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Xiaodong Wang - One of the best experts on this subject based on the ideXlab platform.
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Structure of an Apoptosome-Procaspase-9 CARD Complex
Structure (London England : 1993), 2010Co-Authors: Shujun Yuan, Xiaodong Wang, Maya Topf, Steven J. Ludtke, Christopher W AkeyAbstract:Summary Apaf-1 coassembles with cytochrome c to form the apoptosome, which then binds and activates procaspase-9 (pc-9). We removed pc-9 catalytic domains from the holoapoptosome by site-directed thrombinolysis. A structure of the resulting apoptosome-pc-9 CARD complex was then determined at ∼9.5 A resolution. In our model, the central hub is constructed like other AAA+ protein rings but also contains novel features. At higher radius, the regulatory region of each Apaf-1 is comprised of tandem seven and eight blade β-propellers with cytochrome c docked between them. Remarkably, Apaf-1 CARDs are disordered in the ground state. During activation, each Apaf-1 CARD interacts with a pc-9 CARD and these heterodimers form a flexibly tethered "disk" that sits above the central hub. When taken together, the data reveal conformational changes during Apaf-1 assembly that allow pc-9 activation. The model also provides a plausible explanation for the effects of NOD mutations that have been mapped onto the central hub.
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phapi cas and hsp70 promote apoptosome formation by preventing apaf 1 aggregation and enhancing nucleotide exchange on apaf 1
Molecular Cell, 2008Co-Authors: Hyuneui Kim, Xuejun Jiang, Xiaodong WangAbstract:During apoptosis, cytochrome c is released from mitochondria to the cytosol, where it binds Apaf-1. The Apaf-1/cytochrome c complex then oligomerizes either into heptameric caspase-9-activating apoptosome, which subsequently activates caspase-3 and caspase-7, or bigger inactive aggregates, depending on the availability of nucleotide dATP/ATP. A tumor suppressor protein, PHAPI, enhances caspase-9 activation by promoting apoptosome formation through an unknown mechanism. We report here the identification of cellular apoptosis susceptibility protein (CAS) and heat shock protein 70 (Hsp70) as mediators of PHAPI activity. PHAPI, CAS, and Hsp70 function together to accelerate nucleotide exchange on Apaf-1 and prevent inactive Apaf-1/cytochrome c aggregation. CAS expression is induced by multiple apoptotic stimuli including UV irradiation. Knockdown of CAS by RNA interference (RNAi) in cells attenuates apoptosis induced by UV light and causes endogenous Apaf-1 to form aggregates. These studies indicated that PHAPI, CAS, and Hsp70 play an important regulatory role during apoptosis.
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Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death
Development (Cambridge England), 2006Co-Authors: Fatih Akdemir, Xiaodong Wang, Robert Farkaš, Po Chen, Gábor Juhász, Lucia Medved'ová, Miklós Sass, Lai Wang, Suganthi Chittaranjan, Sharon M. GorskiAbstract:Histolysis refers to a widespread disintegration of tissues that is morphologically distinct from apoptosis and often associated with the stimulation of autophagy. Here, we establish that a component of the apoptosome, and pivotal regulator of apoptosis, is also required for histolytic cell death. Using in vivo and ex vivo assays, we demonstrate a global apoptogenic requirement for dark , the fly ortholog of APAF1, and show that a required focus of dark - organismal lethality maps to the central nervous system. We further demonstrate that the Dark protein itself is a caspase substrate and find that alterations of this cleavage site produced the first hypermorphic point mutation within the APAF1/Ced-4 gene family. In a model of `autophagic cell death9, dark was essential for histolysis but dispensable for characteristic features of the autophagic program, indicating that the induction of autophagy occurs upstream or parallel to histolytic cell death. These results demonstrate that stimulation of autophagy per se is not a `killing event9 and, at the same time, establish that common effector pathways, regulated by the apoptosome, can underlie morphologically distinct forms of programmed cell death.
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formation of apoptosome is initiated by cytochrome c induced datp hydrolysis and subsequent nucleotide exchange on apaf 1
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Hyuneui Kim, Min Fang, Xiaodong WangAbstract:Apoptosis in metazoans is executed by a group of intracellular proteases named caspases. One of the caspase-activating pathways in mammals is initiated by the release of cytochrome c from mitochondria to cytosol, where it binds to Apaf-1 to form a procaspase-9-activating heptameric protein complex named apoptosome. We report here the reconstitution of this pathway with purified recombinant Apaf-1, procaspase-9, procaspase-3, and cytochrome c from horse heart. Apaf-1 contains a dATP as a cofactor. Cytochrome c binding to Apaf-1 induces hydrolysis of dATP to dADP, which is subsequently replaced by exogenous dATP. The dATP hydrolysis and exchange on Apaf-1 are two required steps for apoptosome formation.
