Publications by authors named "Patrick A Curmi"

61 Publications

Publisher Correction: Molecular dynamics of the histamine H3 membrane receptor reveals different mechanisms of GPCR signal transduction.

Sci Rep 2021 Jan 25;11(1):2664. Epub 2021 Jan 25.

UMR‑S U1204, Structure et Activité de Biomolécules Normales et Pathologiques, INSERM/Université d'Evry-Val d'Essonne/Université Paris-Saclay, 91000, Evry, France.

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http://dx.doi.org/10.1038/s41598-021-81885-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835230PMC
January 2021

Molecular dynamics of the histamine H3 membrane receptor reveals different mechanisms of GPCR signal transduction.

Sci Rep 2020 10 9;10(1):16889. Epub 2020 Oct 9.

UMR-S U1204, Structure et Activité de Biomolécules Normales et Pathologiques, INSERM/Université d'Evry-Val d'Essonne/Université Paris-Saclay, 91000, Evry, France.

In this work, we studied the mechanisms of classical activation and inactivation of signal transduction by the histamine H3 receptor, a 7-helix transmembrane bundle G-Protein Coupled Receptor through long-time-scale atomistic molecular dynamics simulations of the receptor embedded in a hydrated double layer of dipalmitoyl phosphatidyl choline, a zwitterionic polysaturated ordered lipid. Three systems were prepared: the apo receptor, representing the constitutively active receptor; and two holo-receptors-the receptor coupled to the antagonist/inverse agonist ciproxifan, representing the inactive state of the receptor, and the receptor coupled to the endogenous agonist histamine and representing the active state of the receptor. An extensive analysis of the simulation showed that the three states of H3R present significant structural and dynamical differences as well as a complex behavior given that the measured properties interact in multiple and interdependent ways. In addition, the simulations described an unexpected escape of histamine from the orthosteric binding site, in agreement with the experimental modest affinities and rapid off-rates of agonists.
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http://dx.doi.org/10.1038/s41598-020-73483-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547658PMC
October 2020

Inhibition of Transcription Induces Phosphorylation of YB-1 at Ser102 and Its Accumulation in the Nucleus.

Cells 2019 Dec 31;9(1). Epub 2019 Dec 31.

Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Russia.

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.
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http://dx.doi.org/10.3390/cells9010104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016903PMC
December 2019

A Single-Molecule Atomic Force Microscopy Study of PARP1 and PARP2 Recognition of Base Excision Repair DNA Intermediates.

J Mol Biol 2019 07 23;431(15):2655-2673. Epub 2019 May 23.

Institute of Chemical Biology and Fundamental Medicine (ICBFM) SB RAS, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia. Electronic address:

Nuclear poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) catalyze the synthesis of poly(ADP-ribose) (PAR) and use NAD as a substrate for the polymer synthesis. Both PARP1 and PARP2 are involved in DNA damage response pathways and function as sensors of DNA breaks, including temporary single-strand breaks formed during DNA repair. Consistently, with a role in DNA repair, PARP activation requires its binding to a damaged DNA site, which initiates PAR synthesis. Here we use atomic force microscopy to characterize at the single-molecule level the interaction of PARP1 and PARP2 with long DNA substrates containing a single damage site and representing intermediates of the short-patch base excision repair (BER) pathway. We demonstrated that PARP1 has higher affinity for early intermediates of BER than PARP2, whereas both PARPs efficiently interact with the nick and may contribute to regulation of the final ligation step. The binding of a DNA repair intermediate by PARPs involved a PARP monomer or dimer depending on the type of DNA damage. PARP dimerization influences the affinity of these proteins to DNA and affects their enzymatic activity: the dimeric form is more effective in PAR synthesis in the case of PARP2 but is less effective in the case of PARP1. PARP2 suppresses PAR synthesis catalyzed by PARP1 after single-strand breaks formation. Our study suggests that the functions of PARP1 and PARP2 overlap in BER after a site cleavage and provides evidence for a role of PARP2 in the regulation of PARP1 activity.
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http://dx.doi.org/10.1016/j.jmb.2019.05.028DOI Listing
July 2019

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

Nucleic Acids Res 2019 04;47(6):3127-3141

SABNP, University of Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.
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http://dx.doi.org/10.1093/nar/gky1303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451097PMC
April 2019

Functional differences of short and long isoforms of spastin harboring missense mutation.

