Publications by authors named "Sylvie Lachkar"

25 Publications

  • Page 1 of 1

Autophagy-mediated metabolic effects of aspirin.

Cell Death Discov 2020 Nov 24;6(1):129. Epub 2020 Nov 24.

Karolinska Institute, Department of Bioscience and Nutrition, Huddinge, Sweden.

Salicylate, the active derivative of aspirin (acetylsalicylate), recapitulates the mode of action of caloric restriction inasmuch as it stimulates autophagy through the inhibition of the acetyltransferase activity of EP300. Here, we directly compared the metabolic effects of aspirin medication with those elicited by 48 h fasting in mice, revealing convergent alterations in the plasma and the heart metabolome. Aspirin caused a transient reduction of general protein acetylation in blood leukocytes, accompanied by the induction of autophagy. However, these effects on global protein acetylation could not be attributed to the mere inhibition of EP300, as determined by epistatic experiments and exploration of the acetyl-proteome from salicylate-treated EP300-deficient cells. Aspirin reduced high-fat diet-induced obesity, diabetes, and hepatosteatosis. These aspirin effects were observed in autophagy-competent mice but not in two different models of genetic (Atg4b or Bcln1) autophagy-deficiency. Aspirin also improved tumor control by immunogenic chemotherapeutics, and this effect was lost in T cell-deficient mice, as well as upon knockdown of an essential autophagy gene (Atg5) in cancer cells. Hence, the health-improving effects of aspirin depend on autophagy.
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http://dx.doi.org/10.1038/s41420-020-00365-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687910PMC
November 2020

Chemical activation of SAT1 corrects diet-induced metabolic syndrome.

Cell Death Differ 2020 10 6;27(10):2904-2920. Epub 2020 May 6.

Centre de Recherche des Cordeliers, INSERM U1138, Team "Metabolism, Cancer & Immunity", Sorbonne Université, Université de Paris, Paris, France.

The pharmacological targeting of polyamine metabolism is currently under the spotlight for its potential in the prevention and treatment of several age-associated disorders. Here, we report the finding that triethylenetetramine dihydrochloride (TETA), a copper-chelator agent that can be safely administered to patients for the long-term treatment of Wilson disease, exerts therapeutic benefits in animals challenged with hypercaloric dietary regimens. TETA reduced obesity induced by high-fat diet, excessive sucrose intake, or leptin deficiency, as it reduced glucose intolerance and hepatosteatosis, but induced autophagy. Mechanistically, these effects did not involve the depletion of copper from plasma or internal organs. Rather, the TETA effects relied on the activation of an energy-consuming polyamine catabolism, secondary to the stabilization of spermidine/spermine N-acetyltransferase-1 (SAT1) by TETA, resulting in enhanced enzymatic activity of SAT. All the positive effects of TETA on high-fat diet-induced metabolic syndrome were lost in SAT1-deficient mice. Altogether, these results suggest novel health-promoting effects of TETA that might be taken advantage of for the prevention or treatment of obesity.
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http://dx.doi.org/10.1038/s41418-020-0550-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494776PMC
October 2020

Immunosuppression by Mutated Calreticulin Released from Malignant Cells.

Mol Cell 2020 02 27;77(4):748-760.e9. Epub 2019 Nov 27.

Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France; Equipe 11 labellisée Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM UMR 1138, Paris, France; Sorbonne Université, Paris, France; Université of Paris, Paris, France; Suzhou Institute for Systems Medicine, Chinese Academy of Sciences, Suzhou, China; Karolinska Institutet, Department of Women's and Children's Health, Stockholm, Sweden; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France. Electronic address:

Mutations affecting exon 9 of the CALR gene lead to the generation of a C-terminally modified calreticulin (CALR) protein that lacks the KDEL endoplasmic reticulum (ER) retention signal and consequently mislocalizes outside of the ER where it activates the thrombopoietin receptor in a cell-autonomous fashion, thus driving myeloproliferative diseases. Here, we used the retention using selective hooks (RUSH) assay to monitor the trafficking of CALR. We found that exon-9-mutated CALR was released from cells in response to the biotin-mediated detachment from its ER-localized hook, in vitro and in vivo. Cellular CALR release was confirmed in suitable mouse models bearing exon-9-mutated hematopoietic systems or tumors. Extracellular CALR mediated immunomodulatory effects and inhibited the phagocytosis of dying cancer cells by dendritic cells (DC), thereby suppressing antineoplastic immune responses elicited by chemotherapeutic agents or by PD-1 blockade. Altogether, our results demonstrate paracrine immunosuppressive effects for exon-9-mutated CALR.
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http://dx.doi.org/10.1016/j.molcel.2019.11.004DOI Listing
February 2020

3,4-Dimethoxychalcone induces autophagy through activation of the transcription factors TFE3 and TFEB.

