Publications by authors named "Rana S Moubarak"

19 Publications

  • Page 1 of 1

SIVA-1 regulates apoptosis and synaptic function by modulating XIAP interaction with the death receptor antagonist FAIM-L.

Cell Death Dis 2020 02 3;11(2):82. Epub 2020 Feb 3.

Cell Signaling and Apoptosis Group, Vall d'Hebron Research Institute (VHIR), 08035, Barcelona, Spain.

The long isoform of Fas apoptosis inhibitory molecule (FAIM-L) is a neuron-specific death receptor antagonist that modulates apoptotic cell death and mechanisms of neuronal plasticity. FAIM-L exerts its antiapoptotic action by binding to X-linked inhibitor of apoptosis protein (XIAP), an inhibitor of caspases, which are the main effectors of apoptosis. XIAP levels are regulated by the ubiquitin-proteasome pathway. FAIM-L interaction with XIAP prevents the ubiquitination and degradation of the latter, thereby allowing it to inhibit caspase activation. This interaction also modulates non-apoptotic functions of caspases, such as the endocytosis of AMPA receptor (AMPAR) in hippocampal long-term depression (LTD). The molecular mechanism of action exerted by FAIM-L is unclear since the consensus binding motifs are still unknown. Here, we performed a two-hybrid screening to discover novel FAIM-L-interacting proteins. We found a functional interaction of SIVA-1 with FAIM-L. SIVA-1 is a proapoptotic protein that has the capacity to interact with XIAP. We describe how SIVA-1 regulates FAIM-L function by disrupting the interaction of FAIM-L with XIAP, thereby promoting XIAP ubiquitination, caspase-3 activation and neuronal death. Furthermore, we report that SIVA-1 plays a role in receptor internalization in synapses. SIVA-1 is upregulated upon chemical LTD induction, and it modulates AMPAR internalization via non-apoptotic activation of caspases. In summary, our findings uncover SIVA-1 as new functional partner of FAIM-L and demonstrate its role as a regulator of caspase activity in synaptic function.
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http://dx.doi.org/10.1038/s41419-020-2282-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997380PMC
February 2020

Epigenetic Silencing of CDR1as Drives IGF2BP3-Mediated Melanoma Invasion and Metastasis.

Cancer Cell 2020 01;37(1):55-70.e15

Department of Pathology, New York University Langone Medical Center, New York, NY, USA; Interdisciplinary Melanoma Cooperative Group, New York University Langone Medical Center, New York, NY, USA. Electronic address:

Metastasis is the primary cause of death of cancer patients. Dissecting mechanisms governing metastatic spread may uncover important tumor biology and/or yield promising therapeutic insights. Here, we investigated the role of circular RNAs (circRNA) in metastasis, using melanoma as a model aggressive tumor. We identified silencing of cerebellar degeneration-related 1 antisense (CDR1as), a regulator of miR-7, as a hallmark of melanoma progression. CDR1as depletion results from epigenetic silencing of LINC00632, its originating long non-coding RNA (lncRNA) and promotes invasion in vitro and metastasis in vivo through a miR-7-independent, IGF2BP3-mediated mechanism. Moreover, CDR1as levels reflect cellular states associated with distinct therapeutic responses. Our study reveals functional, prognostic, and predictive roles for CDR1as and expose circRNAs as key players in metastasis.
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http://dx.doi.org/10.1016/j.ccell.2019.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184928PMC
January 2020

A novel mouse model demonstrates that oncogenic melanocyte stem cells engender melanoma resembling human disease.

Nat Commun 2019 11 4;10(1):5023. Epub 2019 Nov 4.

The Ronald O. Perelman Department of Dermatology, New York University, School of Medicine, New York, NY, 10016, USA.

Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.
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http://dx.doi.org/10.1038/s41467-019-12733-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828673PMC
November 2019

Identification and characterization of new isoforms of human fas apoptotic inhibitory molecule (FAIM).

PLoS One 2017 5;12(10):e0185327. Epub 2017 Oct 5.

Cell Signaling and Apoptosis Group, Vall d´Hebron Institute of Research (VHIR), Barcelona, Spain.

