Santos A Susin

Pierre and Marie Curie University - Paris 6, Lutetia Parisorum, Île-de-France, France

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Publications (129)816.92 Total impact

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    ABSTRACT: The human Apoptosis Inducing Factor (hAIF) is a bifunctional NAD(P)H-dependent flavoreductase involved in both mitochondrial energy metabolism and caspase-independent cell death. Even though several studies indicate that both functions are redox controlled by NADH binding, the exact role of hAIF as a reductase in healthy mitochondria remains unknown. Upon reduction by NADH, hAIF dimerizes and produces very stable flavin:nicotinamide charge transfer complexes (CTC), by stacking of the oxidized nicotinamide moiety of the NAD+ coenzyme against the re-face of the reduced flavin ring of its FAD cofactor, which are critical to restrict the hAIF efficiency as a reductase. The molecular basis of the hAIF reductase activity is here investigated by analyzing the role played by residues contributing to the interaction of the FAD isoalloxazine ring and of the nicotinamide moiety of NADH at the active site. Mutations at K177 and E314 produced drastic effects on the hAIF ability to retain the FAD cofactor, indicating that these residues are important to set up the holo-enzyme active site conformation. Characterization of P173G hAIF indicates that the stacking of P173 against the isoalloxazine ring is significant to determine the flavin environment and to modulate the enzyme affinity for NADH. Finally, the properties of the F310G and H454S hAIF mutants indicate that these two positions contribute to form a compact active site that results essential for NADH binding, CTC stabilization, and NAD+ affinity for the reduced state of hAIF. These features are key determinants of the particular behavior of hAIF as a NADH dependent oxidoreductase.
    No preview · Article · Aug 2015 · Biochemistry
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    ABSTRACT: Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides.
    Full-text · Article · Mar 2015 · PLoS Medicine
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    ABSTRACT: The apoptosis-inducing factor (AIF) is a mitochondrial-flavoprotein that, after cell death induction, is distributed to the nucleus to mediate chromatinolysis. In mitochondria AIF is present in a monomer-dimer equilibrium that after reduction by NADH gets displaced towards the dimer. The crystal structure of the human AIF (hAIF):NAD(H)-bound dimer revealed one FAD and, unexpectedly, two NAD(H) molecules per protomer. A 1:2 hAIF:NAD(H) binding stoichiometry was additionally confirmed in solution by using surface plasmon resonance. The here newly discovered NAD(H)-binding site includes residues mutated in human disorders, and accommodation of the coenzyme in it requires restructuration of an hAIF portion within the 509-560 apoptogenic segment. Disruption of interactions at the dimerization surface by production of the hAIF E413A/R422A/R430A mutant resulted in a non-dimerizable variant considerably less efficiently stabilizing charge-transfer complexes upon coenzyme reduction than WT hAIF. These data reveal that the coenzyme-mediated monomer-dimer transition of hAIF modulates the conformation of its C-terminal proapoptotic domain, as well as its mechanism as reductase. These observations suggest that both the mitochondrial and apoptotic functions of hAIF are interconnected and coenzyme controlled: a key information in the understanding of the physiological role of AIF in the cellular life and death cycle.
    Full-text · Article · Jun 2014 · Biochemistry
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    ABSTRACT: Apoptosis is nowadays what comes first to your scientist mind when someone mentions cellular suicide. However this is not the sole form of programmed cell death and many other alternative or atypical pathways have now been described. These pathways are indeed rather preferred to apoptosis in some instances based on tissue origin, cell type or development stage of the target cell. In this review, we describe many different programmed cell death subtypes according to their characteristics. Discrete, brutal, final or singular cell death pathways all participate in the elimination of unwanted, damaged or dangerous cells in organisms hence contributing to our knowledge of this particular feature of living beings: dying! Through description of anoikis, necroptosis, entosis, netosis, pyroptosis or ferroptosis, we have no choice but to realize that programmed cell death comes in many flavors.
