The Experts below are selected from a list of 7149 Experts worldwide ranked by ideXlab platform
Sabrina Büttner - One of the best experts on this subject based on the ideXlab platform.
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Endonuclease G mediates α synuclein cytotoxicity durinG parkinson s disease
The EMBO Journal, 2013Co-Authors: Sabrina Büttner, Doris Ruli, Lukas Habernig, Filomena Broeskamp, M. Vlachos, Francesca Macchi, Victoria Küttner, Nora F Vogtle, Didac CarmonagutierrezAbstract:MalfunctioninG of the protein α-synuclein is critically involved in the demise of dopaminerGic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaininG this pathoGenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triGGers DNA deGradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminerGic neurons of human Parkinson-diseased patients, while EndoG depletion larGely reduces α-synuclein-induced cell death in human neuroblastoma cells. XenoGenic expression of human α-synuclein in yeast cells triGGers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA deGradation throuGh a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven deGeneration of dopaminerGic neurons. Moreover, the locomotion and survival of α-synuclein-expressinG flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phyloGenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.
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Endonuclease G mediates α‐synuclein cytotoxicity durinG Parkinson's disease
The EMBO journal, 2013Co-Authors: Sabrina Büttner, Didac Carmona-gutierrez, Doris Ruli, Lukas Habernig, Filomena Broeskamp, F.-nora Vögtle, M. Vlachos, Francesca Macchi, Victoria Küttner, Tobias EisenbergAbstract:MalfunctioninG of the protein α-synuclein is critically involved in the demise of dopaminerGic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaininG this pathoGenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triGGers DNA deGradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminerGic neurons of human Parkinson-diseased patients, while EndoG depletion larGely reduces α-synuclein-induced cell death in human neuroblastoma cells. XenoGenic expression of human α-synuclein in yeast cells triGGers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA deGradation throuGh a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven deGeneration of dopaminerGic neurons. Moreover, the locomotion and survival of α-synuclein-expressinG flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phyloGenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.
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Depletion of Endonuclease G selectively kills polyploid cells.
Cell cycle (Georgetown Tex.), 2007Co-Authors: Sabrina Büttner, Didac Carmona-gutierrez, Ilio Vitale, Maria Castedo, Doris Ruli, Tobias Eisenberg, Guido Kroemer, Frank MadeoAbstract:Endonuclease G is a mitochondrio-nuclear located nuclease with dual-vital and lethal-functions. Besides its role in apoptosis execution, we have recently shown that depletion of Endonuclease G leads to necrotic cell death in yeast. Here, we present further mechanistic elucidation of Endonuclease G's vital functions. The deletion of the yeast Endonuclease G Gene causes the complete elimination of tetraploid cells durinG exponential Growth. Consistently, conditional knockdown of mammalian Endonuclease G selectively kills tetraploid but not diploid clones of the human HCT116 colon carcinoma cell line. We conclude that Endonuclease G is important for the viability of polyploid mammalian and yeast cells.
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Endonuclease G ReGulates BuddinG Yeast Life and Death
Molecular cell, 2007Co-Authors: Sabrina Büttner, Didac Carmona-gutierrez, Doris Ruli, Tobias Eisenberg, Heide Knauer, Christoph Ruckenstuhl, Carola B. Sigrist, Silke Wissing, Manfred Kollroser, Kai-uwe FröhlichAbstract:Summary Endonuclease G (EndoG) is located in mitochondria yet translocates into the nucleus of apoptotic cells durinG human deGenerative diseases. Nonetheless, a direct involvement of EndoG in cell-death execution remains equivocal, and the mechanism for mitochondrio-nuclear translocation is not known. Here, we show that the yeast homoloG of EndoG (Nuc1p) can efficiently triGGer apoptotic cell death when excluded from mitochondria. Nuc1p induces apoptosis in yeast independently of metacaspase or of apoptosis inducinG factor. Instead, the permeability transition pore, karyopherin Kap123p, and histone H2B interact with Nuc1p and are required for cell death upon Nuc1p overexpression, suGGestinG a pathway in which mitochondrial pore openinG, nuclear import, and chromatin association are successively involved in EndoG-mediated death. Deletion of NUC1 diminishes apoptotic death when mitochondrial respiration is increased but enhances necrotic death when oxidative phosphorylation is repressed, pointinG to dual—lethal and vital—roles for EndoG.