Dis Model Mech 2018 09 10;11(9). Epub 2018 Sep 10.

Structure and Activity of Normal and Pathological Biomolecules, INSERM U1204, Université Paris Saclay, Université d'Evry, 91000 Evry, France

Mutations of the () gene encoding for spastin protein are the main causes of hereditary spastic paraplegia. Spastin binds to microtubules and severs them through the enzymatic activity of its AAA domain. Several missense mutations located in this domain lead to stable, nonsevering spastins that decorate a subset of microtubules, suggesting a possible negative gain-of-function mechanism for these mutants. Of the two main isoforms of spastin, only mutations of the long isoform, M1, are supposed to be involved in the onset of the pathology, leaving the role of the ubiquitously expressed shorter one, M87, not fully investigated and understood. Here, we show that two isoforms of spastin harboring the same missense mutation bind and bundle different subsets of microtubules in HeLa cells, and likely stabilize them by increasing the level of acetylated tubulin. However, only mutated M1 has the ability to interact with wild-type M1, and decorates a subset of perinuclear microtubules associated with the endoplasmic reticulum that display higher resistance to microtubule depolymerization and increased intracellular ionic strength, compared with those decorated by mutated M87. We further show that only mutated M1 decorates microtubules of proximal axons and dendrites, and strongly impairs axonal transport in cortical neurons through a mechanism likely independent of the microtubule-severing activity of this protein.
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http://dx.doi.org/10.1242/dmm.033704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177001PMC
September 2018

Microtubules as platforms for probing liquid-liquid phase separation in cells - application to RNA-binding proteins.

J Cell Sci 2018 06 11;131(11). Epub 2018 Jun 11.

SABNP Lab, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France

Liquid-liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides having critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 (also known as TARDBP) and FUS because of their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here, we developed a method to analyze the mixing and demixing of two different phases in a cellular context. The principle is the following: RNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA-binding proteins, HuR (also known as ELAVL1), G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing and demixing behavior of mRNA-binding proteins in cells. Taken together, we show that microtubules can be used as platforms to understand the mechanisms underlying liquid-liquid phase separation and their deregulation in human diseases.
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http://dx.doi.org/10.1242/jcs.214692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031325PMC
June 2018

Multiplex epithelium dysfunction due to CLDN10 mutation: the HELIX syndrome.

Genet Med 2018 02 3;20(2):190-201. Epub 2017 Aug 3.

Qatar Biomedical Research Institute, Hamad Ben Khalifa University, Doha, Qatar.

PurposeWe aimed to identify the genetic cause to a clinical syndrome encompassing hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX syndrome), and to comprehensively delineate the phenotype.MethodsWe performed homozygosity mapping, whole-genome sequencing, gene sequencing, expression studies, functional tests, protein bioinformatics, and histological characterization in two unrelated families with HELIX syndrome.ResultsWe identified biallelic missense mutations (c.386C>T, p.S131L and c.2T>C, p.M1T) in CLDN10B in six patients from two unrelated families. CLDN10B encodes Claudin-10b, an integral tight junction (TJ) membrane-spanning protein expressed in the kidney, skin, and salivary glands. All patients had hypohidrosis, renal loss of NaCl with secondary hyperaldosteronism and hypokalemia, as well as hypolacrymia, ichthyosis, xerostomia, and severe enamel wear. Functional testing revealed that patients had a decreased NaCl absorption in the thick ascending limb of the loop of Henle and a severely decreased secretion of saliva. Both mutations resulted in reduced or absent Claudin-10 at the plasma membrane of epithelial cells.ConclusionCLDN10 mutations cause a dysfunction in TJs in several tissues and, subsequently, abnormalities in renal ion transport, ectodermal gland homeostasis, and epidermal integrity.
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http://dx.doi.org/10.1038/gim.2017.71DOI Listing
February 2018

Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

Biochem Pharmacol 2016 Feb 18;102:45-63. Epub 2015 Dec 18.

Institut National de la Santé et de la Recherche Médicale, UMR1204, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val d'Essonne, Evry 91025, France. Electronic address:

Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research.
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http://dx.doi.org/10.1016/j.bcp.2015.12.013DOI Listing
February 2016

Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.

Nucleic Acids Res 2016 Apr 15;44(6):e60. Epub 2015 Dec 15.