EMBO Mol Med 2019 11 14;11(11):e10469. Epub 2019 Oct 14.

Gustave Roussy Cancer Campus, Villejuif, France.

Caloric restriction mimetics (CRMs) are natural or synthetic compounds that mimic the health-promoting and longevity-extending effects of caloric restriction. CRMs provoke the deacetylation of cellular proteins coupled to an increase in autophagic flux in the absence of toxicity. Here, we report the identification of a novel candidate CRM, namely 3,4-dimethoxychalcone (3,4-DC), among a library of polyphenols. When added to several different human cell lines, 3,4-DC induced the deacetylation of cytoplasmic proteins and stimulated autophagic flux. At difference with other well-characterized CRMs, 3,4-DC, however, required transcription factor EB (TFEB)- and E3 (TFE3)-dependent gene transcription and mRNA translation to trigger autophagy. 3,4-DC stimulated the translocation of TFEB and TFE3 into nuclei both in vitro and in vivo, in hepatocytes and cardiomyocytes. 3,4-DC induced autophagy in vitro and in mouse organs, mediated autophagy-dependent cardioprotective effects, and improved the efficacy of anticancer chemotherapy in vivo. Altogether, our results suggest that 3,4-DC is a novel CRM with a previously unrecognized mode of action.
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http://dx.doi.org/10.15252/emmm.201910469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835206PMC
November 2019

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy.

Cell Death Dis 2019 10 10;10(10):771. Epub 2019 Oct 10.

Cell Biology and Metabolomics Platforms, Gustave Roussy Cancer Center, Villejuif, France.

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.
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http://dx.doi.org/10.1038/s41419-019-2011-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787181PMC
October 2019

Identification of pharmacological inhibitors of conventional protein secretion.

Sci Rep 2018 10 8;8(1):14966. Epub 2018 Oct 8.

Cell Biology and Metabolomics Platforms, Gustave Roussy Cancer Campus, Villejuif, France.

The retention using selective hooks (RUSH) system allows to withhold a fluorescent biosensor such as green fluorescent protein (GFP) fused to a streptavidin-binding peptide (SBP) by an excess of streptavidin molecules that are addressed to different subcellular localizations. Addition of biotin competitively disrupts this interaction, liberating the biosensor from its hook. We constructed a human cell line co-expressing soluble secretory-SBP-GFP (ss-SBP-GFP) and streptavidin within the endoplasmic reticulum (ER) lumen and then used this system to screen a compound library for inhibitors of the biotin-induced release of ss-SBP-GFP via the conventional Golgi-dependent protein secretion pathway into the culture supernatant. We identified and validated a series of molecularly unrelated drugs including antianginal, antidepressant, anthelmintic, antipsychotic, antiprotozoal and immunosuppressive agents that inhibit protein secretion. These compounds vary in their capacity to suppress protein synthesis and to compromise ER morphology and Golgi integrity, as well as in the degree of reversibility of such effects. In sum, we demonstrate the feasibility and utility of a novel RUSH-based phenotypic screening assay.
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http://dx.doi.org/10.1038/s41598-018-33378-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175952PMC
October 2018

Aspirin Recapitulates Features of Caloric Restriction.

Cell Rep 2018 02;22(9):2395-2407

Gustave Roussy Cancer Campus, Villejuif, France; INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Université Pierre et Marie Curie, Paris, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France; Karolinska Institute, Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden. Electronic address:

The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs). Here, we show that aspirin or its active metabolite salicylate induce autophagy by virtue of their capacity to inhibit the acetyltransferase activity of EP300. While salicylate readily stimulates autophagic flux in control cells, it fails to further increase autophagy levels in EP300-deficient cells, as well as in cells in which endogenous EP300 has been replaced by salicylate-resistant EP300 mutants. Accordingly, the pro-autophagic activity of aspirin and salicylate on the nematode Caenorhabditis elegans is lost when the expression of the EP300 ortholog cpb-1 is reduced. Altogether, these findings identify aspirin as an evolutionary conserved CRM.
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http://dx.doi.org/10.1016/j.celrep.2018.02.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848858PMC
February 2018

Inhibitor of growth protein 4 interacts with Beclin 1 and represses autophagy.