Michal Kozubek - One of the best experts on this subject based on the ideXlab platform.
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Endonuclease G interacts with histone H2B and DNA topoisomerase II alpha durinG apoptosis.
Molecular and cellular biochemistry, 2011Co-Authors: Miroslav Vařecha, Pavel Matula, Michaela Potěšilová, Michal KozubekAbstract:Apoptosis is a natural form of cell death involved in many physioloGical chanGes in the cell. Defects in the process of apoptosis can lead to serious diseases. DurinG some apoptotic pathways, proteins apoptosis-inducinG factor (AIF) and Endonuclease G (EndoG) are released from the mitochondria and they translocate into the cell nuclei, where they probably participate in chromatin deGradation toGether with other nuclear proteins. Exact mechanism of EndoG activity in cell nucleus is still unknown. Some interactinG partners like flap Endonuclease 1, DNase I, and exonuclease III were already suGGested, but also other interactinG partners were proposed. We conducted a livinG-cell confocal fluorescence microscopy followed by an imaGe analysis of fluorescence resonance enerGy transfer to analyze the possibility of protein interactions of EndoG with histone H2B and human DNA topoisomerase II alpha (TOPO2a). Our results show that EndoG interacts with both these proteins durinG apoptotic cell death. Therefore, we can conclude that EndoG and TOPO2a may actively participate in apoptotic chromatin deGradation. The possible existence of a deGradation complex consistinG of EndoG and TOPO2a and possibly other proteins like AIF and cyclophilin A have yet to be investiGated.
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interaction of Endonuclease G with histone h2b aif and dna topoisomerase ii alpha durinG apoptosis as revealed by fret imaGinG of livinG cells
2011Co-Authors: Miroslav Vařecha, Pavel Matula, Michaela Potěsilova, Michal KozubekAbstract:Apoptosis is a natural form of cell death involved in many physioloGical chanGes in the cell. DurinG some forms of cell death, proteins Endonuclease G (EndoG) and apoptosis-inducinG factor (AIF) are released from mitochondria, then they translocate into the cell nuclei, where they participate in chromatin deGradation in a caspase-independent way. We have conducted livinG-cell confocal fluorescence microscopy followed by analysis of fluorescence resonance enerGy transfer (FRET) to observe the protein interaction of EndoG with AIF and their interactions with other proteins in human cell nuclei after induction of apoptosis. Our results show that EndoG interacts with histone H2B, AIF, and DNA topoisomerase II alpha (TOPO2a). Also AIF was found to interact with TOPO2a. Therefore we can conclude that EndoG, AIF, and TOPO2a may form a protein complex allowinG chromatin deGradation in apoptotic nucleus.
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Endonuclease G interacts with histone H2B, AIF, and DNAtopoisomerase II alpha durinG apoptosis as revealed by FRETanalysis of livinG cells
2011Co-Authors: Miroslav Vařecha, Pavel Matula, Michaela Potěšilová, Michal KozubekAbstract:Apoptosis is a natural form of cell death involved in many physioloGical chanGes in the cell. DurinG some forms of cell death, proteins Endonuclease G (EndoG) and apoptosis-inducinG factor (AIF) are released from mitochondria and then they translocate into the cell nuclei, where they participate in chromatin deGradation in a caspase-independent way. The C. eleGans homoloG of AIF was shown to induce apoptosis and to interact with a homoloG of EndoG and toGether they mediated chromatin DNA deGradation. Our results show that EndoG interacts with histone H2B, AIF, and DNA topoisomerase II alpha (TOPO2a). Also AIF was found to interact with TOPO2a. Therefore we can conclude that EndoG, AIF, and TOPO2a may form a protein complex allowinG chromatin deGradation in apoptotic nucleus. These results offer an important insiGht into the mechanism of apoptotic cell death, which plays a major role in development and proGression of deGenerative diseases, cancer, and inflammation.
