemokine production. J Biol Chem 2012; 288:4878-90. [PMID: 23275336 DOI: 10.1074/jbc.m112.422410] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings.
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Affiliation(s)
- Conor J Kearney
- Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland
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38
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Leu JH, Chen YC, Chen LL, Chen KY, Huang HT, Ho JM, Lo CF. Litopenaeus vannamei inhibitor of apoptosis protein 1 (LvIAP1) is essential for shrimp survival. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2012; 38:78-87. [PMID: 22564858 DOI: 10.1016/j.dci.2012.04.006] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/04/2012] [Revised: 03/25/2012] [Accepted: 04/12/2012] [Indexed: 05/31/2023]
Abstract
The members of the inhibitor of apoptosis protein (IAP) family are involved in the regulation of diverse cellular processes, including apoptosis, signal transduction and mitosis. Here, we report the cloning and characterization of three IAP genes from Pacific white shrimp Litopenaeus vannamei: LvIAP1, LvIAP2 and LvSurvivin. LvIAP1, the orthologue of Penaeus monodon IAP (PmIAP), consists of three BIR domains and one RING domain; LvIAP2 consists of two BIR domains and LvSurvivin has only one BIR domain. Expression profiling by absolute quantitative real-time RT-PCR revealed that of the three IAP genes, LvIAP1 had the highest expression levels in almost all examined tissues and LvSurvivin had the lowest expression levels. Furthermore, among the examined tissues, the lymphoid organs most strongly expressed all three genes. When LvIAP1 expression was silenced by injection of its corresponding dsRNA, the shrimp died within 48h after injection, whereas injection of the other two dsRNAs did not cause shrimp death. In LvIAP1-silenced shrimp, the number of circulating haemocytes decreased dramatically because of extensive apoptosis. This suggested that LvIAP1 is central to the regulation of shrimp haemocyte apoptosis.
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Affiliation(s)
- Jiann-Horng Leu
- Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, Taiwan, ROC.
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39
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Baculovirus Lymantria dispar multiple nucleopolyhedrovirus IAP2 and IAP3 do not suppress apoptosis, but trigger apoptosis of insect cells in a transient expression assay. Virus Genes 2012; 45:370-9. [PMID: 22798056 DOI: 10.1007/s11262-012-0783-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2012] [Accepted: 07/03/2012] [Indexed: 10/28/2022]
Abstract
Ld652Y cells derived from the gypsy moth, Lymantria dispar, are permissive for productive infection with L. dispar multiple nucleopolyhedrovirus (LdMNPV), but undergo apoptosis upon infection with various other NPVs, including those isolated from Bombyx mori, Hyphantria cunea, Spodoptera exigua, Orgyia pseudotsugata, and Spodoptera litura. In this study, we examined whether LdMNPV-encoded inhibitor of apoptosis 2 (Ld-IAP2) and 3 (Ld-IAP3) are involved in apoptosis suppression in LdMNPV-infected Ld652Y cells. We found that neither Ld-IAP2 nor Ld-IAP3 was able to suppress the apoptosis of Ld652Y cells induced by p35-defective Autographa californica MNPV (vAcΔp35). However, both Ld-IAP2 and Ld-IAP3 induced apoptosis in Ld652Y cells in a transient expression assay. The apoptosis induced by Ld-IAP3 was accompanied by the stimulation of caspase-3-like protease activity and cleavage of the B. mori homolog of the initiator caspase Dronc, and was precluded by the LdMNPV-encoded apoptosis suppressor protein Apsup and H. cunea MNPV IAP3. Inconsistent with the results obtained previously in SpIm, Ld652Y and High Five cells infected with NPVs from H. cunea, O. pseudotsugata, and A. californica, respectively, considerable stimulation of caspase-3-like protease activity was not observed in LdMNPV-infected Ld652Y cells, likely due to the strong apoptosis suppression activity of Apsup. These results, together with the previous finding that RNAi-mediated silencing of apsup induces apoptosis of LdMNPV-infected Ld652Y cells, indicate that Apsup, but not Ld-IAP2 or Ld-IAP3, is primarily responsible for the suppression of apoptosis in LdMNPV-infected Ld652Y cells. However, it remains inconclusive whether Ld-IAP2 and Ld-IAP3 function as pro-apoptotic proteins in LdMNPV-infected Ld652Y cells.