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cytochrome c promotes caspase 9 activation by inducing nucleotide binding to apaf 1
Journal of Biological Chemistry, 2000Co-Authors: Xuejun Jiang, Xiaodong WangAbstract:We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c. The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1. cytochrome c complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochrome c-dependent manner. The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, ADPCP (beta,gamma-methylene adenosine 5'-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1.
Yigong Shi - One of the best experts on this subject based on the ideXlab platform.
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Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.
Genes & development, 2015Co-Authors: Mengying Zhou, Chuangye Yan, Xiao Chen Bai, Weiyun Huang, Sjors H.w. Scheres, Yigong ShiAbstract:The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 A resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.
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Molecular determinants of caspase-9 activation by the Apaf-1 apoptosome
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Wen Chen, Zhen Yan, Chuangye Yan, Qionglin Liang, Yigong ShiAbstract:Autocatalytic activation of an initiator caspase triggers the onset of apoptosis. In dying cells, caspase-9 activation is mediated by a multimeric adaptor complex known as the Apaf-1 apoptosome. The molecular mechanism by which caspase-9 is activated by the Apaf-1 apoptosome remains largely unknown. Here we demonstrate that the previously reported 1:1 interaction between Apaf-1 caspase recruitment domain (CARD) and caspase-9 CARD is insufficient for the activation of caspase-9. Rather, formation of a multimeric CARD:CARD assembly between Apaf-1 and caspase-9, which requires three types of distinct interfaces, underlies caspase-9 activation. Importantly, an additional surface area on the multimeric CARD assembly is essential for caspase-9 activation. Together, these findings reveal mechanistic insights into the activation of caspase-9 by the Apaf-1 apoptosome and support the induced conformation model for initiator caspase activation by adaptor complexes.
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Calcium blocks formation of apoptosome by preventing nucleotide exchange in Apaf-1.
Molecular cell, 2007Co-Authors: Qing Bao, Joshua D. Rabinowitz, Yigong ShiAbstract:Apaf-1 plays an essential role in apoptosis. In the presence of cytochrome c and dATP, Apaf-1 assembles into an oligomeric apoptosome, which is responsible for the activation of procaspase-9 and the maintenance of the enzymatic activity of the processed caspase-9. Regulation of apoptosome assembly by other cellular factors is poorly understood. Here we report that physiological concentrations of calcium ion negatively affect the assembly of apoptosome by inhibiting nucleotide exchange in the monomeric, autoinhibited Apaf-1 protein. Consequently, calcium blocks the ability of Apaf-1 to activate caspase-9. These observations suggest an important role of calcium homeostasis on the Apaf-1-dependent apoptotic pathway.
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structure of the apoptotic protease activating factor 1 bound to adp
Nature, 2005Co-Authors: Stefan J Riedl, Yang Chao, Robert Schwarzenbacher, Yigong ShiAbstract:Apoptosis is executed by caspases, which undergo proteolytic activation in response to cell death stimuli. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation downstream of mitochondria. During apoptosis, Apaf-1 binds to cytochrome c and in the presence of ATP/dATP forms an apoptosome, leading to the recruitment and activation of the initiator caspase, caspase-9 (ref. 2). The mechanisms underlying Apaf-1 function are largely unknown. Here we report the 2.2-A crystal structure of an ADP-bound, WD40-deleted Apaf-1, which reveals the molecular mechanism by which Apaf-1 exists in an inactive state before ATP binding. The amino-terminal caspase recruitment domain packs against a three-layered alpha/beta fold, a short helical motif and a winged-helix domain, resulting in the burial of the caspase-9-binding interface. The deeply buried ADP molecule serves as an organizing centre to strengthen interactions between these four adjoining domains, thus locking Apaf-1 in an inactive conformation. Apaf-1 binds to and hydrolyses ATP/dATP and their analogues. The binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of caspase-9.
Emad S. Alnemri - One of the best experts on this subject based on the ideXlab platform.