Institute of Chemical Biology and Fundamental Medicine, 630090, Novosibirsk, Russian Federation Novosibirsk State University, 630090, Novosibirsk, Russian Federation

PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages.
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http://dx.doi.org/10.1093/nar/gkv1476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824093PMC
April 2016

Probing protein interactions in living mammalian cells on a microtubule bench.

Sci Rep 2015 Nov 27;5:17304. Epub 2015 Nov 27.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM U1204 and Université Evry-Val d'Essonne, Evry, 91025 France.

Microtubules are μm-long cylinders of about 25 nm in diameter which are present in the cytoplasm of eukaryotic cells. Here, we have developed a new method which uses these cylindrical structures as platforms to detect protein interactions in cells. The principle is simple: a protein of interest used as bait is brought to microtubules by fusing it to Tau, a microtubule-associated protein. The presence of a protein prey on microtubules then reveals an interaction between bait and prey. This method requires only a conventional optical microscope and straightforward fluorescence image analysis for detection and quantification of protein interactions. To test the reliability of this detection scheme, we used it to probe the interactions among three mRNA-binding proteins in both fixed and living cells and compared the results to those obtained by pull-down assays. We also tested whether the molecular interactions of Cx43, a membrane protein, can be investigated with this system. Altogether, the results indicate that microtubules can be used as platforms to detect protein interactions in mammalian cells, which should provide a basis for investigating pathogenic protein interactions involved in human diseases.
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http://dx.doi.org/10.1038/srep17304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661529PMC
November 2015

Poly(ADP-ribosyl)ation as a new posttranslational modification of YB-1.

Biochimie 2015 Dec 9;119:36-44. Epub 2015 Oct 9.

Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, 630090, Russia; Novosibirsk State University, Novosibirsk, 630090, Russia. Electronic address:

Multifunctional Y-box binding protein 1 (YB-1) is actively studied as one of the components of cellular response to genotoxic stress. However, the precise role of YB-1 in the process of DNA repair is still obscure. In the present work we report for the first time new posttranslational modification of YB-1 - poly(ADP-ribosyl)ation, catalyzed by one of the main regulatory enzymes of DNA repair - poly(ADP-ribose)polymerase 1 (PARP1) in the presence of model DNA substrate carrying multiple DNA lesions. Therefore, poly(ADP-ribosyl)ation of YB-1 catalyzed with PARP1, can be stimulated by damaged DNA. The observed property of YB-1 underlines its ability to participate in the DNA repair by its involvement in the regulatory cascades of DNA repair.
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http://dx.doi.org/10.1016/j.biochi.2015.10.008DOI Listing
December 2015

Highly delayed systemic translocation of aluminum-based adjuvant in CD1 mice following intramuscular injections.

J Inorg Biochem 2015 Nov 22;152:199-205. Epub 2015 Jul 22.

INSERM U955 E10, Paris Est University, Créteil, France; Faculté des Sciences & Technologie UPEC, Créteil, France.

Concerns regarding vaccine safety have emerged following reports of potential adverse events in both humans and animals. In the present study, alum, alum-containing vaccine and alum adjuvant tagged with fluorescent nanodiamonds were used to evaluate i) the persistence time at the injection site, ii) the translocation of alum from the injection site to lymphoid organs, and iii) the behavior of adult CD1 mice following intramuscular injection of alum (400 μg Al/kg). Results showed for the first time a strikingly delayed systemic translocation of adjuvant particles. Alum-induced granuloma remained for a very long time in the injected muscle despite progressive shrinkage from day 45 to day 270. Concomitantly, a markedly delayed translocation of alum to the draining lymph nodes, major at day 270 endpoint, was observed. Translocation to the spleen was similarly delayed (highest number of particles at day 270). In contrast to C57BL/6J mice, no brain translocation of alum was observed by day 270 in CD1 mice. Consistently neither increase of Al cerebral content, nor behavioral changes were observed. On the basis of previous reports showing alum neurotoxic effects in CD1 mice, an additional experiment was done, and showed early brain translocation at day 45 of alum injected subcutaneously at 200 μg Al/kg. This study confirms the striking biopersistence of alum. It points out an unexpectedly delayed diffusion of the adjuvant in lymph nodes and spleen of CD1 mice, and suggests the importance of mouse strain, route of administration, and doses, for future studies focusing on the potential toxic effects of aluminum-based adjuvants.
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http://dx.doi.org/10.1016/j.jinorgbio.2015.07.004DOI Listing
November 2015

mRNA and DNA selection via protein multimerization: YB-1 as a case study.