    No preview · Article · Dec 2013 · Medecine sciences: M/S
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    S Fulda · E Meyer · C Friesen · S A Susin · G Kroemer · K M Debatin

    Full-text · Dataset · Nov 2013
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    Full-text · Dataset · Nov 2012
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    ABSTRACT: The apoptosis inducing factor (AIF) was first discovered as a caspase-independent apoptosis effector that promoted cell death upon release from the mitochondria (triggered by pro-apoptotic stimuli) and relocalization into the nucleus, where it promotes chromatin condensation and DNA fragmentation. AIF is a mammalian mitochondrial FAD-dependent flavoenzyme, ubiquitous in vertebrate cells, and with orthologs in all eukaryotes. Beyond its role in apoptosis AIF has additional functions in mitochondria, mainly related with the redox function of its flavin adenine dinucleotide cofactor (FAD), which despite being poorly understood are vital. Thus, defects in AIF trigger major dysfunctions in oxidative phosphorylation, and cause severe illnesses related with neurodegeneration as a consequence of mitochondriopathies. AIF folds in three modules: a FAD-binding, a nicotine adenine dinucleotide (NADH)-binding and a C-terminal modules. Upon reduction of the flavin cofactor by NADH, conformational changes leading to AIF dimerization are proposed as a key early event in the mitochondrial sensing/signaling functions of AIF. The recent interest in the design of new therapies to modulate caspase-independent apoptosis pathways also makes AIF a potential pharmacological target to treat pathological disorders related with AIF dependent mitochondriopathies. Therefore, the first step in this direction must be to understand the molecular basis of the AIF redox reactions and their relationship with the apoptotic function. Here, we examine recent research towards the molecular mechanisms linked to the AIF oxido-reduction properties.
    No preview · Article · Oct 2012 · Current pharmaceutical design
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    ABSTRACT: The alkylating DNA-damage agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces a form of caspase-independent necroptosis implicating the mitochondrial flavoprotein apoptosis-inducing factor (AIF). Following the activation of PARP-1 (poly(ADP-ribose) polymerase-1), calpains, BID (BH3 interacting domain death agonist), and BAX (Bcl-2-associated X protein), the apoptogenic form of AIF (tAIF) is translocated to the nucleus where, associated with Ser139-phosphorylated histone H2AX (γH2AX), it creates a DNA-degrading complex that provokes chromatinolysis and cell death by necroptosis. The generation of γH2AX is crucial for this form of cell death, as mutation of H2AX Ser139 to Ala or genetic ablation of H2AX abolish both chromatinolysis and necroptosis. On the contrary, reintroduction of H2AX-wt or the phosphomimetic H2AX mutant (H2AX-S139E) into H2AX(-/-) cells resensitizes to MNNG-triggered necroptosis. Employing a pharmacological approach and gene knockout cells, we also demonstrate in this paper that the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK) mediate γH2AX generation and, consequently, MNNG-induced necroptosis. By contrast, H2AX phosphorylation is not regulated by ATR or other H2AX-related kinases, such as JNK. Interestingly, ATM and DNA-PK phosphorylate H2AX at Ser139 in a synergistic manner with different kinetics of activation. Early after MNNG treatment, ATM generates γH2AX. Further, DNA-PK contributes to H2AX Ser139 phosphorylation. In revealing the pivotal role of PIKKs in MNNG-induced cell death, our data uncover a milestone in the mechanisms regulating AIF-mediated caspase-independent necroptosis.
    Full-text · Article · Sep 2012 · Cell Death & Disease
  • N Zamzani · SA Susin · G Kroemer

    No preview · Article · Aug 2012
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    ABSTRACT: How do effector CD4 T cells escape cell death during the contraction of the immune response (IR) remain largely unknown. CD47, through interactions with thrombospondin-1 (TSP-1) and SIRP-α, is implicated in cell death and phagocytosis of malignant cells. Here, we reported a reduction in SIRP-α-Fc binding to effector memory T cells (T(EM)) and in vitro TCR-activated human CD4 T cells that was linked to TSP-1/CD47-induced cell death. The reduced SIRP-α-Fc binding (CD47(low) status) was not detected when CD4 T cells were stained with two anti-CD47 mAbs, which recognize distinct epitopes. In contrast, increased SIRP-α-Fc binding (CD47(high) status) marked central memory T cells (T(CM)) as well as activated CD4 T cells exposed to IL-2, and correlated with resistance to TSP-1/CD47-mediated killing. Auto-aggressive CD4 effectors, which accumulated in lymph nodes and at mucosal sites of patients with Crohn's disease, displayed a CD47(high) status despite a high level of TSP-1 release in colonic tissues. In mice, CD47 (CD47(low) status) was required on antigen (Ag)-specific CD4 effectors for the contraction of the IR in vivo, as significantly lower numbers of Ag-specific CD47(+/+)CD4 T cells were recovered when compared to Ag-specific CD47(-/-) CD4 T cells. In conclusion, we demonstrate that a transient change in the status of CD47, i.e. from CD47(high) to CD47(low), on CD4 effectors regulates the decision-making process that leads to CD47-mediated cell death and contraction of the IR while maintenance of a CD47(high) status on tissue-destructive CD4 effectors prevents the resolution of the inflammatory response.