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Bioinformatic and imaGe analyses of the cellular localization of the apoptotic proteins Endonuclease G, AIF, and AMID durinG apoptosis in human cells
Apoptosis, 2007Co-Authors: Miroslav Vařecha, Jana Amrichová, Michal Zimmermann, Vladimír Ulman, Emilie Lukášová, Michal KozubekAbstract:We studied the cellular localization of the apoptotic proteins Endonuclease G, AIF, and AMID in silico usinG three prediction tools and in livinG cells usinG both sinGle-cell colocalization imaGe analysis and nuclear translocation analysis. We confirmed the mitochondrial localization of Endonuclease G and AIF by prediction analysis and by sinGle-cell colocalization imaGe analysis. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the membranes of various orGanelles. The hiGhest concentration of AMID was observed associated with the GolGi. Colocalization of AMID with lysosomes was also indirectly confirmed by analysis of AMID-rich vesicle velocity usinG manual trackinG analysis. Bioinformatic analysis also detected nuclear localization siGnals in Endonuclease G and AIF, but not in AMID. A novel analysis of time-lapse fluorescence imaGe data durinG staurosporine-induced apoptosis revealed nuclear translocation only for Endonuclease G and AIF.
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Bioinformatic predictions and imaGe analysis of localizationand interactions of Endonuclease G, AIF, and AMID in humancells
2007Co-Authors: Miroslav Vařecha, Jana Amrichová, Michal Zimmermann, Vladimír Ulman, Pavel Matula, Michal KozubekAbstract:DurinG apoptosis several mitochondrial proteins are released. They participate in caspase-independent nuclear DNA deGradation, namely apoptosis-inducinG factor (AIF, also PDCD8 or AIFM1) and Endonuclease G. Another interestinG protein, which is not located inside mitochondria, was expected to act similarly as AIF due to hiGh sequence homoloGy with AIF. This protein is AIF-homoloGous mitochondrion-associated inducer of death (AMID, also PRG3 or AIFM2). We studied the cellular localization and colocalization of proteins AIF, Endonuclease G and AMID experimentally usinG desiGned mammalian expression vectors, which carry Genes encodinG the proteins of interest fused to the fluorescent proteins and usinG bioinformatic predictions, that analyze the amino acid sequence of the proteins with various alGorithms. We also desiGned and applied the novel method of sinGle-cell imaGe analysis of the translocation of the fluorescent proteins into the nucleus. We confirmed the colocalization of AIF and Endonuclease G in the mitochondria of human cells. We also analyzed their translocation from mitochondria to the nucleus durinG apoptosis. AMID was found to be cytoplasmic protein, bound to various cellular surfaces from their cytoplasmic side. Overexpression of fusion protein AMID-HcRed-tandem was not lethal to the cells or mitochondria. We did not observe its translocation into the nucleus durinG staurosporine-induced apoptosis. The possible role of AMID in apoptosis was not observed. Bioinformatic predictions and time-lapse FRET experiments were conducted to analyze the interactions of the studied proteins in livinG and fixed human cells. Our results contribute to the comprehension of localization, interactions and functions of AMID, AIF, and Endonuclease G in human cells.
Piotr Widlak - One of the best experts on this subject based on the ideXlab platform.
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reGulation of the human apoptotic dnase rnase Endonuclease G involvement of hsp70 and atp
Apoptosis, 2005Co-Authors: Magdalena Kalinowska, W. Garncarz, Monika Pietrowska, William T. Garrard, Piotr WidlakAbstract:Endonuclease G (EndoG) is a mitochondrial enzyme that becomes an apoptotic nuclease when released from the mitochondrial intermembrane space. EndoG will diGest either DNA or RNA, but at physioloGical ionic strenGth, RNA is a much more favorable substrate as compared to chromatin. This indicates that EndoG’s major in vivo function(s) may be: (i) an apoptotic RNase, and/or (ii) an apoptotic DNase in the presence of additional co-activators. In the present study we have searched for factors that modulate the activity of human EndoG on DNA substrates. We demonstrate that EndoG forms complexes with AIF and FEN-1 but not with PCNA. InterestinGly, heat shock proteins 70 interact with EndoG and are involved in the reGulation of its activity. Purified Hsp70 prevented stimulation of EndoG DNase activity by other nuclear factors in the ATP-dependent manner.
