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Kelvin Cain - One of the best experts on this subject based on the ideXlab platform.

  • In Vitro Assembly and Analysis of the Apoptosome Complex
    Cold Spring Harbor protocols, 2015
    Co-Authors: Claudia Langlais, Kelvin Cain, Michelle A. Hughes, Marion Macfarlane
    Abstract:

    This protocol describes an in vitro model for studying the mechanisms of caspase activation and native Apoptosome complex assembly in cell-free extracts. Active caspases in dATP-activated lysates are detected by fluorimetry using a tetrapeptide substrate (DEVD) tagged with a fluorophore (AFC), which, when released, produces a real-time readout for caspase-3 and -7 (DEVDase) activity. Gel filtration is used to isolate the Apoptosome complex from the activated lysates, and assembly of Apaf-1 and caspase-9 from their monomeric forms into the multiprotein Apoptosome can be confirmed via western blot. Apoptosome complex activity can be shown by incubation with exogenous procaspase-3 and -7 followed by fluorimetric bioassay (to confirm functionality of the processed effector caspases) and/or western blotting (for detection of cleaved caspase-3 and -7). A method for preparation of free procaspases for the bioassay is also described.

  • caspase 7 is directly activated by the 700 kda Apoptosome complex and is released as a stable xiap caspase 7 200 kda complex
    Journal of Biological Chemistry, 2006
    Co-Authors: Davina Twiddy, Gerald M. Cohen, Marion Macfarlane, Kelvin Cain
    Abstract:

    Abstract MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 Apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating Apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce ∼1.4-MDa and ∼700-kDa Apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This ∼700-kDa Apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the Apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the Apoptosome and forms a stable ∼200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the Apoptosome and also forms a stable ∼200-kDa complex with active caspase-7.

  • Caspase-7 Is Directly Activated by the ∼700-kDa Apoptosome Complex and Is Released as a Stable XIAP-Caspase-7 ∼200-kDa Complex
    The Journal of biological chemistry, 2005
    Co-Authors: Davina Twiddy, Gerald M. Cohen, Marion Macfarlane, Kelvin Cain
    Abstract:

    MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 Apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating Apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce approximately 1.4-MDa and approximately 700-kDa Apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This approximately 700-kDa Apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the Apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the Apoptosome and forms a stable approximately 200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the Apoptosome and also forms a stable approximately 200-kDa complex with active caspase-7.

  • Caspase-7 Is Directly Activated by the ∼700-kDa Apoptosome Complex and Is Released as a Stable XIAP-Caspase-7 ∼200-kDa Complex
    The Journal of biological chemistry, 2005
    Co-Authors: Davina Twiddy, Gerald M. Cohen, Marion Macfarlane, Kelvin Cain
    Abstract:

    Abstract MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 Apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating Apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce ∼1.4-MDa and ∼700-kDa Apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This ∼700-kDa Apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the Apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the Apoptosome and forms a stable ∼200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the Apoptosome and also forms a stable ∼200-kDa complex with active caspase-7.

  • pro apoptotic proteins released from the mitochondria regulate the protein composition and caspase processing activity of the native apaf 1 caspase 9 Apoptosome complex
    Journal of Biological Chemistry, 2004
    Co-Authors: Davina Twiddy, Colin Adrain, Seamus J Martin, Gerald M. Cohen, Marion Macfarlane, David G Brown, Rebekah Jukes, Kelvin Cain
    Abstract:

    Abstract The Apoptosome is a large caspase-activating (∼700–1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is ∼135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the Apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp91–130) containing the CARD domain of caspase-9-(1–130), which binds to the CARD domain of Apaf-1 when it is in the Apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified Apoptosome complex contained only Apaf-1XL and GST-casp91–130, demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active Apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This Apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified Apoptosome complex increased 6–8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional Apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.

Gerald M. Cohen - One of the best experts on this subject based on the ideXlab platform.