    Full-text · Article · Aug 2012 · PLoS ONE
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    ABSTRACT: T cell memory is the hallmark of adaptive immunity. Central questions are to determine which cells among proliferating effector T cells will live beyond the crash of the immune response (IR) and develop into functional memory T cells. CD47, considered as a marker of self, is implicated in cell death, cell elimination, and in the inflammatory response. We report in this article that CD47 expression was transiently regulated on Ag-specific CD4 T cells, that is, from CD47(high) to CD47(low) to CD47(high), during the course of the in vivo IR. Specifically, CD47(high) status marked central memory CD4 T cell precursors at an early time point of the IR. By contrast, cytokine production was a functional attribute restricted to CD47(high), but not CD47(low), polyclonal effector CD4 T cells during recall responses in an experimental model of chronic airway inflammatory disease. Passive transfer of CD47(high), but not CD47(low), CD4 T cells in nonlymphopenic naive mice generated long-lived memory T cells capable of anamnestic responses. We conclude that CD47(high) status on CD4 T cells identifies functional long-lived memory T cell progenitors.
    Preview · Article · Mar 2012 · The Journal of Immunology
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    ABSTRACT: Alkylating DNA-damage agents such as N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) trigger necroptosis, a newly defined form of programmed cell death (PCD) managed by receptor interacting protein kinases. This caspase-independent mode of cell death involves the sequential activation of poly(ADP-ribose) polymerase-1 (PARP-1), calpains, BAX and AIF, which redistributes from mitochondria to the nucleus to promote chromatinolysis. We have previously demonstrated that the BAX-mediated mitochondrial release of AIF is a critical step in MNNG-mediated necroptosis. However, the mechanism regulating BAX activation in this PCD is poorly understood. Employing mouse embryonic knockout cells, we reveal that BID controls BAX activation in AIF-mediated necroptosis. Indeed, BID is a link between calpains and BAX in this mode of cell death. Therefore, even if PARP-1 and calpains are activated after MNNG treatment, BID genetic ablation abolishes both BAX activation and necroptosis. These PCD defects are reversed by reintroducing the BID-wt cDNA into the BID(-/-) cells. We also demonstrate that, after MNNG treatment, BID is directly processed into tBID by calpains. In this way, calpain non-cleavable BID proteins (BID-G70A or BID-Δ68-71) are unable to promote BAX activation and necroptosis. Once processed, tBID localizes in the mitochondria of MNNG-treated cells, where it can facilitate BAX activation and PCD. Altogether, our data reveal that, as in caspase-dependent apoptosis, BH3-only proteins are key regulators of caspase-independent necroptosis.
    Full-text · Article · Jul 2011 · Cell death and differentiation
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    ABSTRACT: Cell death has been initially divided into apoptosis, in which the cell plays an active role, and necrosis, which is considered a passive cell death program. Intense research performed in the last decades has concluded that "programmed" cell death (PCD) is a more complex physiological process than initially thought. Indeed, although in most cases the PCD process is achieved via a family of Cys proteases known as caspases, an important number of regulated PCD pathways do not involve this family of proteases. As a consequence, active forms of PCD are initially referred to as caspase-dependent and caspase-independent. More recent data has revealed that there are also active caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). The existence of necroptotic forms of death was corroborated by the discovery of key executioners such as the kinase RIP1 or the mitochondrial protein apoptosis-inducing factor (AIF). AIF is a Janus protein with a redox activity in the mitochondria and a pro-apoptotic function in the nucleus. We have recently described a particular form of AIF-mediated caspase-independent necroptosis that also implicates other molecules such as PARP-1, calpains, Bax, Bcl-2, histone H2AX, and cyclophilin A. From a therapeutic point of view, the unraveling of this new form of PCD poses a question: is it possible to modulate this necroptotic pathway independently of the classical apoptotic paths? Because the answer is yes, a wider understanding of AIF-mediated necroptosis could theoretically pave the way for the development of new drugs that modulate PCD. To this end, we present here an overview of the current knowledge of AIF and AIF-mediated necroptosis. We also summarize the state of the art in some of the most interesting therapeutic strategies that could target AIF or the AIF-mediated necroptotic pathway.