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negative regulation of cytochrome c mediated oligomerization of apaf 1 and activation of procaspase 9 by heat shock protein 90
The EMBO Journal, 2000Co-Authors: Pramod Pandey, Srinivasa M. Srinivasula, Emad S. Alnemri, Ayman Saleh, Atsuko Nakazawa, Shailendra Kumar, Vijay Kumar, Ralph R Weichselbaum, Carlo Nalin, Donald KufeAbstract:The release of cytochrome c from mitochondria results in the formation of an Apaf-1–caspase-9 apoptosome and induces the apoptotic protease cascade by activation of procaspase-3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf-1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf-1 and thereby inhibits cytochrome c-mediated activation of caspase-9. Addition of purified Apaf-1 to Hsp90-depleted cytosolic extracts restores cytochrome c-mediated activation of procaspase-9. We also show that Hsp90 inhibits cytochrome c-mediated oligomerization of Apaf-1 and thereby activation of procaspase-9. Furthermore, treatment of cells with diverse DNA-damaging agents dissociates the Hsp90–Apaf-1 complex and relieves the inhibition of procaspase-9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c-dependent apoptosis and for involvement of a chaperone in the caspase cascade.
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Negative regulation of the Apaf-1 apoptosome by Hsp70
Nature cell biology, 2000Co-Authors: Ayman Saleh, Srinivasa M. Srinivasula, Levent Balkir, Paul D. Robbins, Emad S. AlnemriAbstract:Release of cytochrome c from mitochondria by apoptotic signals induces ATP/dATP-dependent formation of the oligomeric Apaf-1-caspase-9 apoptosome. Here we show that the documented anti-apoptotic effect of the principal heat-shock protein, Hsp70, is mediated through its direct association with the caspase-recruitment domain (CARD) of Apaf-1 and through inhibition of apoptosome formation. The interaction between Hsp70 and Apaf-1 prevents oligomerization of Apaf-1 and association of Apaf-1 with procaspase-9. On the basis of these results, we propose that resistance to apoptosis exhibited by stressed cells and some tumours, which constitutively express high levels of Hsp70, may be due in part to modulation of Apaf-1 function by Hsp70.
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cytochrome c and datp mediated oligomerization of apaf 1 is a prerequisite for procaspase 9 activation
Journal of Biological Chemistry, 1999Co-Authors: Ayman Saleh, Srinivasa M. Srinivasula, Samir Acharya, Richard Fishel, Emad S. AlnemriAbstract:Abstract To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we produced recombinant full-length Apaf-1 and purified it to complete homogeneity. Here we show using gel filtration that full-length Apaf-1 exists as a monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochromec in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochromec-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5′-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex.
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Autoactivation of Procaspase-9 by Apaf-1-Mediated Oligomerization
Molecular cell, 1998Co-Authors: Srinivasa M. Srinivasula, Manzoor Ahmad, Teresa Fernandes-alnemri, Emad S. AlnemriAbstract:Abstract Activation of procaspase-9 by Apaf-1 in the cytochrome c/dATP–dependent pathway requires proteolytic cleavage to generate the mature caspase molecule. To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we designed an in vitro Apaf-1–procaspase-9 activation system using recombinant components. Here, we show that deletion of the Apaf-1 WD-40 repeats makes Apaf-1 constitutively active and capable of processing procaspase-9 independent of cytochrome c and dATP. Apaf-1-mediated processing of procaspase-9 occurs at Asp-315 by an intrinsic autocatalytic activity of procaspase-9 itself. We provide evidence that Apaf-1 can form oligomers and may facilitate procaspase-9 autoactivation by oligomerizing its precursor molecules. Once activated, caspase-9 can initiate a caspase cascade involving the downstream executioners caspase-3, -6, and -7.
Gabriel Nunez - One of the best experts on this subject based on the ideXlab platform.
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Expression and Functional Analysis of Apaf-1 Isoforms: EXTRA WD-40 REPEAT IS REQUIRED FOR CYTOCHROME cBINDING AND REGULATED ACTIVATION OF PROCASPASE-9
The Journal of biological chemistry, 2000Co-Authors: Mary A Benedict, Naohiro Inohara, Gabriel NunezAbstract:Abstract Apaf-1 is an important apoptotic signaling molecule that can activate procaspase-9 in a cytochromec/dATP-dependent fashion. Alternative splicing can create an NH2-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate procaspase-9 remain poorly characterized. We performed analysis of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms and identified Apaf-1XL, containing both the NH2-terminal and COOH-terminal inserts, as the major RNA form expressed in all tissues tested. We also identified another expressed isoform, Apaf-1LN, containing the NH2-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated procaspase 9 in vitro in response to cytochrome c and dATP, while the NH2-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate procaspase-9 in response to cytochrome c and dATP.