Fas Apoptosis Inhibitory Molecule (FAIM) is an evolutionarily highly conserved death receptor antagonist, widely expressed and known to participate in physiological and pathological processes. Two FAIM transcript variants have been characterized to date, namely FAIM short (FAIM-S) and FAIM long (FAIM-L). FAIM-S is ubiquitously expressed and serves as an anti-apoptotic protein in the immune system. Furthermore, in neurons, this isoform promotes NGF-induced neurite outgrowth through NF-кB and ERK signaling. In contrast FAIM-L is found only in neurons, where it exerts anti-apoptotic activity against several stimuli. In addition to these two variants, in silico studies point to the existence of two additional isoforms, neither of which have been characterized to date. In this regard, here we confirm the presence of these two additional FAIM isoforms in human fetal brain, fetal and adult testes, and placenta tissues. We named them FAIM-S_2a and FAIM-L_2a since they have the same sequence as FAIM-S and FAIM-L, but include exon 2a. PCR and western blot revealed that FAIM-S_2a shows ubiquitous expression in all the tissues and cellular models tested, while FAIM-L_2a is expressed exclusively in tissues of the nervous system. In addition, we found that, when overexpressed in non-neuronal cells, the splicing factor nSR100 induces the expression of the neuronal isoforms, thus identifying it as responsible for the generation of FAIM-L and FAIM-L_2a. Functionally, FAIM-S_2a and FAIM-L_2a increased neurite outgrowth in response to NGF stimulation in a neuronal model. This observation thus, supports the notion that these two isoforms are involved in neuronal differentiation. Furthermore, subcellular fractionation experiments revealed that, in contrast to FAIM-S and FAIM-L, FAIM-S_2a and FAIM-L_2a are able to localize to the nucleus, where they may have additional functions. In summary, here we report on two novel FAIM isoforms that may have relevant roles in the physiology and pathology of the nervous system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185327PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628826PMC
October 2017

FAIM-L regulation of XIAP degradation modulates Synaptic Long-Term Depression and Axon Degeneration.

Sci Rep 2016 10 21;6:35775. Epub 2016 Oct 21.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Spain.

Caspases have recently emerged as key regulators of axonal pruning and degeneration and of long-term depression (LTD), a long-lasting form of synaptic plasticity. However, the mechanism underlying these functions remains unclear. In this context, XIAP has been shown to modulate these processes. The neuron-specific form of FAIM protein (FAIM-L) is a death receptor antagonist that stabilizes XIAP protein levels, thus preventing death receptor-induced neuronal apoptosis. Here we show that FAIM-L modulates synaptic transmission, prevents chemical-LTD induction in hippocampal neurons, and thwarts axon degeneration after nerve growth factor (NGF) withdrawal. Additionally, we demonstrate that the participation of FAIM-L in these two processes is dependent on its capacity to stabilize XIAP protein levels. Our data reveal FAIM-L as a regulator of axonal degeneration and synaptic plasticity.
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http://dx.doi.org/10.1038/srep35775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073314PMC
October 2016

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells.

J Biol Chem 2016 Jan 18;291(3):1221-34. Epub 2015 Nov 18.

From the Cell Signaling and Apoptosis Group and the Institut de Neurociències, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain,

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.
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http://dx.doi.org/10.1074/jbc.M115.677682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714210PMC
January 2016

TNFα sensitizes neuroblastoma cells to FasL-, cisplatin- and etoposide-induced cell death by NF-κB-mediated expression of Fas.

Mol Cancer 2015 Mar 19;14:62. Epub 2015 Mar 19.

Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain.

Background: Patients with high-risk neuroblastoma (NBL) tumors have a high mortality rate. Consequently, there is an urgent need for the development of new treatments for this condition. Targeting death receptor signaling has been proposed as an alternative to standard chemo- and radio-therapies in various tumors. In NBL, this therapeutic strategy has been largely disregarded, possibly because ~50-70% of all human NBLs are characterized by caspase-8 silencing. However, the expression of caspase-8 is detected in a significant group of NBL patients, and they could therefore benefit from treatments that induce cell death through death receptor activation. Given that cytokines, such as TNFα, are able to upregulate Fas expression, we sought to address the therapeutic relevance of co-treatment with TNFα and FasL in NBL.

Methods: For the purpose of the study we used a set of eight NBL cell lines. Here we explore the cell death induced by TNFα, FasL, cisplatin, and etoposide, or a combination thereof by Hoechst staining and calcein viability assay. Further assessment of the signaling pathways involved was performed by caspase activity assays and Western blot experiments. Characterization of Fas expression levels was achieved by qRT-PCR, cell surface biotinylation assays, and cytometry.