    Preview · Article · Apr 2011 · International Union of Biochemistry and Molecular Biology Life
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    ABSTRACT: Blood Cancer Journal is a peer-reviewed, open access online journal publishing pre-clinical and clinical work in the field of hematology with ramifications into translational biology research down to new therapies
    Full-text · Article · Feb 2011 · Blood Cancer Journal
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    ABSTRACT: Caspase-independent programmed necrosis is a highly regulated cellular demise that displays morphological and biochemical necrotic hallmarks, such as an earlier permeability of the plasma membrane and lactate dehydrogenase (LDH) leakiness. This form of programmed cell death (PCD) is regulated by AIF, a FAD-dependent oxidoreductase, which is released from the mitochondria to the nucleus where it induces chromatin tcondensation and DNA fragmentation. Some years ago, it was established that the sequential activation of poly(ADP-ribose) polymerase- 1 (PARP-1), calpains and Bax regulates the mitochondrial AIF release associated to programmed necrosis. But, what happens when AIF is in the nucleus? How does this protein induce chromatinolysis and programmed necrosis? Recently, we have unraveled some of the mechanisms underlying the nuclear action of AIF in this type of caspase-independent cell death. Indeed, AIF plays a key role in programmed necrosis by its ability to organize a DNA-degrading complex with H2AX and Cyclophiline A (CypA). The AIF/H2AX link is indeed a critical event and explains the nuclear AIF apoptogenic action. In the present article, we outline the current knowledge on cell death by programmed necrosis and discuss the relevance of the AIF/H2AX/CypA DNA-degrading complex in the regulation of this original form of cell death.
    Full-text · Article · Aug 2010 · Cell cycle (Georgetown, Tex.)
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    ABSTRACT: Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.
    Full-text · Article · Apr 2010 · The EMBO Journal
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    ABSTRACT: Bcl-2 family proteins are key regulators of the intrinsic apoptotic pathway, either facilitating (Bax, Bak, BH3-only) or inhibiting (Bcl-2, Bcl-x(L), Mcl-1, A1) mitochondrial release of apoptogenic factors. The role of caspases in this process is a matter of controversy. We have analyzed the relative contribution of caspases and Bcl-2 family of proteins in the induction phase of apoptosis triggered by doxorubicin in two p53-deficient leukemia cell lines, Jurkat and U937. First, we have found that caspases are dispensable for the induction phase of doxorubicin-induced apoptosis in both cell lines but they are needed to speed up the execution phase in Jurkat cells, not expressing Bax. Thus, down-regulation of Bak expression by siRNA significantly prevented doxorubicin-induced apoptosis in Jurkat but not in U937 cells. Reduction of Mcl-1 protein levels with siRNA increased sensitivity to apoptosis in both cell lines. Moreover, our results indicate that the contribution of BH3-only proteins to apoptosis is cell line specific. In Jurkat cells simultaneous silencing of Bim and PUMA was necessary to reduce doxorubicin-induced apoptosis. In U937 cells silencing of Bim or Noxa reduced sensitivity to doxorubicin. Immunoprecipitation experiments discarded an interaction between Mcl-1 and Bak in both cell lines and underscored the role of Bim and PUMA as mediators of Bax/Bak activation.
    Full-text · Article · Feb 2010 · Biochemical pharmacology
  • Slavica Krantic · Santos A. Susin
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    ABSTRACT: Historically, cell death has been divided into two generic categories: apoptosis, which requires energy and in which the cell plays an active role, and necrosis, which occurs accidentally, does not require energy consumption and is considered as a passive, uncontrolled cell death program. Among the conceptually opposite cell death forms, apoptosis is the best understood. This death program has been defined as developmentally programmed and ordered cellular response. Apoptosis is initiated by cell rounding and subsequent detachment from the surrounding cells. Chromatin condenses into “crescent-like” forms abutting the inner nuclear membrane. Plasma membrane convolutes and gives rise to characteristic vesicles containing cellular organelles and cytoplasm, known as the “apoptotic bodies.” Apoptosis is generally not accompanied by inflammation since macrophages or neighbouring cells engulf the formed apoptotic bodies before the loss of plasma membrane integrity (Kerr et al. 1972). In contrast to apoptosis, necrosis is characterized by disruption of the plasma membrane with a subsequent water influx and leakage of cell content to the surroundings. Cell death by necrosis can elicit an inflammatory response (Edinger and Thompson 2004).