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Discovery, reGulation, and action of the major apoptotic nucleases DFF40/CAD and Endonuclease G.
Journal of cellular biochemistry, 2005Co-Authors: Piotr Widlak, William T. GarrardAbstract:Toward the end of the 20th and beGinninG of the 21st centuries, clever in vitro biochemical complementation experiments and Genetic screens from the laboratories of XiaodonG WanG, ShiGekazu NaGata, and DinG Xue led to the discovery of two major apoptotic nucleases, termed DNA fraGmentation factor (DFF) or caspase-activated DNase (CAD) and Endonuclease G (Endo G). Both Endonucleases attack chromatin to yield 3′-hydroxyl Groups and 5′-phosphate residues, first at the level of 50–300 kb cleavaGe products and next at the level of internucleosomal DNA fraGmentation, but these nucleases possess completely different cellular locations in normal cells and are reGulated in vastly different ways. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45) [also called inhibitor of CAD (ICAD-L)] and a 40 kD latent nuclease subunit (DFF40/CAD). Apoptotic activation of caspase-3 or -7 results in the cleavaGe of DFF45/ICAD and release of active DFF40/CAD nuclease. DFF40's nuclease activity is further activated by specific chromosomal proteins, such as histone H1, HMGB1/2, and topoisomerase II. DFF is reGulated by multiple pre- and post-activation fail-safe steps, which include the requirements for DFF45/ICAD, Hsp70, and Hsp40 proteins to mediate appropriate foldinG durinG translation to Generate a potentially activatable nuclease, and the synthesis in stoichiometric excess of the inhibitors (DFF45/35; ICAD-S/L). By contrast, Endo G resides in the mitochondrial intermembrane space in normal cells, and is released into the nucleus upon apoptotic disruption of mitochondrial membrane permeability in association with co-activators such as apoptosis-inducinG factor (AIF). UnderstandinG further reGulatory check-points involved in safeGuardinG non-apoptotic cells aGainst accidental activation of these nucleases remain as future challenGes, as well as desiGninG ways to selectively activate these nucleases in tumor cells. © 2005 Wiley-Liss, Inc.
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discovery reGulation and action of the major apoptotic nucleases dff40 cad and Endonuclease G
Journal of Cellular Biochemistry, 2005Co-Authors: Piotr Widlak, William T. GarrardAbstract:Toward the end of the 20th and beGinninG of the 21st centuries, clever in vitro biochemical complementation experiments and Genetic screens from the laboratories of XiaodonG WanG, ShiGekazu NaGata, and DinG Xue led to the discovery of two major apoptotic nucleases, termed DNA fraGmentation factor (DFF) or caspase-activated DNase (CAD) and Endonuclease G (Endo G). Both Endonucleases attack chromatin to yield 3′-hydroxyl Groups and 5′-phosphate residues, first at the level of 50–300 kb cleavaGe products and next at the level of internucleosomal DNA fraGmentation, but these nucleases possess completely different cellular locations in normal cells and are reGulated in vastly different ways. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45) [also called inhibitor of CAD (ICAD-L)] and a 40 kD latent nuclease subunit (DFF40/CAD). Apoptotic activation of caspase-3 or -7 results in the cleavaGe of DFF45/ICAD and release of active DFF40/CAD nuclease. DFF40's nuclease activity is further activated by specific chromosomal proteins, such as histone H1, HMGB1/2, and topoisomerase II. DFF is reGulated by multiple pre- and post-activation fail-safe steps, which include the requirements for DFF45/ICAD, Hsp70, and Hsp40 proteins to mediate appropriate foldinG durinG translation to Generate a potentially activatable nuclease, and the synthesis in stoichiometric excess of the inhibitors (DFF45/35; ICAD-S/L). By contrast, Endo G resides in the mitochondrial intermembrane space in normal cells, and is released into the nucleus upon apoptotic disruption of mitochondrial membrane permeability in association with co-activators such as apoptosis-inducinG factor (AIF). UnderstandinG further reGulatory check-points involved in safeGuardinG non-apoptotic cells aGainst accidental activation of these nucleases remain as future challenGes, as well as desiGninG ways to selectively activate these nucleases in tumor cells. © 2005 Wiley-Liss, Inc.