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41
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Hedskog L, Zhang S, Ankarcrona M. Strategic role for mitochondria in Alzheimer's disease and cancer. Antioxid Redox Signal 2012; 16:1476-91. [PMID: 21902456 DOI: 10.1089/ars.2011.4259] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
SIGNIFICANCE Detailed knowledge about cell death and cell survival mechanisms and how these pathways are impaired in neurodegenerative disorders and cancer forms the basis for future drug development for these diseases that affect millions of people around the world. RECENT ADVANCES In neurodegenerative disorders such as Alzheimer's disease (AD), cell death pathways are inappropriately activated, resulting in neuronal cell death. In contrast, cancer cells develop resistance to apoptosis by regulating anti-apoptotic proteins signaling via mitochondria. Mounting evidence shows that mitochondrial function is central in both cancer and AD. Cancer cells typically shut down oxidative phosphorylation (OXPHOS) in mitochondria and switch to glycolysis for ATP production, making them resistant to hypoxia. In AD, for example, amyloid-β peptide (Aβ) and reactive oxygen species impair mitochondrial function. Neurons therefore also switch to glycolysis to maintain ATP production and to produce molecules involved in antioxidant metabolism in an attempt to survive. CRITICAL ISSUES One critical difference between cancer cells and neurons is that cancer cells can survive without OXPHOS, while neurons are dependent on OXPHOS for long-term survival. FUTURE DIRECTIONS This review will focus on these abnormalities of mitochondrial function shared in AD and cancer and discuss the potential mechanisms underlying links that may be key steps in the development of therapeutic strategies.
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Affiliation(s)
- Louise Hedskog
- Department of Neurobiology, Care Sciences and Society (NVS), KI-Alzheimer Disease Research Center, Karolinska Institutet, Stockholm, Sweden
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Piacentino III V, Milano CA, Bolanos M, Schroder J, Messina E, Cockrell AS, Jones E, Krol A, Bursac N, Mao L, Devi GR, Samulski RJ, Bowles DE. X-linked inhibitor of apoptosis protein-mediated attenuation of apoptosis, using a novel cardiac-enhanced adeno-associated viral vector. Hum Gene Ther 2012; 23:635-46. [PMID: 22339372 PMCID: PMC3392616 DOI: 10.1089/hum.2011.186] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2011] [Accepted: 02/09/2012] [Indexed: 12/31/2022] Open
Abstract
Successful amelioration of cardiac dysfunction and heart failure through gene therapy approaches will require a transgene effective at attenuating myocardial injury, and subsequent remodeling, using an efficient and safe delivery vehicle. Our laboratory has established a well-curated, high-quality repository of human myocardial tissues that we use as a discovery engine to identify putative therapeutic transgene targets, as well as to better understand the molecular basis of human heart failure. By using this rare resource we were able to examine age- and sex-matched left ventricular samples from (1) end-stage failing human hearts and (2) nonfailing human hearts and were able to identify the X-linked inhibitor of apoptosis protein (XIAP) as a novel target for treating cardiac dysfunction. We demonstrate that XIAP is diminished in failing human hearts, indicating that this potent inhibitor of apoptosis may be central in protecting the human heart from cellular injury culminating in heart failure. Efforts to ameliorate heart failure through delivery of XIAP compelled the design of a novel adeno-associated viral (AAV) vector, termed SASTG, that achieves highly efficient transduction in mouse heart and in cultured neonatal rat cardiomyocytes. Increased XIAP expression achieved with the SASTG vector inhibits caspase-3/7 activity in neonatal cardiomyocytes after induction of apoptosis through three common cardiac stresses: protein kinase C-γ inhibition, hypoxia, or β-adrenergic receptor agonist. These studies demonstrate the potential benefit of XIAP to correct heart failure after highly efficient delivery to the heart with the rationally designed SASTG AAV vector.