Oncotarget 2017 Oct 6;8(52):89527-89538. Epub 2017 Jul 6.

Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

Beclin 1 (BECN1) is a multifunctional protein that activates the pro-autophagic class III phosphatidylinositol 3-kinase (PIK3C3, best known as VPS34), yet also interacts with multiple negative regulators. Here we report that BECN1 interacts with inhibitor of growth family member 4 (ING4), a tumor suppressor protein that is best known for its capacity to interact with the tumor suppressor protein p53 (TP53) and the acetyltransferase E1A binding protein p300 (EP300). Removal of TP53 or EP300 did not affect the BECN1/ING4 interaction, which however was lost upon culture of cells in autophagy-inducing, nutrient free conditions. Depletion of ING4 stimulated the enzymatic activity of PIK3C3, as visualized by means of a red fluorescent protein-tagged short peptide (FYVE) that specifically binds to phosphatidylinositol-3-phosphate (PI3P)-containing subcellular vesicles and enhanced autophagy, as indicated by an enhanced lipidation of microtubule-associated proteins 1A/1B light chain 3 beta (LC3B) and the redistribution of a green-fluorescent protein (GFP)-LC3B fusion protein to cytoplasmic puncta. The generation of GFP-LC3B puncta stimulated by ING4 depletion was reduced by simultaneous depletion, or pharmacological inhibition, of PIK3C3/VPS34. In conclusion, ING4 acts as a negative regulator of the lipid kinase activity of the BECN1 complex, and starvation-induced autophagy is accompanied by the dissociation of the ING4/BECN1 interaction.
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http://dx.doi.org/10.18632/oncotarget.19033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685689PMC
October 2017

Identification of pharmacological agents that induce HMGB1 release.

Sci Rep 2017 11 2;7(1):14915. Epub 2017 Nov 2.

Cell Biology and Metabolomics Platforms, Gustave Roussy Cancer Campus, Villejuif, France.

The translocation of the protein high mobility group box 1 (HMGB1) from the nucleus to the cytoplasm and its secretion or passive release through the permeabilized plasma membrane, constitutes a major cellular danger signal. Extracellular HMGB1 can interact with pattern recognition receptors to stimulate pro-inflammatory and immunostimulatory pathways. Here, we developed a screening assay to identify pharmacological agents endowed with HMGB1 releasing properties. For this, we took advantage of the "retention using selective hooks" (RUSH) system in which a streptavidin-NLS3 fusion protein was used as a nuclear hook to sequestrate streptavidin-binding peptide (SBP) fused with HMGB1 and green fluorescent protein (GFP). When combined with biotin, which competitively disrupts the interaction between streptavidin-NLS3 and HMGB1-SBP-GFP, immunogenic cell death (ICD) inducers such as anthracyclines were able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. This system, was used in a high-content screening (HCS) campaign for the identification of HMGB1 releasing agents. Hits fell into three functional categories: known ICD inducers, microtubule inhibitors and epigenetic modifiers. These agents induced ICD through a panoply of distinct mechanisms. Their effective action was confirmed by multiple methods monitoring nuclear, cytoplasmic and extracellular HMGB1 pools, both in cultured human or murine cells, as well as in mouse plasma.
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http://dx.doi.org/10.1038/s41598-017-14848-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668281PMC
November 2017

Metabolic interactions between cysteamine and epigallocatechin gallate.

Cell Cycle 2017 Feb 6;16(3):271-279. Epub 2017 Jan 6.

a Equipe 11 labellisée Ligue contre le Cancer, Centre de Recherche des Cordeliers , INSERM U 1138, Paris , France.

Phase II clinical trials indicate that the combination of cysteamine plus epigallocatechin gallate (EGCG) is effective against cystic fibrosis in patients bearing the most frequent etiological mutation (CFTRΔF508). Here, we investigated the interaction between both agents on cultured respiratory epithelia cells from normal and CFTRΔF508-mutated donors. We observed that the combination of both agents affected metabolic circuits (and in particular the tricarboxylic acid cycle) in a unique way and that cysteamine plus EGCG reduced cytoplasmic protein acetylation more than each of the 2 components alone. In a cell-free system, protein cross-linking activity of EGCG was suppressed by cysteamine. Finally, EGCG was able to enhance the conversion of cysteamine into taurine in metabolic flux experiments. Altogether, these results indicate that multiple pharmacological interactions occur between cysteamine and EGCG, suggesting that they contribute to the unique synergy of both agents in restoring the function of mutated CFTRΔF508.
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http://dx.doi.org/10.1080/15384101.2016.1249550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323035PMC
February 2017

Caloric Restriction Mimetics Enhance Anticancer Immunosurveillance.