  • caspase 7 is directly activated by the 700 kda Apoptosome complex and is released as a stable xiap caspase 7 200 kda complex
    Journal of Biological Chemistry, 2006
    Co-Authors: Davina Twiddy, Gerald M. Cohen, Marion Macfarlane, Kelvin Cain
    Abstract:

    Abstract MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 Apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating Apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce ∼1.4-MDa and ∼700-kDa Apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This ∼700-kDa Apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the Apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the Apoptosome and forms a stable ∼200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the Apoptosome and also forms a stable ∼200-kDa complex with active caspase-7.

  • Caspase-7 Is Directly Activated by the ∼700-kDa Apoptosome Complex and Is Released as a Stable XIAP-Caspase-7 ∼200-kDa Complex
    The Journal of biological chemistry, 2005
    Co-Authors: Davina Twiddy, Gerald M. Cohen, Marion Macfarlane, Kelvin Cain
    Abstract:

    MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 Apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating Apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce approximately 1.4-MDa and approximately 700-kDa Apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This approximately 700-kDa Apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the Apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the Apoptosome and forms a stable approximately 200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the Apoptosome and also forms a stable approximately 200-kDa complex with active caspase-7.

  • Caspase-7 Is Directly Activated by the ∼700-kDa Apoptosome Complex and Is Released as a Stable XIAP-Caspase-7 ∼200-kDa Complex
    The Journal of biological chemistry, 2005
    Co-Authors: Davina Twiddy, Gerald M. Cohen, Marion Macfarlane, Kelvin Cain
    Abstract:

    Abstract MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 Apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating Apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce ∼1.4-MDa and ∼700-kDa Apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This ∼700-kDa Apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the Apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the Apoptosome and forms a stable ∼200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the Apoptosome and also forms a stable ∼200-kDa complex with active caspase-7.

  • pro apoptotic proteins released from the mitochondria regulate the protein composition and caspase processing activity of the native apaf 1 caspase 9 Apoptosome complex
    Journal of Biological Chemistry, 2004
    Co-Authors: Davina Twiddy, Colin Adrain, Seamus J Martin, Gerald M. Cohen, Marion Macfarlane, David G Brown, Rebekah Jukes, Kelvin Cain
    Abstract:

    Abstract The Apoptosome is a large caspase-activating (∼700–1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is ∼135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the Apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp91–130) containing the CARD domain of caspase-9-(1–130), which binds to the CARD domain of Apaf-1 when it is in the Apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified Apoptosome complex contained only Apaf-1XL and GST-casp91–130, demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active Apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This Apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified Apoptosome complex increased 6–8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional Apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.

  • The Apaf-1 Apoptosome: a large caspase-activating complex.
    Biochimie, 2002
    Co-Authors: Kelvin Cain, Shawn B. Bratton, Gerald M. Cohen
    Abstract:

    Abstract It is increasingly recognized that many key biological processes, including apoptosis, are carried out within very large multi-protein complexes. Apoptosis can be initiated by activation of death receptors or perturbation of the mitochondria causing the release of apoptogenic proteins, which result in the activation of caspases which are responsible for most of the biochemical and morphological changes observed during apoptosis. Caspases are normally inactive and require proteolytic processing for activity and this is achieved by the formation of large protein complexes known as the DISC (death inducing signalling complex) and the Apoptosome. In the case of the latter complex, the central scaffold protein is a mammalian CED-4 homologue known as Apaf-1. This is an ∼130 kDa protein, which in the presence of cytochrome c and dATP oligomerizes to form a very large (∼700–1400 kDa) Apoptosome complex. The Apoptosome recruits and processes caspase-9 to form a holoenzyme complex, which in turn recruits and activates the effector caspases. The Apoptosome has been described in cells undergoing apoptosis, in dATP activated cell lysates and in reconstitution studies with recombinant proteins. Recent studies show that formation and function of the Apoptosome can be regulated by a variety of factors including intracellular levels of K + , inhibitor of apoptosis proteins (IAPs), heat shock proteins and Smac/Diablo. These various factors thus ensure that the Apoptosome complex is only fully assembled and functional when the cell is irrevocably destined to die.

Shawn B. Bratton - One of the best experts on this subject based on the ideXlab platform.