    No preview · Chapter · Dec 2009
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    ABSTRACT: Programmed cell death has been traditionally related with caspase activation. However, it is now accepted that caspase-independent forms of programmed cell death also regulate cell death. In chronic lymphocytic leukemia, CD47 ligation induces one of these alternative forms of cell death: type III programmed cell death. This poorly understood process is characterized by cytoplasmic hallmarks, such as mitochondrial damage. To gain insights into the molecular pathways regulating type III programmed cell death in chronic lymphocytic leukemia, we performed extensive biochemical and cell biology assessments. After CD47 triggering, purified B-cells from 20 patients with chronic lymphocytic leukemia were studied by flow cytometry, immunofluorescence and three-dimensional imaging, immunoblotting, electron microscopy, and fibrillar/globular actin measurements. Finally, we subjected CD47-treated chronic lymphocytic leukemia cells to a phagocytosis assay. We first confirmed that induction of type III programmed cell death is an efficient means of triggering cell death in chronic lymphocytic leukemia. Further, we demonstrated that the signaling events induced by CD47 ligation provoked a reduction in cell size. This alteration is related to F-actin disruption, as the two other cytoskeleton networks, microtubules and intermediate filaments, remain undisturbed in type III programmed cell death. Strikingly, we revealed that the pharmacological modulation of F-actin dynamics regulated this type of death. Finally, our data delineated a new programmed cell death pathway in chronic lymphocytic leukemia initiated by CD47 triggering, and followed by serine protease activation, F-actin rearrangement, mitochondrial damage, phosphatidylserine exposure, and cell clearance. Our work reveals a key molecular tool in the modulation of cell death in chronic lymphocytic leukemia: F-actin. By assessing the regulation of F-actin and type III programmed cell death, this analysis provides new options for destroying chronic lymphocytic leukemia cells, such as a combination of therapies based on apoptosis regulators (e.g., caspases, Bcl-2, Bax) along with alternative therapies based on type III death effectors (e.g., F-actin).
    Full-text · Article · May 2009 · Haematologica
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    H Merle-Béral · S Barbier · G Roué · M Bras · M Sarfati · S A Susin
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    ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
    Full-text · Article · Dec 2008 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

Publication Stats

26k Citations
816.92 Total Impact Points


  • 2008-2015
    • Pierre and Marie Curie University - Paris 6
      • Centre de recherche des Cordeliers - UMR_S 872
      Lutetia Parisorum, Île-de-France, France
    • Université de Montréal
      • Center for Mathematical Research
      Montréal, Quebec, Canada
  • 2014
    • Centre de Recherche des Cordeliers de Jussieu (CRC)
      Lutetia Parisorum, Île-de-France, France
  • 2012
    • Université Paris-Sorbonne - Paris IV
      Lutetia Parisorum, Île-de-France, France
    • Université René Descartes - Paris 5
      Lutetia Parisorum, Île-de-France, France
  • 2010
    • Unité Inserm U1077
      Caen, Lower Normandy, France
  • 2000-2008
    • Institut Pasteur
      • Department of Virology
      Lutetia Parisorum, Île-de-France, France
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France
    • McMaster University
      • Department of Chemistry and Chemical Biology
      Hamilton, Ontario, Canada
  • 1995-2006
    • French National Centre for Scientific Research
      • Institute for Molecular and Cellular Biology (IBMC)
      Lutetia Parisorum, Île-de-France, France
  • 2003
    • Kagoshima University
      • Department of Ophthalmology
      Kagosima, Kagoshima, Japan
  • 2002
    • Kyushu University
      • Department of Ophthalmology
      Hukuoka, Fukuoka, Japan
  • 2001
    • St. Jude Children's Research Hospital
      • Department of Infectious Diseases
      Memphis, Tennessee, United States
    • Assistance Publique – Hôpitaux de Paris
      Lutetia Parisorum, Île-de-France, France
    • University of Zaragoza
      • Faculty of Sciences (CIENCIAS)
      Caesaraugusta, Aragon, Spain
  • 2000-2001
    • Institut de Cancérologie Gustave Roussy
      Villejuif, Île-de-France, France
    • University of Toronto
      Toronto, Ontario, Canada
  • 1999
    • Université de Technologie de Compiègne
      Compiègne, Picardie, France
  • 1998
    • Universität Konstanz
      Constance, Baden-Württemberg, Germany
  • 1997
    • University of Innsbruck
      Innsbruck, Tyrol, Austria