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role of cytochrome c and datp atp hydrolysis in apaf 1 mediated caspase 9 activation and apoptosis
The EMBO Journal, 1999Co-Authors: Mary A Benedict, Liyun Ding, Gabriel NunezAbstract:Apaf-1 plays a critical role in apoptosis by binding to and activating procaspase-9. We have identified a novel Apaf-1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activated procaspase9 in a cytochrome c and dATP/ATP-dependent manner. We used this Apaf-1 to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to procaspase-9. A P-loop mutant (Apaf-1K160R) was unable to associate with Apaf-1 or bind to procaspase-9. Mutation of Met368 to Leu enabled Apaf-1 to self-associate and bind procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibited greater ability to induce apoptosis compared with the wild-type Apaf-1. We also show that procaspase-9 can recruit procaspase3 to the Apaf-1‐procaspase-9 complex. Apaf-1(1‐570), a mutant lacking the WD-40 repeats, associated with and activated procaspase-9, but failed to recruit procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in procaspase-9-mediated procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate procaspase-9 and induce apoptosis.
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WD-40 Repeat Region Regulates Apaf-1 Self-association and Procaspase-9 Activation
The Journal of biological chemistry, 1998Co-Authors: Liyun Ding, David M. Spencer, Gabriel NunezAbstract:The casp9 protein plays a critical role in apoptosis induced by a variety of death stimuli. A regulator of apoptosis, Apaf-1, binds to and activates pro-casp9 in the presence of cytochrome c and dATP, a requirement that is bypassed by deletion of the WD-40 repeats located in the C-terminal half of Apaf-1. In this report, we used constitutively active Apaf-1 mutant lacking the WD-40 repeat region to study the mechanism and regulation of pro-casp9 activation. Mutational analysis revealed that only a small portion of the CED-4 homologous region (residues 456–559) could be deleted without destroying the ability of Apaf-1-(1–559) to activate pro-casp9. Apaf-1 can self-associate to form oligomers. Disruption of Apaf-1 self-association by deletion (Δ109–559) or mutation of the P-loop region (K149R) abrogated Apaf-1-mediated pro-casp9 activation. Forced oligomerization of the caspase recruitment domain of Apaf-1 was sufficient for pro-casp9 activation. Dimerization of chimeric Fpk-pro-casp9 protein with the dimerizer drug FK1012 induced pro-casp9 processing and apoptosis in cells. Significantly, the C-terminal region containing WD-40 repeats interacted with its N-terminal CED-4 homologous region, as determined by immunoprecipitation experiments. Importantly, expression of the WD-40 repeat region inhibited Apaf-1 self-association and proteolytic activation of pro-casp9. These studies provide a mechanism by which Apaf-1 promotes autoactivation of pro-casp9 through Apaf-1 self-association, a process that is negatively regulated by the WD-40 repeats.
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bcl xl interacts with apaf 1 and inhibits apaf 1 dependent caspase 9 activation
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Mary A Benedict, Gabriel NunezAbstract:Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.
Hiroki Yoshida - One of the best experts on this subject based on the ideXlab platform.
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APAF1 plays a negative regulatory role in T cell responses by suppressing activation of antigen-stimulated T cells
2018Co-Authors: Honglian Tong, Yasunobu Miyake, Fumika Mi-ichi, Yoichiro Iwakura, Hiromitsu Hara, Hiroki YoshidaAbstract:APAF1 is a critical component of the apoptosome and initiates apoptosis downstream mitochondrial damages. Although the importance of APAF1 in embryonic development was shown, the role of APAF1 in immune responses, especially T cell responses, has yet to be elucidated. We generated T cell-specific APAF1-deficient mice (Lck-Cre-APAF1f/f mice) and examined the antigen-specific delayed-type hypersensitivity (DTH). Lck-Cre-APAF1f/f mice exhibited exacerbation of DTH responses as compared with APAF1-sufficient control mice. In Lck-Cre-APAF1f/f mice, antigen-specific T cells proliferated more, and produced more inflammatory cytokines than control T cells. APAF1-deficient T cells from antigen-immunized mice showed higher percentages of activation phenotypes upon restimulation in vitro. APAF1-deficient T cells from naive (non-immunized) mice also showed higher proliferation activity and cytokine production over control cells. The impact of APAF1-deficiency in T cells, however, was not restored by a pan-caspase inhibitor, suggesting that the role of APAF1 in T cell responses was caspase-independent/non-apoptotic. These data collectively demonstrated that APAF1 is a negative regulator of T cell responses and implicated APAF1 as a potential target for immunosuppressive drug discovery.
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Generation of T cell-specific APAF1-deficient mice.