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ReGulation of the human apoptotic DNase/RNase Endonuclease G: involvement of Hsp70 and ATP.
Apoptosis : an international journal on programmed cell death, 2005Co-Authors: Magdalena Kalinowska, W. Garncarz, Monika Pietrowska, William T. Garrard, Piotr WidlakAbstract:Endonuclease G (EndoG) is a mitochondrial enzyme that becomes an apoptotic nuclease when released from the mitochondrial intermembrane space. EndoG will diGest either DNA or RNA, but at physioloGical ionic strenGth, RNA is a much more favorable substrate as compared to chromatin. This indicates that EndoG’s major in vivo function(s) may be: (i) an apoptotic RNase, and/or (ii) an apoptotic DNase in the presence of additional co-activators. In the present study we have searched for factors that modulate the activity of human EndoG on DNA substrates. We demonstrate that EndoG forms complexes with AIF and FEN-1 but not with PCNA. InterestinGly, heat shock proteins 70 interact with EndoG and are involved in the reGulation of its activity. Purified Hsp70 prevented stimulation of EndoG DNase activity by other nuclear factors in the ATP-dependent manner.
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Action of recombinant human apoptotic Endonuclease G on naked DNA and chromatin substrates: cooperation with exonuclease and DNase I.
The Journal of biological chemistry, 2001Co-Authors: Piotr Widlak, Xiaodong Wang, William T. GarrardAbstract:Abstract Endonuclease G (endoG) is released from mitochondria durinG apoptosis and is in part responsible for internucleosomal DNA cleavaGe. Here we report the action of the purified human recombinant form of this Endonuclease on naked DNA and chromatin substrates. The addition of the protein to isolated nuclei from non-apoptotic cells first induces hiGher order chromatin cleavaGe into DNA fraGments ≥ 50 kb in lenGth, followed by inter- and intranucleosomal DNA cleavaGes with products possessinG siGnificant internal sinGle-stranded nicks spaced at nucleosomal (∼190 bases) and subnucleosomal (∼10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of endoG to Generate double-stranded DNA cleavaGe products at physioloGical ionic strenGths, suGGestinG that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown.
Tobias Eisenberg - One of the best experts on this subject based on the ideXlab platform.
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Endonuclease G mediates α‐synuclein cytotoxicity durinG Parkinson's disease
The EMBO journal, 2013Co-Authors: Sabrina Büttner, Didac Carmona-gutierrez, Doris Ruli, Lukas Habernig, Filomena Broeskamp, F.-nora Vögtle, M. Vlachos, Francesca Macchi, Victoria Küttner, Tobias EisenbergAbstract:MalfunctioninG of the protein α-synuclein is critically involved in the demise of dopaminerGic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaininG this pathoGenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triGGers DNA deGradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminerGic neurons of human Parkinson-diseased patients, while EndoG depletion larGely reduces α-synuclein-induced cell death in human neuroblastoma cells. XenoGenic expression of human α-synuclein in yeast cells triGGers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA deGradation throuGh a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven deGeneration of dopaminerGic neurons. Moreover, the locomotion and survival of α-synuclein-expressinG flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phyloGenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.
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Depletion of Endonuclease G selectively kills polyploid cells.
Cell cycle (Georgetown Tex.), 2007Co-Authors: Sabrina Büttner, Didac Carmona-gutierrez, Ilio Vitale, Maria Castedo, Doris Ruli, Tobias Eisenberg, Guido Kroemer, Frank MadeoAbstract:Endonuclease G is a mitochondrio-nuclear located nuclease with dual-vital and lethal-functions. Besides its role in apoptosis execution, we have recently shown that depletion of Endonuclease G leads to necrotic cell death in yeast. Here, we present further mechanistic elucidation of Endonuclease G's vital functions. The deletion of the yeast Endonuclease G Gene causes the complete elimination of tetraploid cells durinG exponential Growth. Consistently, conditional knockdown of mammalian Endonuclease G selectively kills tetraploid but not diploid clones of the human HCT116 colon carcinoma cell line. We conclude that Endonuclease G is important for the viability of polyploid mammalian and yeast cells.