Results: We have found that TNFα is able to increase FasL-induced cell death by a mechanism that involves the NF-κB-mediated induction of the Fas receptor. Moreover, TNFα sensitized NBL cells to DNA-damaging agents (i.e. cisplatin and etoposide) that induce the expression of FasL. Priming to FasL-, cisplatin-, and etoposide-induced cell death could only be achieved in NBLs that display TNFα-induced upregulation of Fas. Further analysis denotes that the high degree of heterogeneity between NBLs is also manifested in Fas expression and modulation thereof by TNFα.

Conclusions: In summary, our findings reveal that TNFα sensitizes NBL cells to FasL-induced cell death by NF-κB-mediated upregulation of Fas and unveil a new mechanism through which TNFα enhances the efficacy of currently used NBL treatments, cisplatin and etoposide.
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http://dx.doi.org/10.1186/s12943-015-0329-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407790PMC
March 2015

FBXW7 modulates cellular stress response and metastatic potential through ​HSF1 post-translational modification.

Nat Cell Biol 2015 Mar;17(3):322-332

Howard Hughes Medical Institute and Department of Pathology, NYU School of Medicine, New York, NY 10016, USA.

​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate ​HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the ​HSF1 transcriptional program both in the presence of exogenous stress and in cancer.
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http://dx.doi.org/10.1038/ncb3121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4401662PMC
March 2015

FAIM-L is an IAP-binding protein that inhibits XIAP ubiquitinylation and protects from Fas-induced apoptosis.

J Neurosci 2013 Dec;33(49):19262-75

Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron (VHIR), 08035 Barcelona, Spain, Institut de Neurociències, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra Spain, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Spain, Departament de Ciències Experimentals i de la Salut, Cell Signalling Unit, Universitat Pompeu Fabra, 08003 Barcelona, Spain, Laboratoris Clinics, Hospital Universitari Vall d'Hebron, 08035 Barcelona, Spain, and Institut de Recerca Biomèdica de Lleida, Universitat de Lleida, 25198 Lleida, Spain.

The neuronal long isoform of Fas Apoptotic Inhibitory Molecule (FAIM-L) protects from death receptor (DR)-induced apoptosis, yet its mechanism of protection remains unknown. Here, we show that FAIM-L protects rat neuronal Type II cells from Fas-induced apoptosis. XIAP has previously emerged as a molecular discriminator that is upregulated in Type II and downregulated in Type I apoptotic signaling. We demonstrate that FAIM-L requires sustained endogenous levels of XIAP to protect Type II cells as well as murine cortical neurons from Fas-induced apoptosis. FAIM-L interacts with the BIR2 domain of XIAP through an IAP-binding motif, the mutation of which impairs the antiapoptotic function of FAIM-L. Finally, we report that FAIM-L inhibits XIAP auto-ubiquitinylation and maintains its stability, thus conferring protection from apoptosis. Our results bring new understanding of the regulation of endogenous XIAP by a DR antagonist, pointing out at FAIM-L as a promising therapeutic tool for protection from apoptosis in pathological situations where XIAP levels are decreased.
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http://dx.doi.org/10.1523/JNEUROSCI.2479-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618789PMC
December 2013

X-linked inhibitor of apoptosis protein negatively regulates neuronal differentiation through interaction with cRAF and Trk.

Sci Rep 2013 ;3:2397

Institut de Neurociències and Dpt. Bioquímica and Biología Molecular, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain.

X-linked Inhibitor of apoptosis protein (XIAP) has been classically identified as a cell death regulator. Here, we demonstrate a novel function of XIAP as a regulator of neurite outgrowth in neuronal cells. In PC12 cells, XIAP overexpression prevents NGF-induced neuronal differentiation, whereas NGF treatment induces a reduction of endogenous XIAP levels concomitant with the induction of neuronal differentiation. Accordingly, downregulation of endogenous XIAP protein levels strongly increases neurite outgrowth in PC12 cells as well as axonal and dendritic length in primary cortical neurons. The effects of XIAP are mediated by the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (ERKs) pathway since blocking this pathway completely prevents the neuritogenesis mediated by XIAP downregulation. In addition, we found that XIAP binds to cRaf and Trk receptors. Our results demonstrate that XIAP plays a new role as a negative regulator of neurotrophin-induced neurite outgrowth and neuronal differentiation in developing neurons.
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http://dx.doi.org/10.1038/srep02397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739015PMC
February 2014

A pathway involving HDAC5, cFLIP and caspases regulates expression of the splicing regulator polypyrimidine tract binding protein in the heart.