Cancer Cell 2016 07;30(1):147-160

Gustave Roussy Cancer Campus, 94800 Villejuif, France; INSERM, U1138, 75006 Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, 75006 Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, 75006 Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, 94800 Villejuif, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, 75015 Paris, France; Department of Women's and Children's Health, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden; INSERM U1138, Centre de Recherche des Cordeliers, 75006 Paris, France. Electronic address:

Caloric restriction mimetics (CRMs) mimic the biochemical effects of nutrient deprivation by reducing lysine acetylation of cellular proteins, thus triggering autophagy. Treatment with the CRM hydroxycitrate, an inhibitor of ATP citrate lyase, induced the depletion of regulatory T cells (which dampen anticancer immunity) from autophagy-competent, but not autophagy-deficient, mutant KRAS-induced lung cancers in mice, thereby improving anticancer immunosurveillance and reducing tumor mass. Short-term fasting or treatment with several chemically unrelated autophagy-inducing CRMs, including hydroxycitrate and spermidine, improved the inhibition of tumor growth by chemotherapy in vivo. This effect was only observed for autophagy-competent tumors, depended on the presence of T lymphocytes, and was accompanied by the depletion of regulatory T cells from the tumor bed.
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http://dx.doi.org/10.1016/j.ccell.2016.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715805PMC
July 2016

Interaction between AIF and CHCHD4 Regulates Respiratory Chain Biogenesis.

Mol Cell 2015 Jun 21;58(6):1001-14. Epub 2015 May 21.

Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, 75006 Paris, France; INSERM, UMRS 1138, 75006 Paris, France; Gustave Roussy Comprehensive Cancer Center, 94805 Villejuif, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, 75006 Paris, France; Université Pierre et Marie Curie, 75006 Paris, France. Electronic address:

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.
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http://dx.doi.org/10.1016/j.molcel.2015.04.020DOI Listing
June 2015

Direct interaction between STAT3 and EIF2AK2 controls fatty acid-induced autophagy.

Autophagy 2013 Mar 7;9(3):415-7. Epub 2012 Dec 7.

INSERM, U848, Villejuif, France.

A chemical screen designed to identify novel inducers of autophagy led to the discovery that signal transducer and activator of transcription 3 (STAT3) inhibitors can potently stimulate the autophagic flux. Although STAT3 is best known as a pro-inflammatory and oncogenic transcription factor, mechanistic analyses revealed that autophagy is regulated by the cytoplasmic, not nuclear, pool of STAT3. Cytoplasmic STAT3 normally interacts with the eukaryotic translation initiation factor 2, subunit 1α, 35kDa (EIF2S1/eIF2α) kinase 2/protein kinase, RNA-activated (EIF2AK2/PKR), a sensor of double-stranded RNA. This interaction, which could be recapitulated using recombinant proteins in pull-down experiments, involves the catalytic domain of EIF2AK2 as well as the SH2 domain of STAT3, which can adopt a fold similar to that of EIF2S1. Thus, STAT3 may act as a competitive inhibitor of EIF2AK2. Indeed, pharmacological or genetic inhibition of STAT3 stimulates EIF2AK2-dependent EIF2S1 phosphorylation and autophagy. Conversely, the overexpression of wild-type STAT3 as well as of STAT3 mutants that cannot be phosphorylated by JAK2 or are excluded from the nucleus inhibits autophagy. However, STAT3 mutants that fail to interact with EIF2AK2 are unable to suppress autophagy. Both STAT3-targeting agents (i.e., Stattic, JSI-124 and WP1066) and EIF2AK2 activators (such as the double-strand RNA mimetic polyinosinic:polycytidylic acid) are capable of disrupting the inhibitory interaction between STAT3 and EIF2AK2 in cellula, yet only the latter does so in cell-free systems in vitro. A further screen designed to identify EIF2AK2-dependent autophagy inducers revealed that several fatty acids including palmitate trigger autophagy via a pathway that involves the disruption of the STAT3-EIF2AK2 complex as well as the phosphorylation of mitogen-activated protein kinase 8/c-Jun N-terminal kinase 1 (MAPK8/JNK1) and EIF2S1. These results reveal an unsuspected crosstalk between cellular metabolism (fatty acids), pro-inflammatory signaling (STAT3), innate immunity (EIF2AK2), and translational control (EIF2S1) that regulates autophagy.
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http://dx.doi.org/10.4161/auto.22910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590262PMC
March 2013

Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity.