2018Co-Authors: Honglian Tong, Yasunobu Miyake, Fumika Mi-ichi, Yoichiro Iwakura, Hiromitsu Hara, Hiroki YoshidaAbstract:(A) Southern blot analysis of genomic DNA from APAF1f-Neo/+ thymocytes (left), APAF1f/+ thymocytes (right), and APAF1f/+ ES cells, in which Neo gene was removed by transient expression of FLPe recombinase (middle). DNA was digested with EcoRI and detected with the probe at exon 4. (See S1 Fig for detail.) (B) Western blot analysis of proteins from LN T cells and thymocytes of APAF1f/f mice and Lck-Cre-APAF1f/f mice. (C) Thymocytes from APAF1f/f mice (open columns) or Lck-Cre-APAF1f/f mice (closed columns) were stimulated with indicated doses of anti-Fas antibodies plus cycloheximide (αFas + CHX), dexamethasone (Dex), staurosporine (Stauro), γ-irradiation, or left untreated. Apoptotic cells were detected by flow cytometry. (D) Purified T cells from LN of APAF1f/f (open columns) or Lck-Cre-APAF1f/f (closed columns) were activated for 48 hours with anti-CD3ε antibody plus anti-CD28 antibody. Activated cells, after removal of dead cells, were cultured in the presence of conditioned medium (CM), in the fresh medium for growth factor deprivation (Media), or re-stimulated with anti-CD3ε antibody in fresh medium for activation-induced cell death, for 20 hours. Apoptotic cells were detected by flow cytometry. Data show means + SD of triplicated samples. Experiments were repeated three times with similar results. *; p
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Caspase-independent role of APAF1 in T cell activation.
2018Co-Authors: Honglian Tong, Yasunobu Miyake, Fumika Mi-ichi, Yoichiro Iwakura, Hiromitsu Hara, Hiroki YoshidaAbstract:LN cells from immunized APAF1f/f-OTII or Lck-Cre-APAF1f/f -OTII mice were stimulated with indicated doses of OVA or anti-CD3ε antibody in the presence or absence of z-VAD-fmk (z-VAD) for 48 hours. (A) Cell proliferation, cell viability (Annexin V-negative and PI-negative), production of IFN-γ and IL-17, or expression of CD69, CD44, and CD62L were analyzed. Open columns; APAF1f/f-OTII mice and closed columns; Lck-Cre-APAF1f/f-OTII mice. Data show means + SD of triplicated samples. Experiments were repeated three times with similar results. #; p’
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Enhanced ex vivo recall responses of APAF1-deficient T cells.
2018Co-Authors: Honglian Tong, Yasunobu Miyake, Fumika Mi-ichi, Yoichiro Iwakura, Hiromitsu Hara, Hiroki YoshidaAbstract:(A and B) LN cells from OVA-immunized APAF1f/f-OTII or Lck-Cre-APAF1f/f-OTII mice were assessed for expression of CD69, CD44 and CD62L. Percentages (A) and absolute numbers (B) of CD69+ cells and CD44highCD62Llow cells in TCR Tg-positive population (OTII population) are shown. (C) LN cells from immunized mice were re-stimulated with OVA or anti-CD3ε antibody at the indicated concentration for 48 hours. Percentages of CD69+ cells and CD44highCD62Llow cells are shown. Open columns; APAF1f/f-OTII mice and closed columns; Lck-Cre-APAF1f/f-OTII mice. Shown are mean+SD of triplicated samples. Experiments were repeated three times with similar results. *; p
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Enhanced proliferation and cytokine production by APAF1-deficient naïve T cells.
2018Co-Authors: Honglian Tong, Yasunobu Miyake, Fumika Mi-ichi, Yoichiro Iwakura, Hiromitsu Hara, Hiroki YoshidaAbstract:(A and B) LN cells from unimmunized APAF1f/f-OTII or Lck-Cre-APAF1f/f -OTII mice were labeled with CFSE and stimulated with anti-CD3ε antibody or OVA peptide at the indicated concentrations. CFSE intensity was analyzed and cells with lower intensity (in cell division) were measured. Representative figures with percentages of cells with lower CFSE intensity are shown in A and results with triplicated samples are shown in B. (C) IFN-γ production by LN cells from unimmunized mice stimulated with anti-CD3ε antibody or OVA peptide. Open columns; APAF1f/f-OTII mice and closed columns; Lck-Cre-APAF1f/f-OTII mice. Experiments were repeated three times with similar results. Shown are mean+SD. Experiments were repeated three times with similar results. *; p