  • the apaf 1 procaspase 9 Apoptosome complex functions as a proteolytic based molecular timer
    The EMBO Journal, 2009
    Co-Authors: Srinivas Malladi, Howard O. Fearnhead, Madhavi Challamalladi, Shawn B. Bratton
    Abstract:

    During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 Apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that Apoptosome‐mediated cleavage of procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector procaspase‐3, procaspase‐9 cannot be processed by the Apoptosome as a typical substrate. Indeed, procaspase‐9 possessed higher affinity for the Apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase‐9 per se contributed little to the activation of procaspase‐3. Thus, the Apaf‐1 Apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of procaspase‐9 sets the overall duration of the timer, procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate procaspase‐3) dictates how fast the timer ‘ticks’ over.

  • The Apaf‐1•procaspase‐9 Apoptosome complex functions as a proteolytic‐based molecular timer
    The EMBO journal, 2009
    Co-Authors: Srinivas Malladi, Madhavi Challa-malladi, Howard O. Fearnhead, Shawn B. Bratton
    Abstract:

    During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 Apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that Apoptosome‐mediated cleavage of procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector procaspase‐3, procaspase‐9 cannot be processed by the Apoptosome as a typical substrate. Indeed, procaspase‐9 possessed higher affinity for the Apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase‐9 per se contributed little to the activation of procaspase‐3. Thus, the Apaf‐1 Apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of procaspase‐9 sets the overall duration of the timer, procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate procaspase‐3) dictates how fast the timer ‘ticks’ over.

  • intracellular nucleotides act as critical prosurvival factors by binding to cytochrome c and inhibiting Apoptosome
    Cell, 2006
    Co-Authors: Dhyan Chandra, Howard O. Fearnhead, Shawn B. Bratton, Angel G Martin, Mary Ayres, Varsha Gandhi, Maria D Person, Yanan Tian, Dean G. Tang
    Abstract:

    Cytochrome c (CC)-initiated Apaf-1 Apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect Apoptosome activation remains unclear. Here we show that physiological levels of nucleotides inhibit the CC-initiated Apoptosome formation and caspase-9 activation by directly binding to CC on several key lysine residues and thus preventing CC interaction with Apaf-1. We show that in various apoptotic systems caspase activation is preceded or accompanied by decreases in overall intracellular NTP pools. Microinjection of nucleotides inhibits whereas experimentally reducing NTP pools enhances both CC and apoptotic stimuli-induced cell death. Our results thus suggest that the intracellular nucleotides represent critical prosurvival factors by functioning as natural inhibitors of Apoptosome formation and a barrier that cells must overcome the nucleotide barrier to undergo apoptosis cell death.

  • The Apaf-1 Apoptosome: a large caspase-activating complex.
    Biochimie, 2002
    Co-Authors: Kelvin Cain, Shawn B. Bratton, Gerald M. Cohen
    Abstract:

    Abstract It is increasingly recognized that many key biological processes, including apoptosis, are carried out within very large multi-protein complexes. Apoptosis can be initiated by activation of death receptors or perturbation of the mitochondria causing the release of apoptogenic proteins, which result in the activation of caspases which are responsible for most of the biochemical and morphological changes observed during apoptosis. Caspases are normally inactive and require proteolytic processing for activity and this is achieved by the formation of large protein complexes known as the DISC (death inducing signalling complex) and the Apoptosome. In the case of the latter complex, the central scaffold protein is a mammalian CED-4 homologue known as Apaf-1. This is an ∼130 kDa protein, which in the presence of cytochrome c and dATP oligomerizes to form a very large (∼700–1400 kDa) Apoptosome complex. The Apoptosome recruits and processes caspase-9 to form a holoenzyme complex, which in turn recruits and activates the effector caspases. The Apoptosome has been described in cells undergoing apoptosis, in dATP activated cell lysates and in reconstitution studies with recombinant proteins. Recent studies show that formation and function of the Apoptosome can be regulated by a variety of factors including intracellular levels of K + , inhibitor of apoptosis proteins (IAPs), heat shock proteins and Smac/Diablo. These various factors thus ensure that the Apoptosome complex is only fully assembled and functional when the cell is irrevocably destined to die.