Mol Cell 2012 Dec 17;48(5):667-80. Epub 2012 Oct 17.

INSERM, U848, 94805 Villejuif, France.

In a screen designed to identify novel inducers of autophagy, we discovered that STAT3 inhibitors potently stimulate the autophagic flux. Accordingly, genetic inhibition of STAT3 stimulated autophagy in vitro and in vivo, while overexpression of STAT3 variants, encompassing wild-type, nonphosphorylatable, and extranuclear STAT3, inhibited starvation-induced autophagy. The SH2 domain of STAT3 was found to interact with the catalytic domain of the eIF2α kinase 2 EIF2AK2, best known as protein kinase R (PKR). Pharmacological and genetic inhibition of STAT3 stimulated the activating phosphorylation of PKR and consequent eIF2α hyperphosphorylation. Moreover, PKR depletion inhibited autophagy as initiated by chemical STAT3 inhibitors or free fatty acids like palmitate. STAT3-targeting chemicals and palmitate caused the disruption of inhibitory STAT3-PKR interactions, followed by PKR-dependent eIF2α phosphorylation, which facilitates autophagy induction. These results unravel an unsuspected mechanism of autophagy control that involves STAT3 and PKR as interacting partners.
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http://dx.doi.org/10.1016/j.molcel.2012.09.013DOI Listing
December 2012

Specific serine-proline phosphorylation and glycogen synthase kinase 3β-directed subcellular targeting of stathmin 3/Sclip in neurons.

J Biol Chem 2012 Jun 10;287(26):22341-53. Epub 2012 May 10.

INSERM U839, Paris F-75005, France.

During nervous system development, neuronal growth, migration, and functional morphogenesis rely on the appropriate control of the subcellular cytoskeleton including microtubule dynamics. Stathmin family proteins play major roles during the various stages of neuronal differentiation, including axonal growth and branching, or dendritic development. We have shown previously that stathmins 2 (SCG10) and 3 (SCLIP) fulfill distinct, independent and complementary regulatory roles in axonal morphogenesis. Although the two proteins have been proposed to display the four conserved phosphorylation sites originally identified in stathmin 1, we show here that they possess distinct phosphorylation sites within their specific proline-rich domains (PRDs) that are differentially regulated by phosphorylation by proline-directed kinases involved in the control of neuronal differentiation. ERK2 or CDK5 phosphorylate the two proteins but with different site specificities. We also show for the first time that, unlike stathmin 2, stathmin 3 is a substrate for glycogen synthase kinase (GSK) 3β both in vitro and in vivo. Interestingly, stathmin 3 phosphorylated at its GSK-3β target site displays a specific subcellular localization at neuritic tips and within the actin-rich peripheral zone of the growth cone of differentiating hippocampal neurons in culture. Finally, pharmacological inhibition of GSK-3β induces a redistribution of stathmin 3, but not stathmin 2, from the periphery toward the Golgi region of neurons. Stathmin proteins can thus be either regulated locally or locally targeted by specific phosphorylation, each phosphoprotein of the stathmin family fulfilling distinct and specific roles in the control of neuronal differentiation.
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http://dx.doi.org/10.1074/jbc.M112.344044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381194PMC
June 2012

Direct molecular interactions between Beclin 1 and the canonical NFκB activation pathway.

Autophagy 2012 Feb 1;8(2):268-70. Epub 2012 Feb 1.

INSERM, Villejuif, France.