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Affiliation(s)
- Valentino Piacentino III
- Cardiothoracic Division, Department of Surgery, Duke University Medical Center, Durham, NC 27710
| | - Carmelo A. Milano
- Cardiothoracic Division, Department of Surgery, Duke University Medical Center, Durham, NC 27710
| | - Michael Bolanos
- Cardiothoracic Division, Department of Surgery, Duke University Medical Center, Durham, NC 27710
| | - Jacob Schroder
- Cardiothoracic Division, Department of Surgery, Duke University Medical Center, Durham, NC 27710
| | - Emily Messina
- Cardiothoracic Division, Department of Surgery, Duke University Medical Center, Durham, NC 27710
| | - Adam S. Cockrell
- Carolina Vaccine Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Edward Jones
- Cardiothoracic Division, Department of Surgery, Duke University Medical Center, Durham, NC 27710
| | - Ava Krol
- Department of Biomedical Engineering, Duke University, Durham, NC 27710
| | - Nenad Bursac
- Department of Biomedical Engineering, Duke University, Durham, NC 27710
| | - Lan Mao
- Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, NC 27710
| | - Gayathri R. Devi
- Department of Pathology, Duke University Medical Center, Durham, NC 27710
- Division of Surgical Sciences, Department of Surgery, Duke University Medical Center, Durham, NC 27710
| | - R. Jude Samulski
- Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Dawn E. Bowles
- Division of Surgical Sciences, Department of Surgery, Duke University Medical Center, Durham, NC 27710
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Nguyen Q, Palfreyman RW, Chan LCL, Reid S, Nielsen LK. Transcriptome sequencing of and microarray development for a Helicoverpa zea cell line to investigate in vitro insect cell-baculovirus interactions. PLoS One 2012; 7:e36324. [PMID: 22629315 PMCID: PMC3356360 DOI: 10.1371/journal.pone.0036324] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2012] [Accepted: 03/30/2012] [Indexed: 11/18/2022] Open
Abstract
The Heliothine insect complex contains some of the most destructive pests of agricultural crops worldwide, including the closely related Helicoverpa zea and H. armigera. Biological control using baculoviruses is practiced at a moderate level worldwide. In order to enable more wide spread use, a better understanding of cell-virus interactions is required. While many baculoviruses have been sequenced, none of the Heliothine insect genomes have been available. In this study, we sequenced, assembled and functionally annotated 29,586 transcripts from cultured H. zea cells using Illumina 100 bps and paired-end transcriptome sequencing (RNA-seq). The transcript sequences had high assembly coverage (64.5 times). 23,401 sequences had putative protein functions, and over 13,000 sequences had high similarities to available sequences in other insect species. The sequence database was estimated to cover at least 85% of all H. zea genes. The sequences were used to construct a microarray, which was evaluated on the infection of H. zea cells with H. Armigera single-capsid nucleopolyhedrovirus (HearNPV). The analysis revealed that up-regulation of apoptosis genes is the main cellular response in the early infection phase (18 hours post infection), while genes linked to four major immunological signalling pathways (Toll, IMD, Jak-STAT and JNK) were down-regulated. Only small changes (generally downwards) were observed for central carbon metabolism. The transcriptome and microarray platform developed in this study represent a greatly expanded resource base for H. zea insect- HearNPV interaction studies, in which key cellular pathways such as those for metabolism, immune response, transcription and replication have been identified. This resource will be used to develop better cell culture-based virus production processes, and more generally to investigate the molecular basis of host range and susceptibility, virus infectivity and virulence, and the ecology and evolution of baculoviruses.
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Affiliation(s)
- Quan Nguyen
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia
| | - Robin W. Palfreyman
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia
| | - Leslie C. L. Chan
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia
| | - Steven Reid
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia
- * E-mail:
| | - Lars K. Nielsen
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia
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Muñoz-Pinedo C. Signaling pathways that regulate life and cell death: evolution of apoptosis in the context of self-defense. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2012; 738:124-43. [PMID: 22399377 DOI: 10.1007/978-1-4614-1680-7_8] [Citation(s) in RCA: 79] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Programmed Cell Death is essential for the life cycle of many organisms. Cell death in multicellular organisms can occur as a consequence of massive damage (necrosis) or in a controlled form, through engagement of diverse biochemical programs. The best well known form of programmed cell death is apoptosis. Apoptosis occurs in animals as a consequence of a variety of stimuli including stress and social signals and it plays essential roles in morphogenesis and immune defense. The machinery of apoptosis is well conserved among animals and it is composed of caspases (the proteases which execute cell death), adapter proteins (caspase activators), Bcl-2 family proteins and Inhibitor of Apoptosis Proteins (IAPs). We will describe in this chapter the main apoptotic pathways in animals: the extrinsic (death receptor-mediated), the intrinsic/mitochondrial and the Granzyme B pathway. Other forms of non-apoptotic Programmed Cell Death which occur in animals will also be discussed. We will summarize the current knowledge about apoptotic-like and other forms of cell death in other organisms such as plants and protists.Additionally, we will discuss the hypothesis that apoptosis originated as part of a host defense mechanism. We will explore the similarities between the protein complexes which mediate apoptosis (apoptosomes) and complexes involved in immunity: inflammasomes. Additional functions of apoptotic proteins related to immune function will be summarized, in an effort to explore the evolutionary origins of cell death.