  • The Apaf-1 Apoptosome: a large caspase-activating complex.
    Biochimie, 2002
    Co-Authors: Kelvin Cain, Shawn B. Bratton, Gerald M. Cohen
    Abstract:

    It is increasingly recognized that many key biological processes, including apoptosis, are carried out within very large multi-protein complexes. Apoptosis can be initiated by activation of death receptors or perturbation of the mitochondria causing the release of apoptogenic proteins, which result in the activation of caspases which are responsible for most of the biochemical and morphological changes observed during apoptosis. Caspases are normally inactive and require proteolytic processing for activity and this is achieved by the formation of large protein complexes known as the DISC (death inducing signalling complex) and the Apoptosome. In the case of the latter complex, the central scaffold protein is a mammalian CED-4 homologue known as Apaf-1. This is an approximately 130 kDa protein, which in the presence of cytochrome c and dATP oligomerizes to form a very large (approximately 700-1400 kDa) Apoptosome complex. The Apoptosome recruits and processes caspase-9 to form a holoenzyme complex, which in turn recruits and activates the effector caspases. The Apoptosome has been described in cells undergoing apoptosis, in dATP activated cell lysates and in reconstitution studies with recombinant proteins. Recent studies show that formation and function of the Apoptosome can be regulated by a variety of factors including intracellular levels of K(+), inhibitor of apoptosis proteins (IAPs), heat shock proteins and Smac/Diablo. These various factors thus ensure that the Apoptosome complex is only fully assembled and functional when the cell is irrevocably destined to die.

Howard O. Fearnhead - One of the best experts on this subject based on the ideXlab platform.

  • The Lumiptosome, an engineered luminescent form of the Apoptosome can report cell death by using the same Apaf-1 dependent pathway.
    Journal of cell science, 2020
    Co-Authors: Elaheh Sadat Hosseini, Howard O. Fearnhead, Maryam Nikkhah, Amir Ali Hamidieh, Jean-paul Concordet, Saman Hosseinkhani
    Abstract:

    ABSTRACT Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and Apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the Apoptosome that we named ‘Lumiptosome’. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the Apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the Apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c. Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.

  • Apoptosome dependent myotube formation involves activation of caspase 3 in differentiating myoblasts
    Cell Death and Disease, 2020
    Co-Authors: Mahshid H Dehkordi, Amin Tashakor, Enda Oconnell, Howard O. Fearnhead
    Abstract:

    : Caspase-2, -9, and -3 are reported to control myoblast differentiation into myotubes. This had been previously explained by phosphatidylserine exposure on apoptotic myoblasts inducing differentiation in neighboring cells. Here we show for the first time that caspase-3 is activated in the myoblasts undergoing differentiation. Using RNAi, we also demonstrate that differentiation requires both cytochrome c and Apaf-1, and by using a new pharmacological approach, we show that Apoptosome formation is required. We also show that Bid, whose cleavage links caspase-2 to the mitochondrial death pathway, was required for differentiation, and that the caspase cleavage product, tBid, was generated during differentiation. Taken together, these data suggest that myoblast differentiation requires caspase-2 activation of the mitochondrial death pathway, and that this occurs in the cells that differentiate. Our data also reveal a hierarchy of caspases in differentiation with caspase-2 upstream of Apoptosome activation, and exerting a more profound control of differentiation, while caspases downstream of the Apoptosome primarily control cell fusion.

  • A new split-luciferase complementation assay identifies pentachlorophenol as an inhibitor of Apoptosome formation.
    FEBS open bio, 2019
    Co-Authors: Amin Tashakor, Saman Hosseinkhani, Mahshid H‐dehkordi, Enda O’connell, Sergi Gomez Ganau, Rafael Gozalbes, Leif A. Eriksson, Howard O. Fearnhead
    Abstract:

    The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase-based assay to screen a library of 177 toxicants for inhibitors of Apoptosome formation. The Apoptosome contains seven Apoptotic Protease-Activating Factor-1 (Apaf-1) molecules and induces cell death by activating caspase-9. Apaf-1-dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf-1 fused to an N-terminal fragment of luciferase binds to Apaf-1 fused to a C-terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits Apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill-defined mechanisms. Although the data show that PCP inhibited Apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying Apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants.