General (macro)autophagy and the activation of NFκB constitute prominent responses to a large array of intracellular and extracellular stress conditions. The depletion of any of the three subunits of the inhibitor of NFκB (IκB) kinase (IKKα, IKKβ, IKKγ/NEMO), each of which is essential for the canonical NFκB activation pathway, limits autophagy induction by physiological or pharmacological triggers, while constitutive active IKK subunits suffice to stimulate autophagy. The activation of IKK usually relies on TGFβ-activated kinase 1 (TAK1), which is also necessary for the optimal induction of autophagy in multiple settings. TAK1 interacts with two structurally similar co-activators, TAK1-binding proteins 2 and 3 (TAB2 and TAB3). Importantly, in resting conditions both TAB2 and TAB3 bind the essential autophagic factor Beclin 1, but not TAK1. In response to pro-autophagic stimuli, TAB2 and TAB3 dissociate from Beclin 1 and engage in stimulatory interactions with TAK1. The inhibitory interaction between TABs and Beclin 1 is mediated by their coiled-coil domains (CCDs). Accordingly, the overexpression of either TAB2 or TAB3 CCD stimulates Beclin 1- and TAK1-dependent autophagy. These results point to the existence of a direct molecular crosstalk between the canonical NFκB activation pathway and the autophagic core machinery that guarantees the coordinated induction of these processes in response to stress.
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http://dx.doi.org/10.4161/auto.8.2.18845DOI Listing
February 2012

Inhibition of autophagy by TAB2 and TAB3.

EMBO J 2011 Nov 11;30(24):4908-20. Epub 2011 Nov 11.

INSERM, U848, Villejuif, France.

Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory 'switch' whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.
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http://dx.doi.org/10.1038/emboj.2011.413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243630PMC
November 2011

Nodes of ranvier and paranodes in chronic acquired neuropathies.

PLoS One 2011 Jan 18;6(1):e14533. Epub 2011 Jan 18.

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche en Santé 839, Paris, France.

Chronic acquired neuropathies of unknown origin are classified as chronic inflammatory demyelinating polyneuropathies (CIDP) and chronic idiopathic axonal polyneuropathies (CIAP). The diagnosis can be very difficult, although it has important therapeutic implications since CIDP can be improved by immunomodulating treatment. The aim of this study was to examine the possible abnormalities of nodal and paranodal regions in these two types of neuropathies. Longitudinal sections of superficial peroneal nerves were obtained from biopsy material from 12 patients with CIDP and 10 patients with CIAP and studied by immunofluorescence and in some cases electron microscopy. Electron microscopy revealed multiple alterations in the nodal and paranodal regions which predominated in Schwann cells in CIDP and in axons in CIAP. In CIDP paranodin/Caspr immunofluorescence was more widespread than in control nerves, extending along the axon in internodes where it appeared intense. Nodal channels Nav and KCNQ2 were less altered but were also detected in the internodes. In CIAP paranodes, paranodin labeling was irregular and/or decreased. To test the consequences of acquired primary Schwann cells alteration on axonal proteins, we used a mouse model based on induced deletion of the transcription factor Krox-20 gene. In the demyelinated sciatic nerves of these mice we observed alterations similar to those found in CIDP by immunofluorescence, and immunoblotting demonstrated increased levels of paranodin. Finally we examined whether the alterations in paranodin immunoreactivity could have a diagnosis value. In a sample of 16 biopsies, the study of paranodin immunofluorescence by blind evaluators led to correct diagnosis in 70 ± 4% of the cases. This study characterizes for the first time the abnormalities of nodes of Ranvier in CIAP and CIDP, and the altered expression and distribution of nodal and paranodal proteins. Marked differences were observed between CIDP and CIAP and the alterations in paranodin immunofluorescence may be an interesting tool for their differential diagnosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014533PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022580PMC
January 2011

Drosophila stathmins bind tubulin heterodimers with high and variable stoichiometries.

J Biol Chem 2010 Apr 9;285(15):11667-80. Epub 2010 Feb 9.

INSERM U 839, Paris F-75005, France.

In vertebrates, stathmins form a family of proteins possessing two tubulin binding repeats (TBRs), which each binds one soluble tubulin heterodimer. The stathmins thus sequester two tubulins in a phosphorylation-dependent manner, providing a link between signal transduction and microtubule dynamics. In Drosophila, we show here that a single stathmin gene (stai) encodes a family of D-stathmin proteins. Two of the D-stathmins are maternally deposited and then restricted to germ cells, and the other two are detected in the nervous system during embryo development. Like in vertebrates, the nervous system-enriched stathmins contain an N-terminal domain involved in subcellular targeting. All the D-stathmins possess a domain containing three or four predicted TBRs, and we demonstrate here, using complementary biochemical and biophysical methods, that all four predicted TBR domains actually bind tubulin. D-stathmins can indeed bind up to four tubulins, the resulting complex being directly visualized by electron microscopy. Phylogenetic analysis shows that the presence of regulated multiple tubulin sites is a conserved characteristic of stathmins in invertebrates and allows us to predict key residues in stathmin for the binding of tubulin. Altogether, our results reveal that the single Drosophila stathmin gene codes for a stathmin family similar to the multigene vertebrate one, but with particular tubulin binding properties.
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http://dx.doi.org/10.1074/jbc.M109.096727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857042PMC
April 2010

The PN2-3 domain of centrosomal P4.1-associated protein implements a novel mechanism for tubulin sequestration.