J Cell Sci 2013 Apr 19;126(Pt 7):1682-91. Epub 2013 Feb 19.

Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida - IRBLLEIDA, Av Rovira Roure, 80, Lleida, 25198 Spain.

Polypyrimidine tract binding protein (PTB) regulates pre-mRNA splicing, having special relevance for determining gene expression in the differentiating muscle. We have previously shown that PTB protein abundance is progressively reduced during heart development without reduction of its own transcript. Simultaneous reduction of histone deacetylase (HDAC) expression prompted us to investigate the potential link between these events. HDAC5-deficient mice have reduced cardiac PTB protein abundance, and HDAC inhibition in myocytes causes a reduction in endogenous expression of cellular FLICE-like inhibitory protein (cFLIP) and caspase-dependent cleavage of PTB. In agreement with this, cardiac PTB expression is abnormally high in mice with cardiac-specific executioner caspase deficiency, and cFLIP overexpression prevents PTB cleavage in vitro. Caspase-dependent cleavage triggers further fragmentation of PTB, and these fragments accumulate in the presence of proteasome inhibitors. Experimental modification of the above processes in vivo and in vitro results in coherent changes in the alternative splicing of genes encoding tropomyosin-1 (TPM1), tropomyosin-2 (TPM2) and myocyte enhancer factor-2 (MEF2). Thus, we report a pathway connecting HDAC, cFLIP and caspases regulating the progressive disappearance of PTB, which enables the expression of the adult variants of proteins involved in the regulation of contraction and transcription during cardiac muscle development.
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http://dx.doi.org/10.1242/jcs.121384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647441PMC
April 2013

NF-κB activation fails to protect cells to TNFα-induced apoptosis in the absence of Bcl-xL, but not Mcl-1, Bcl-2 or Bcl-w.

Biochim Biophys Acta 2013 May 28;1833(5):1085-95. Epub 2013 Jan 28.

Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain.

TNFα can promote either cell survival or cell death. The activation of NF-κB plays a central role in cell survival while its inhibition makes TNFα-triggered cytotoxicity possible. Here, we report that the overexpression of a non-degradable mutant of the inhibitor of NF-κB (super-repressor (SR)-IκBα) sensitizes HeLa cells towards TNFα-induced apoptosis, involving caspases activation and cytocrome C release from the mitochondria. Interestingly, we describe that the specific knockdown of Bcl-xL, but not that of Bcl-2, Bcl-w or Mcl-1, renders cells sensitive to TNFα-induced apoptosis. This cytotoxic effect occurs without altering the activation of NF-κB. Then, the activation of the NF-κB pathway is not sufficient to protect Bcl-xL-downregulated cells from TNFα-induced cell death, meaning that TNFα is not able to promote cell survival in the absence of Bcl-xL. In addition, Bcl-xL silencing does not potentiate the cytotoxicity afforded by the cytokine in SR-IκBα-overexpressing cells. This indicates that TNFα-induced apoptosis in SR-IκBα-overexpressing cells relies on the protein levels of Bcl-xL. We have corroborated these findings using RD and DU-145 cells, which also become sensitive to TNFα-induced apoptosis after Bcl-xL knockdown despite that NF-κB remains activated. Altogether, our results point out that the impairment of the anti-apoptotic function of Bcl-xL should make cells sensitive towards external insults circumventing the TNFα-triggered NF-κB-mediated cytoprotective effect. Hence, the specific inhibition of Bcl-xL could be envisaged as a promising alternative strategy against NF-κB-dependent highly chemoresistant proliferative malignancies.
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http://dx.doi.org/10.1016/j.bbamcr.2013.01.014DOI Listing
May 2013

The death receptor antagonist FLIP-L interacts with Trk and is necessary for neurite outgrowth induced by neurotrophins.

J Neurosci 2010 Apr;30(17):6094-105

Institut de Neurociències, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Campus de Bellaterra (Edifici M), 08193 Bellaterra, Spain.

FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5-15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor kappaB (NF-kappaB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75(NTR), in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-kappaB-mediated control of neurite growth in developing neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.0537-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632611PMC
April 2010

BCL-XL regulates TNF-alpha-mediated cell death independently of NF-kappaB, FLIP and IAPs.

Cell Res 2008 Oct;18(10):1020-36

Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain.

Upon activation, tumor necrosis factor alpha (TNF-alpha) receptor can engage apoptotic or survival pathways. Inhibition of macromolecular synthesis is known to sensitize cells to TNF-alpha-induced cell death. It is believed that this sensitization is due to the transcriptional blockade of genes regulated by NF-kappaB. Nevertheless, such evidence has remained elusive in the nervous system. Here, we show that TNF-alpha cannot normally induce apoptosis in PC12 cells or cortical neurons. However, cells treated with Actinomycin D (ActD) become susceptible to TNF-alpha-induced cell death through the activation of caspase-8, generation of tBid and activation of caspase-9 and -3. Analysis of several proteins involved in TNF-alpha receptor signaling showed no significant downregulation of NF-kappaB target genes, such as IAPs or FLIP, under such conditions. However, Bcl-x(L) protein levels, but not those of Bcl-2, Bax and Bak, are reduced by ActD or TNF-alpha/ActD treatments. Moreover, Bcl-x(L) overexpression fully protects cells against TNF-alpha/ActD-induced cell death. When endogenous levels of Bcl-x(L) are specifically downregulated by lentiviral-based RNAi, cells no longer require ActD to be sensitive to TNF-alpha-triggered apoptosis. Furthermore, Bcl-x(L) downregulation does not affect TNF-alpha-mediated NF-kappaB activation. Altogether, our results demonstrate that Bcl-x(L), and not Bcl-2, FLIP or IAPs, acts as the endogenous regulator of neuronal resistance/sensitivity to TNF-alpha-induced apoptosis in an NF-kappaB-independent manner.
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http://dx.doi.org/10.1038/cr.2008.76DOI Listing
October 2008

The long form of Fas apoptotic inhibitory molecule is expressed specifically in neurons and protects them against death receptor-triggered apoptosis.

J Neurosci 2007 Oct;27(42):11228-41

Cell Signaling and Apoptosis Group, Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida-Hospital Universitari Arnau de Vilanova, 25198 Lleida, Spain.

Death receptors (DRs) and their ligands are expressed in developing nervous system. However, neurons are generally resistant to death induction through DRs and rather their activation promotes neuronal outgrowth and branching. These results suppose the existence of DRs antagonists expressed in the nervous system. Fas apoptosis inhibitory molecule (FAIM(S)) was first identified as a Fas antagonist in B-cells. Soon after, a longer alternative spliced isoform with unknown function was identified and named FAIM(L). FAIM(S) is widely expressed, including the nervous system, and we have shown previously that it promotes neuronal differentiation but it is not an anti-apoptotic molecule in this system. Here, we demonstrate that FAIM(L) is expressed specifically in neurons, and its expression is regulated during the development. Expression could be induced by NGF through the extracellular regulated kinase pathway in PC12 (pheochromocytoma cell line) cells. Contrary to FAIM(S), FAIM(L) does not increase the neurite outgrowth induced by neurotrophins and does not interfere with nuclear factor kappaB pathway activation as FAIM(S) does. Cells overexpressing FAIM(L) are resistant to apoptotic cell death induced by DRs such as Fas or tumor necrosis factor R1. Reduction of endogenous expression by small interfering RNA shows that endogenous FAIM(L) protects primary neurons from DR-induced cell death. The detailed analysis of this antagonism shows that FAIM(L) can bind to Fas receptor and prevent the activation of the initiator caspase-8 induced by Fas. In conclusion, our results indicate that FAIM(L) could be responsible for maintaining initiator caspases inactive after receptor engagement protecting neurons from the cytotoxic action of death ligands.
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http://dx.doi.org/10.1523/JNEUROSCI.3462-07.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673028PMC
October 2007

Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis.

Mol Cell Biol 2007 Jul 30;27(13):4844-62. Epub 2007 Apr 30.

Apoptose et Système Immunitaire, CNRS-URA 1961, Institut Pasteur, 25 Rue du Dr. Roux, 75015 Paris, France.

Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N'-nitro-N'-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.
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http://dx.doi.org/10.1128/MCB.02141-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951482PMC
July 2007

Identification and characterization of AIFsh2, a mitochondrial apoptosis-inducing factor (AIF) isoform with NADH oxidase activity.

J Biol Chem 2006 Jul 27;281(27):18507-18. Epub 2006 Apr 27.

Apoptose et Système Immunitaire, CNRS-URA 1961, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France.

Apoptosis-inducing factor (AIF) is a bifunctional NADH oxidase involved in mitochondrial respiration and caspase-independent apoptosis. Three alternatively spliced mRNA isoforms of AIF have been identified previously: AIF, AIF-exB, and AIFsh. Here, we report the cloning and the biochemical characterization of a new isoform named AIF short 2 (AIFsh2). AIFsh2 transcript includes a previously unknown exon placed between exons 9 and 10 of AIF. The resulting AIFsh2 protein, which localizes in mitochondria, corresponds to the oxidoreductase domain of AIF. In this way, AIFsh2 exhibits similar NADH oxidase activity to AIF and generates reactive oxygen species. Like AIF, AIFsh2 is released from mitochondria to cytosol after an apoptotic insult in a calpain or cathepsin-dependent manner. However, in contrast to AIF, AIFsh2 does not induce nuclear apoptosis. Thus, it seems that the reactive oxygen species produced by the oxidoreductase domain of AIF/AIFsh2 are not important for AIF-dependent nuclear apoptosis. In addition, we demonstrate that the AIFsh2 mRNA is absent in normal brain tissue, whereas it is expressed in neuroblastoma-derived cells, suggesting a different regulation in normal and transformed cells from the brain lineage. Together, our results reveal that AIF yields an original and independent genetic regulation of the two AIF functions. This is an important issue to understand the physiological role of this protein.
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http://dx.doi.org/10.1074/jbc.M601751200DOI Listing
July 2006

AIFsh, a novel apoptosis-inducing factor (AIF) pro-apoptotic isoform with potential pathological relevance in human cancer.

J Biol Chem 2006 Mar 19;281(10):6413-27. Epub 2005 Dec 19.

Apoptose et Système Immunitaire, CNRS-URA 1961 and Plateforme 5-Production de Protéines Recombinantes et d'Anticorps, Institut Pasteur, 25, rue du Dr. Roux, 75015 Paris, France.

AIF is a main mediator of caspase-independent cell death. It is encoded by a single gene located on chromosome X, region q25-26 and A6 in humans and mice, respectively. Previous studies established that AIF codes for two isoforms of the protein, AIF and AIF-exB. Here, we identify a third AIF isoform resulting from an alternate transcriptional start site located at intron 9 of AIF. The resulting mRNA encodes a cytosolic protein that corresponds to the C-terminal domain of AIF (amino acids 353-613). We named this new isoform AIFshort (AIFsh). AIFsh overexpression in HeLa cells results in nuclear translocation and caspase-independent cell death. Once in the nucleus, AIFsh provokes the same effects than AIF, namely chromatin condensation and large scale (50 kb) DNA fragmentation. In contrast, these apoptogenic effects are not precluded by the AIF-inhibiting protein Hsp70. These findings identify AIFsh as a new pro-apoptotic isoform of AIF, and also reveal that the first N-terminal 352 amino acids of AIF are not required for its apoptotic activity. In addition, we demonstrate that AIFsh is strongly down-regulated in tumor cells derived from kidney, vulva, skin, thyroid, and pancreas, whereas, gamma-irradiation treatment provokes AIFsh up-regulation. Overall, our results identify a novel member of the AIF-dependent pathway and shed new light on the role of caspase-independent cell death in tumor formation/suppression.
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http://dx.doi.org/10.1074/jbc.M509884200DOI Listing
March 2006

The contribution of apoptosis-inducing factor, caspase-activated DNase, and inhibitor of caspase-activated DNase to the nuclear phenotype and DNA degradation during apoptosis.

J Biol Chem 2005 Oct 27;280(42):35670-83. Epub 2005 Jul 27.

Department of Ciències Mèdiques Bàsiques, Cell Signalling and Apoptosis Group, Facultat de Medicina, Universitat de Lleida, Montserrat Roig, 2, E-25008 Lleida, Spain.

We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process.
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http://dx.doi.org/10.1074/jbc.M504015200DOI Listing
October 2005