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Labbé K, McIntire CR, Doiron K, Leblanc PM, Saleh M. Cellular inhibitors of apoptosis proteins cIAP1 and cIAP2 are required for efficient caspase-1 activation by the inflammasome. Immunity 2012; 35:897-907. [PMID: 22195745 DOI: 10.1016/j.immuni.2011.10.016] [Citation(s) in RCA: 133] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2011] [Revised: 10/05/2011] [Accepted: 10/21/2011] [Indexed: 01/05/2023]
Abstract
Pathogen and danger recognition by the inflammasome activates inflammatory caspases that mediate inflammation and cell death. The cellular inhibitor of apoptosis proteins (cIAPs) function in apoptosis and innate immunity, but their role in modulating the inflammasome and the inflammatory caspases is unknown. Here we report that the cIAPs are critical effectors of the inflammasome and are required for efficient caspase-1 activation. cIAP1, cIAP2, and the adaptor protein TRAF2 interacted with caspase-1-containing complexes and mediated the activating nondegradative K63-linked polyubiquitination of caspase-1. Deficiency in cIAP1 (encoded by Birc2) or cIAP2 (Birc3) impaired caspase-1 activation after spontaneous or agonist-induced inflammasome assembly, and Birc2(-/-) or Birc3(-/-) mice or mice administered with an IAP antagonist had a dampened response to inflammasome agonists and were resistant to peritonitis. Our results describe a role for the cIAPs in innate immunity and further demonstrate the evolutionary conservation between cell death and inflammation mechanisms.
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Affiliation(s)
- Katherine Labbé
- Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3G 0B1, Canada
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Abstract
Proper embryonic development and normal tissue homeostasis require a series of molecular processes, regulating cell growth, differentiation and apoptosis. Perturbation in any of these processes invariably contributes to the development of cancer. In particular, defects in apoptosis are seen in virtually all types of human cancers. The Notch pathway plays an important role in cell fate determination in both embryonic development and organ homeostasis. Not surprisingly, Notch also plays a role in cancer when it is dysregulated. In this chapter, we will explore how Notch signaling interacts with key pathways that regulate apoptosis in cancer. Particularly, we will focus on the relationship between Notch and proteins responsible for activation of the caspase pathway. Notch regulates apoptosis through extensive networks, involving cell cycle, growth and survival pathways. Thus, we will also examine how apoptosis is modulated by the crosstalk between Notch and other signaling pathways such as p53, NF-κB and PI3K-Akt pathways.
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Thiyagarajan P, Senthil Murugan R, Kavitha K, Anitha P, Prathiba D, Nagini S. Dietary chlorophyllin inhibits the canonical NF-κB signaling pathway and induces intrinsic apoptosis in a hamster model of oral oncogenesis. Food Chem Toxicol 2011; 50:867-76. [PMID: 22210229 DOI: 10.1016/j.fct.2011.12.019] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2011] [Revised: 11/21/2011] [Accepted: 12/13/2011] [Indexed: 01/24/2023]
Abstract
Chlorophyllin, a water-soluble, semi-synthetic derivative of the ubiquitous green pigment chlorophyll is shown to exert potent anticarcinogenic effects. In the present study, we investigated the chemopreventive effects of chlorophyllin on 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis by analyzing the expression of NF-κB family members and markers of intrinsic apoptosis. Dietary administration of chlorophyllin (4 mg/kg bw) suppressed the development of HBP carcinomas by inhibiting the canonical NF-κB signaling pathway by downregulating IKKβ, preventing the phosphorylation of IκB-α, and reducing the expression of nuclear NF-κB. Inactivation of NF-κB signaling by chlorophyllin was associated with the induction of intrinsic apoptosis as evidenced by modulation of Bcl-2 family proteins, enforced nuclear localization of survivin, upregulation of apoptogenic molecules, activation of caspases, and cleavage of PARP. The results of the present study demonstrate that chlorophyllin inhibits the development of DMBA-induced HBP carcinogenesis by targeting NF-κB and the intrinsic apoptotic pathway. Thus, dietary agents such as chlorophyllin that simultaneously target divergent pathways of cell survival and cell death are novel candidates for cancer chemoprevention.