J Biol Chem 2009 Mar 7;284(11):6909-17. Epub 2009 Jan 7.

Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Bâtiment 34, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.

Microtubules are cytoskeletal components involved in multiple cell functions such as mitosis, motility, or intracellular traffic. In vivo, these polymers made of alphabeta-tubulin nucleate mostly from the centrosome to establish the interphasic microtubule network or, during mitosis, the mitotic spindle. Centrosomal P4.1-associated protein (CPAP; also named CENPJ) is a centrosomal protein involved in the assembly of centrioles and important for the centrosome function. This protein contains a microtubule-destabilizing region referred to as PN2-3. Here we decrypt the microtubule destabilization activity of PN2-3 at the molecular level and show that it results from the sequestration of tubulin by PN2-3 in a non-polymerizable 1:1 complex. We also map the tubulin/PN2-3 interaction both on the PN2-3 sequence and on the tubulin surface. NMR and CD data on free PN2-3 in solution show that this is an intrinsically unstructured protein that comprises a 23-amino acid residue alpha-helix. This helix is embedded in a 76-residue region that interacts strongly with tubulin. The interference of PN2-3 with well characterized tubulin properties, namely GTPase activity, nucleotide exchange, vinblastine-induced self-assembly, and stathmin family protein binding, highlights the beta subunit surface located at the intermolecular longitudinal interface when tubulin is embedded in a microtubule as a tubulin/PN2-3 interaction area. These findings characterize the PN2-3 fragment of CPAP as a protein with an unprecedented tubulin sequestering mechanism distinct from that of stathmin family proteins.
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http://dx.doi.org/10.1074/jbc.M808249200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652345PMC
March 2009

N-terminal stathmin-like peptides bind tubulin and impede microtubule assembly.

Biochemistry 2005 Nov;44(44):14616-25

Laboratoire Structure et Reconnaissance des Biomolécules, EA3637, Université Evry-Val d'Essonne, Evry, F-91025 France.

Microtubules are major cytoskeletal components involved in numerous cellular functions such as mitosis, cell motility, or intracellular traffic. These cylindrical polymers of alphabeta-tubulin assemble in a closely regulated dynamic manner. We have shown that the stathmin family proteins sequester tubulin in a nonpolymerizable ternary complex, through their stathmin-like domains (SLD) and thus contribute to the regulation of microtubule dynamics. We demonstrate here that short peptides derived from the N-terminal part of SLDs impede tubulin polymerization with various efficiencies and that phosphorylation of the most potent of these peptides reduces its efficiency as in full-length stathmin. To understand the mechanism of action of these peptides, we undertook a NMR-based structural analysis of the peptide-tubulin interaction with the most efficient peptide (I19L). Our results show that, while disordered when free in solution, I19L folds into a beta-hairpin upon binding to tubulin. We further identified, by means of saturation transfer difference NMR, hydrophobic residues located on the beta2-strand of I19L that are involved in its tubulin binding. These structural data were used together with tubulin atomic coordinates from the tubulin/RB3-SLD crystal structure to model the I19L/tubulin interaction. The model agrees with I19L acting through an autonomous tubulin capping capability to impede tubulin polymerization and provides information to help understand the variation of efficiency against tubulin polymerization among the peptides tested. Altogether these results enlighten the mechanism of tubulin sequestration by SLDs, while they pave the way for the development of protein-based compounds aimed at interfering with tubulin polymerization.
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http://dx.doi.org/10.1021/bi0512492DOI Listing
November 2005

Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain.

Nature 2004 Mar;428(6979):198-202

European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, BP 181, 38042 Grenoble Cedex 9, France.

Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.
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http://dx.doi.org/10.1038/nature02393DOI Listing
March 2004

A synergistic relationship between three regions of stathmin family proteins is required for the formation of a stable complex with tubulin.