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Endonuclease G ReGulates BuddinG Yeast Life and Death
Molecular cell, 2007Co-Authors: Sabrina Büttner, Didac Carmona-gutierrez, Doris Ruli, Tobias Eisenberg, Heide Knauer, Christoph Ruckenstuhl, Carola B. Sigrist, Silke Wissing, Manfred Kollroser, Kai-uwe FröhlichAbstract:Summary Endonuclease G (EndoG) is located in mitochondria yet translocates into the nucleus of apoptotic cells durinG human deGenerative diseases. Nonetheless, a direct involvement of EndoG in cell-death execution remains equivocal, and the mechanism for mitochondrio-nuclear translocation is not known. Here, we show that the yeast homoloG of EndoG (Nuc1p) can efficiently triGGer apoptotic cell death when excluded from mitochondria. Nuc1p induces apoptosis in yeast independently of metacaspase or of apoptosis inducinG factor. Instead, the permeability transition pore, karyopherin Kap123p, and histone H2B interact with Nuc1p and are required for cell death upon Nuc1p overexpression, suGGestinG a pathway in which mitochondrial pore openinG, nuclear import, and chromatin association are successively involved in EndoG-mediated death. Deletion of NUC1 diminishes apoptotic death when mitochondrial respiration is increased but enhances necrotic death when oxidative phosphorylation is repressed, pointinG to dual—lethal and vital—roles for EndoG.
Miroslav Vařecha - One of the best experts on this subject based on the ideXlab platform.
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Endonuclease G interacts with histone H2B and DNA topoisomerase II alpha durinG apoptosis.
Molecular and cellular biochemistry, 2011Co-Authors: Miroslav Vařecha, Pavel Matula, Michaela Potěšilová, Michal KozubekAbstract:Apoptosis is a natural form of cell death involved in many physioloGical chanGes in the cell. Defects in the process of apoptosis can lead to serious diseases. DurinG some apoptotic pathways, proteins apoptosis-inducinG factor (AIF) and Endonuclease G (EndoG) are released from the mitochondria and they translocate into the cell nuclei, where they probably participate in chromatin deGradation toGether with other nuclear proteins. Exact mechanism of EndoG activity in cell nucleus is still unknown. Some interactinG partners like flap Endonuclease 1, DNase I, and exonuclease III were already suGGested, but also other interactinG partners were proposed. We conducted a livinG-cell confocal fluorescence microscopy followed by an imaGe analysis of fluorescence resonance enerGy transfer to analyze the possibility of protein interactions of EndoG with histone H2B and human DNA topoisomerase II alpha (TOPO2a). Our results show that EndoG interacts with both these proteins durinG apoptotic cell death. Therefore, we can conclude that EndoG and TOPO2a may actively participate in apoptotic chromatin deGradation. The possible existence of a deGradation complex consistinG of EndoG and TOPO2a and possibly other proteins like AIF and cyclophilin A have yet to be investiGated.
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interaction of Endonuclease G with histone h2b aif and dna topoisomerase ii alpha durinG apoptosis as revealed by fret imaGinG of livinG cells
2011Co-Authors: Miroslav Vařecha, Pavel Matula, Michaela Potěsilova, Michal KozubekAbstract:Apoptosis is a natural form of cell death involved in many physioloGical chanGes in the cell. DurinG some forms of cell death, proteins Endonuclease G (EndoG) and apoptosis-inducinG factor (AIF) are released from mitochondria, then they translocate into the cell nuclei, where they participate in chromatin deGradation in a caspase-independent way. We have conducted livinG-cell confocal fluorescence microscopy followed by analysis of fluorescence resonance enerGy transfer (FRET) to observe the protein interaction of EndoG with AIF and their interactions with other proteins in human cell nuclei after induction of apoptosis. Our results show that EndoG interacts with histone H2B, AIF, and DNA topoisomerase II alpha (TOPO2a). Also AIF was found to interact with TOPO2a. Therefore we can conclude that EndoG, AIF, and TOPO2a may form a protein complex allowinG chromatin deGradation in apoptotic nucleus.