  • the apaf 1 procaspase 9 Apoptosome complex functions as a proteolytic based molecular timer
    The EMBO Journal, 2009
    Co-Authors: Srinivas Malladi, Howard O. Fearnhead, Madhavi Challamalladi, Shawn B. Bratton
    Abstract:

    During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 Apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that Apoptosome‐mediated cleavage of procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector procaspase‐3, procaspase‐9 cannot be processed by the Apoptosome as a typical substrate. Indeed, procaspase‐9 possessed higher affinity for the Apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase‐9 per se contributed little to the activation of procaspase‐3. Thus, the Apaf‐1 Apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of procaspase‐9 sets the overall duration of the timer, procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate procaspase‐3) dictates how fast the timer ‘ticks’ over.

  • The Apaf‐1•procaspase‐9 Apoptosome complex functions as a proteolytic‐based molecular timer
    The EMBO journal, 2009
    Co-Authors: Srinivas Malladi, Madhavi Challa-malladi, Howard O. Fearnhead, Shawn B. Bratton
    Abstract:

    During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 Apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that Apoptosome‐mediated cleavage of procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector procaspase‐3, procaspase‐9 cannot be processed by the Apoptosome as a typical substrate. Indeed, procaspase‐9 possessed higher affinity for the Apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase‐9 per se contributed little to the activation of procaspase‐3. Thus, the Apaf‐1 Apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of procaspase‐9 sets the overall duration of the timer, procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate procaspase‐3) dictates how fast the timer ‘ticks’ over.

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  • The Lumiptosome, an engineered luminescent form of the Apoptosome can report cell death by using the same Apaf-1 dependent pathway.
    Journal of cell science, 2020
    Co-Authors: Elaheh Sadat Hosseini, Howard O. Fearnhead, Maryam Nikkhah, Amir Ali Hamidieh, Jean-paul Concordet, Saman Hosseinkhani
    Abstract:

    ABSTRACT Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and Apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the Apoptosome that we named ‘Lumiptosome’. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the Apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the Apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c. Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.

  • A new split-luciferase complementation assay identifies pentachlorophenol as an inhibitor of Apoptosome formation.
    FEBS open bio, 2019
    Co-Authors: Amin Tashakor, Saman Hosseinkhani, Mahshid H‐dehkordi, Enda O’connell, Sergi Gomez Ganau, Rafael Gozalbes, Leif A. Eriksson, Howard O. Fearnhead
    Abstract:

    The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase-based assay to screen a library of 177 toxicants for inhibitors of Apoptosome formation. The Apoptosome contains seven Apoptotic Protease-Activating Factor-1 (Apaf-1) molecules and induces cell death by activating caspase-9. Apaf-1-dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf-1 fused to an N-terminal fragment of luciferase binds to Apaf-1 fused to a C-terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits Apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill-defined mechanisms. Although the data show that PCP inhibited Apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying Apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants.

  • Apoptosome assay by Split-luciferase constructs
    2018
    Co-Authors: Fatemeh Rabbania, Farangis Ataei, Saman Hosseinkhani
    Abstract:

    Introduction: Apoptosis is a process of programmed cell death that plays several critical roles in normal biological events happening in multi cellular organisms. Tissue homeostasis, defense against pathogens and involvement in development by controlling the number of cells are some of these critical roles. The two best-described activation mechanisms are the intrinsic (also called the mitochondrial pathway) and the extrinsic pathways. The formation of a supramolecular complex called Apoptosome in mammals is tightly linked to ignition of the intrinsic pathway. This complex mainly consists of Apaf-1 molecules (apoptotic factor protease activating 1). The assembled Apaf-1 in Apoptosome leads to the formation of functional caspase-9 that it further triggers the caspase cascade, a fundamental cascade that subsequently causes cell death. So detecting the formation of the Apoptosome complex will help to screen the drugs and substances inducing intrinsic pathways also it helps in cell death related researches. Methods and Results: we utilized previously developed split luciferase biosensor to investigate Apoptosome activity of cells that were treated with Tunicamycin. Tunicamycin is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. Tunicamycin causes accumulation of unfolded proteins in cell endoplasmic reticulum (ER) and induces ER stress. ER stress is an essential mechanism for cellular homeostasis which has a role in cell death via reprogramming of protein processing, regulation of autophagy and apoptosis. Therefore, it can trigger apoptosis by induction of protein release such as cytochrome c that stimulates Apoptosome formation. The biosensor consists of two separate constructs, N-terminal luciferase-Apaf-1 and C-terminal luciferase-Apaf-1. These constructs are cotransfected into mouse embryonic fibroblasts cells by polyethyleneimine (PEI). When Apoptosome complex forms the assembling of Apaf-1 proteins brings the Nlucs and Clubs in spatial proximity that enables the enzyme to catalyst its substrate luciferin and bioluminescence. Split luciferase activity measured in several times after induction by Tunicamycin Conclusions: Apoptosome activity has fluctuation mode and we can control this complex activity by pharmacokinetic features of related drugs. Key words: Apoptosis; Apoptosome; Apaf-1; Caspase; Split Luciferase; Doxorubicin