Biochem J 2004 Mar;378(Pt 3):877-88

Signalisation et Différenciation Cellulaires dans les Systèmes Nerveux et Musculaire, U440 Institut National de la Santé et de la Recherche Médicale/Université Pierre et Marie Curie, Paris, France.

Stathmin is a ubiquitous 17 kDa cytosolic phosphoprotein proposed to play a general role in the integration and relay of intracellular signalling pathways. It is believed to regulate microtubule dynamics by sequestering tubulin in a complex made of two tubulin heterodimers per stathmin molecule (T2S complex). The other proteins of the stathmin family can also bind two tubulin heterodimers through their SLD (stathmin-like domain), but the different tubulin:SLD complexes display varying stabilities. In this study, we analysed the relative influence of three regions of SLDs on the interaction with tubulin and the mechanistic processes that lead to its sequestration. Tubulin-binding properties of fragments and chimaeras of stathmin and RB3(SLD) were studied in vitro by tubulin polymerization, size-exclusion chromatography and surface plasmon resonance assays. Our results show that the N-terminal region of SLDs favours the binding of the first tubulin heterodimer and that the second C-terminal tubulinbinding site confers the specific stability of a given tubulin:SLD complex. Our results highlight the molecular processes by which tubulin co-operatively interacts with the SLDs. This knowledge may contribute to drug development aimed at disturbing microtubules that could be used for the treatment of cancer.
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http://dx.doi.org/10.1042/BJ20031413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1224029PMC
March 2004

Quantitative RT-PCR reveals a ubiquitous but preferentially neural expression of the KIS gene in rat and human.

Brain Res Mol Brain Res 2003 May;114(1):55-64

Laboratoire de Génétique Moléculaire-UPRES EA 3618, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes-Paris V, 75006 Paris, France.

KIS is the only known protein kinase that possesses an RNA recognition motif. This original structure indicates a role for KIS in the maturation of RNAs possibly by phosphorylating and regulating the activities of RNA associated factors. Another function of KIS has recently been unravelled--it negatively regulates the cdk inhibitor p27Kip1 and thus promotes cell cycle progression through G1. In order to explore the functional expression of this kinase, we quantified its mRNA in a wide range of rat and human tissues, during development and in tumors. In both species, the highest level of KIS gene expression was in adult neural tissues. Interestingly, within the adult rat brain, KIS mRNA is enriched in several areas including the substantia nigra compacta and nuclei of the brain stem. Furthermore, KIS gene expression increases dramatically during brain development. Altogether our results point to a ubiquitous function for KIS together with a particular implication during neural differentiation or in the function of mature neural cells. No dysregulation of KIS gene expression was detected in human tumors from breast, bladder, prostate, liver and kidney origins. On the other hand, the KIS gene was overexpressed in NF1-associated plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) as compared to dermal neurofibroma which suggests a possible implication of KIS in the genesis of NF1-associated tumors.
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http://dx.doi.org/10.1016/s0169-328x(03)00132-3DOI Listing
May 2003

Expression of stathmin family genes in human tissues: non-neural-restricted expression for SCLIP.

Genomics 2003 Apr;81(4):400-10

Laboratoire de Génétique Moléculaire, UPRES JE 2195, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes-Paris V, Paris, France.

The stathmin family consists of phosphoproteins highly conserved in vertebrates and thought to be implicated in the development and functional regulation of various organs, most notably the nervous system. This family includes stathmin, SCG10, SCLIP, and RB3, phosphoproteins that are related by structural and functional homologies. They all sequester tubulin and interfere with microtubule dynamics, a property due to their shared stathmin-like domain. Little is known about the expression of the stathmin gene family in humans. Herein, we describe for the first time, for a collection of human tissues, the expression of each member of this family, using real-time quantitative RT-PCR. We found that stathmin is ubiquitously expressed, whereas SCG10 and RB3 are neural enriched, expression patterns similar to those reported for other mammals. Surprisingly, SCLIP, whose expression is thought to be neural-specific, exhibits a broader tissue distribution. Analyses of the SCLIP gene (approved symbol STMN3) show that it contains several NRSE-like elements that display low or no affinity for the cognate binding protein NRSF. The substantial expression of SCLIP in most tissues points out a novel function for this protein outside the nervous system and raises the possibility that its coexpression with stathmin could provide some degree of functional redundancy.
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http://dx.doi.org/10.1016/s0888-7543(03)00031-4DOI Listing
April 2003
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