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Endonuclease G interacts with histone H2B, AIF, and DNAtopoisomerase II alpha durinG apoptosis as revealed by FRETanalysis of livinG cells
2011Co-Authors: Miroslav Vařecha, Pavel Matula, Michaela Potěšilová, Michal KozubekAbstract:Apoptosis is a natural form of cell death involved in many physioloGical chanGes in the cell. DurinG some forms of cell death, proteins Endonuclease G (EndoG) and apoptosis-inducinG factor (AIF) are released from mitochondria and then they translocate into the cell nuclei, where they participate in chromatin deGradation in a caspase-independent way. The C. eleGans homoloG of AIF was shown to induce apoptosis and to interact with a homoloG of EndoG and toGether they mediated chromatin DNA deGradation. Our results show that EndoG interacts with histone H2B, AIF, and DNA topoisomerase II alpha (TOPO2a). Also AIF was found to interact with TOPO2a. Therefore we can conclude that EndoG, AIF, and TOPO2a may form a protein complex allowinG chromatin deGradation in apoptotic nucleus. These results offer an important insiGht into the mechanism of apoptotic cell death, which plays a major role in development and proGression of deGenerative diseases, cancer, and inflammation.
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Bioinformatic and imaGe analyses of the cellular localization of the apoptotic proteins Endonuclease G, AIF, and AMID durinG apoptosis in human cells
Apoptosis, 2007Co-Authors: Miroslav Vařecha, Jana Amrichová, Michal Zimmermann, Vladimír Ulman, Emilie Lukášová, Michal KozubekAbstract:We studied the cellular localization of the apoptotic proteins Endonuclease G, AIF, and AMID in silico usinG three prediction tools and in livinG cells usinG both sinGle-cell colocalization imaGe analysis and nuclear translocation analysis. We confirmed the mitochondrial localization of Endonuclease G and AIF by prediction analysis and by sinGle-cell colocalization imaGe analysis. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the membranes of various orGanelles. The hiGhest concentration of AMID was observed associated with the GolGi. Colocalization of AMID with lysosomes was also indirectly confirmed by analysis of AMID-rich vesicle velocity usinG manual trackinG analysis. Bioinformatic analysis also detected nuclear localization siGnals in Endonuclease G and AIF, but not in AMID. A novel analysis of time-lapse fluorescence imaGe data durinG staurosporine-induced apoptosis revealed nuclear translocation only for Endonuclease G and AIF.
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Bioinformatic predictions and imaGe analysis of localizationand interactions of Endonuclease G, AIF, and AMID in humancells
2007Co-Authors: Miroslav Vařecha, Jana Amrichová, Michal Zimmermann, Vladimír Ulman, Pavel Matula, Michal KozubekAbstract:DurinG apoptosis several mitochondrial proteins are released. They participate in caspase-independent nuclear DNA deGradation, namely apoptosis-inducinG factor (AIF, also PDCD8 or AIFM1) and Endonuclease G. Another interestinG protein, which is not located inside mitochondria, was expected to act similarly as AIF due to hiGh sequence homoloGy with AIF. This protein is AIF-homoloGous mitochondrion-associated inducer of death (AMID, also PRG3 or AIFM2). We studied the cellular localization and colocalization of proteins AIF, Endonuclease G and AMID experimentally usinG desiGned mammalian expression vectors, which carry Genes encodinG the proteins of interest fused to the fluorescent proteins and usinG bioinformatic predictions, that analyze the amino acid sequence of the proteins with various alGorithms. We also desiGned and applied the novel method of sinGle-cell imaGe analysis of the translocation of the fluorescent proteins into the nucleus. We confirmed the colocalization of AIF and Endonuclease G in the mitochondria of human cells. We also analyzed their translocation from mitochondria to the nucleus durinG apoptosis. AMID was found to be cytoplasmic protein, bound to various cellular surfaces from their cytoplasmic side. Overexpression of fusion protein AMID-HcRed-tandem was not lethal to the cells or mitochondria. We did not observe its translocation into the nucleus durinG staurosporine-induced apoptosis. The possible role of AMID in apoptosis was not observed. Bioinformatic predictions and time-lapse FRET experiments were conducted to analyze the interactions of the studied proteins in livinG and fixed human cells. Our results contribute to the comprehension of localization, interactions and functions of AMID, AIF, and Endonuclease G in human cells.