  • Apoptosome formation upon overexpression of native and truncated Apaf-1 in cell-free and cell-based systems
    Archives of biochemistry and biophysics, 2018
    Co-Authors: Ali Reza Noori, Maryam Nikkhah, Elaheh Sadat Hosseini, Saman Hosseinkhani
    Abstract:

    Apaf-1 is a cytosolic multi-domain protein in the apoptosis regulatory network. When cytochrome c releases from mitochondria; it binds to WD-40 repeats of Apaf-1 molecule and induces oligomerization of Apaf-1. Here in, a split luciferase assay was used to compare Apoptosome formation in cell-free and cell-based systems. This assay uses Apaf-1 tagged with either N-terminal fragment or C-terminal fragment of P. pyralis luciferase. In cell based-system, the Apoptosome formation is induced inside the cells which express Apaf-1 tagged with complementary fragments of luciferase while in cell-free system, the Apoptosome formation is induced in extracts of the cells. In cell-free system, cytochrome c dependent luciferase activity was observed with full length Apaf-1. However, luciferase activity due to Apoptosome formation was much higher in cell based system compared to cell-free system. The truncated Apaf-1 which lacks WD-40 repeats (ΔApaf-1) interacted with endogenous Apaf-1 in a different fashion compared to native form as confirmed by different retention time of eluate in gel filtration and binding to affinity column. The interactions between endogenous Apaf-1 and ΔApaf-1 is stronger than its interaction with native exogenous Apaf-1 as indicated by dominant negative effect of ΔApaf-1 on caspase-3 processing.

  • role of the salt bridge between glutamate 546 and arginine 907 in preservation of autoinhibited form of apaf 1
    International Journal of Biological Macromolecules, 2015
    Co-Authors: Raheleh Shakeri, Jamshid Davoodi, Mayur V Jain, Artur Cieślarpobuda, Mehrdad Rafat, Saman Hosseinkhani, Sussan Kabudanian Ardestani
    Abstract:

    Abstract Apaf-1, the key element of apoptotic mitochondrial pathway, normally exists in an auto-inhibited form inside the cytosol. WRD-domain of Apaf-1 has a critical role in the preservation of auto-inhibited form; however the underlying mechanism is unclear. It seems the salt bridges between WRD and NOD domains are involved in maintaining the inactive conformation of Apaf-1. At the present study, we have investigated the effect of E546-R907 salt bridge on the maintenance of auto-inhibited form of human Apaf-1. E546 is mutated to glutamine (Q) and arginine (R). Over-expression of wild type Apaf-1 and its E546Q and E546R variants in HEK293T cells does not induce apoptosis unlike – HL-60 cancer cell line. In vitro Apoptosome formation assay showed that all variants are cytochrome c and dATP dependent to form Apoptosome and activate endogenous procaspase-9 in Apaf-1-knockout MEF cell line. These results suggest that E546 is not a critical residue for preservation of auto-inhibited Apaf-1. Furthermore, the behavior of Apaf-1 variants for in vitro Apoptosome formation in HEK293T cell is similar to exogenous wild type Apaf-1. Wild type and its variants can form Apoptosome in HEK293T cell with different procaspase-3 processing pattern in the presence and absence of exogenous cytochrome c and dATP.