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Affiliation(s)
- P Thiyagarajan
- Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
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Drosophila IAP1-mediated ubiquitylation controls activation of the initiator caspase DRONC independent of protein degradation. PLoS Genet 2011; 7:e1002261. [PMID: 21909282 PMCID: PMC3164697 DOI: 10.1371/journal.pgen.1002261] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2011] [Accepted: 07/06/2011] [Indexed: 02/07/2023] Open
Abstract
Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition (“undead” cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in “undead” cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs. The Drosophila inhibitor of apoptosis 1 (DIAP1) readily promotes ubiquitylation of the CASPASE-9–like initiator caspase DRONC in vitro and in vivo. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by effector caspase inhibition—producing so-called “undead” cells—it has been proposed that DIAP1-mediated ubiquitylation would target full-length DRONC for proteasomal degradation, ensuring survival of normal cells. However, this has never been tested rigorously in vivo. By examining loss and gain of diap1 function, we show that DIAP1-mediated ubiquitylation does not trigger degradation of full-length DRONC. Our analysis demonstrates that DIAP1-mediated ubiquitylation controls DRONC processing and activation in a non-proteolytic manner. Interestingly, once DRONC is processed and activated, it has reduced protein stability. We also demonstrate that “undead” cells induce transcription of dronc, explaining increased protein levels of DRONC in these cells. This study re-defines the mechanism by which IAP-mediated ubiquitylation regulates caspase activity.
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Ikeda M, Yamada H, Ito H, Kobayashi M. Baculovirus IAP1 induces caspase-dependent apoptosis in insect cells. J Gen Virol 2011; 92:2654-2663. [PMID: 21795471 DOI: 10.1099/vir.0.033332-0] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Baculoviruses encode inhibitors of apoptosis (IAPs), which are classified into five groups, IAP1-5, based on their sequence homology. Most of the baculovirus IAPs with anti-apoptotic functions belong to the IAP3 group, with certain exceptions. The functional roles of IAPs from other groups during virus infection have not been well established. We have previously shown that Hyphantria cunea multiple nucleopolyhedrovirus (HycuMNPV) encodes three iap genes, hycu-iap1, hycu-iap2 and hycu-iap3, and that only Hycu-IAP3 has anti-apoptotic activity against actinomycin D-induced apoptosis of Spodoptera frugiperda Sf9 cells. In the present study, we demonstrate that transient expression of Hycu-IAP1 is capable of inducing apoptosis and/or stimulating caspase-3-like protease activity in various lepidopteran and dipteran cell lines. Transient-expression assay analysis also demonstrates that not only Hycu-IAP1 but also IAP1s from Autographa californica MNPV, Bombyx mori NPV and Orgyia pseudotsugata MNPV (OpMNPV) are capable of inducing apoptosis, and that apoptosis induced by Hycu-IAP1 is precluded by the functional anti-apoptotic baculovirus protein Hycu-IAP3. In HycuMNPV-infected Spilosoma imparilis (SpIm) cells and OpMNPV-infected Ld652Y cells, caspase-3-like protease activity is markedly stimulated during the late stages of infection, and the caspase-3-like protease activity stimulated in HycuMNPV-infected SpIm cells is repressed by RNA interference-mediated silencing of hycu-iap1. In addition, initiator caspase Bm-Dronc, the B. mori homologue of Dronc, is cleaved upon transfection of BM-N cells with a plasmid expressing Hycu-IAP1. These results provide the first evidence that baculovirus IAP1s act to induce caspase-dependent apoptosis, possibly by replacing the cellular IAP1 that prevents Dronc activation.
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Affiliation(s)
- Motoko Ikeda
- Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
| | - Hayato Yamada
- Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
| | - Hiroyuki Ito
- Laboratory of Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
| | - Michihiro Kobayashi
- Laboratory of Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
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Active depletion of host cell inhibitor-of-apoptosis proteins triggers apoptosis upon baculovirus DNA replication. J Virol 2011; 85:8348-58. [PMID: 21653668 DOI: 10.1128/jvi.00667-11] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda, respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects.
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