Nucleic Acids Res 2015 Oct 13;43(19):9457-73. Epub 2015 Aug 13.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM U1204 and Université Evry-Val d'Essonne, Evry, 91025 France

Translation is tightly regulated in cells for keeping adequate protein levels, this task being notably accomplished by dedicated mRNA-binding proteins recognizing a specific set of mRNAs to repress or facilitate their translation. To select specific mRNAs, mRNA-binding proteins can strongly bind to specific mRNA sequences/structures. However, many mRNA-binding proteins rather display a weak specificity to short and redundant sequences. Here we examined an alternative mechanism by which mRNA-binding proteins could inhibit the translation of specific mRNAs, using YB-1, a major translation regulator, as a case study. Based on a cooperative binding, YB-1 forms stable homo-multimers on some mRNAs while avoiding other mRNAs. Via such inhomogeneous distribution, YB-1 can selectively inhibit translation of mRNAs on which it has formed stable multimers. This novel mechanistic view on mRNA selection may be shared by other proteins considering the elevated occurrence of multimerization among mRNA-binding proteins. Interestingly, we also demonstrate how, by using the same mechanism, YB-1 can form multimers on specific DNA structures, which could provide novel insights into YB-1 nuclear functions in DNA repair and multi-drug resistance.
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http://dx.doi.org/10.1093/nar/gkv822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627072PMC
October 2015

Fluorescent nanodiamonds as a relevant tag for the assessment of alum adjuvant particle biodisposition.

BMC Med 2015 Jun 17;13:144. Epub 2015 Jun 17.

Institut National de la Santé et de la Recherche Médicale (INSERM) - UMR 1204, Université Evry-Val d'Essonne, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Evry, France.

Background: Aluminum oxyhydroxide (alum) is a crystalline compound widely used as an immunologic adjuvant of vaccines. Concerns linked to alum particles have emerged following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion in patients with myalgic encephalomyelitis, revealing an unexpectedly long-lasting biopersistence of alum within immune cells and a fundamental misconception of its biodisposition. Evidence that aluminum-coated particles phagocytozed in the injected muscle and its draining lymph nodes can disseminate within phagocytes throughout the body and slowly accumulate in the brain further suggested that alum safety should be evaluated in the long term. However, lack of specific staining makes difficult the assessment of low quantities of bona fide alum adjuvant particles in tissues.

Methods: We explored the feasibility of using fluorescent functionalized nanodiamonds (mfNDs) as a permanent label of alum (Alhydrogel(®)). mfNDs have a specific and perfectly photostable fluorescence based on the presence within the diamond lattice of nitrogen-vacancy centers (NV centers). As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term. We thus developed fluorescent nanodiamonds functionalized by hyperbranched polyglycerol (mfNDs) allowing good coupling and stability of alum:mfNDs (AluDia) complexes. Specificities of AluDia complexes were comparable to the whole reference vaccine (anti-hepatitis B vaccine) in terms of particle size and zeta potential.

Results: In vivo, AluDia injection was followed by prompt phagocytosis and AluDia particles remained easily detectable by the specific signal of the fND particles in the injected muscle, draining lymph nodes, spleen, liver and brain. In vitro, mfNDs had low toxicity on THP-1 cells and AluDia showed cell toxicity similar to alum alone. Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes.

Conclusions: The fluorescent nanodiamond technology is able to overcome the limitations of previously used organic fluorophores, thus appearing as a choice methodology for studying distribution, persistence and long-term neurotoxicity of alum adjuvants and beyond of other types of nanoparticles.
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http://dx.doi.org/10.1186/s12916-015-0388-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4482291PMC
June 2015

Inhibition of abasic site cleavage in bubble DNA by multifunctional protein YB-1.

J Mol Recognit 2015 Feb 21;28(2):117-23. Epub 2015 Jan 21.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk, 630090, Russia.

Y-box binding protein 1 (YB-1) is widely known to participate in a multiple DNA and RNA processing events in the living cell. YB-1 is also regarded as a putative component of DNA repair. This possibility is supported by relocalization of YB-1 into the nucleus following genotoxic stress. Increased affinity of YB-1 for damaged DNA, especially in its single-stranded form, and its functional interaction with proteins responsible for the initiation of apurinic/apyrimidinic (AP) site repair, namely, AP endonuclease 1 and DNA glycosylase NEIL1, suggest that YB-1 could be involved in the repair of AP sites as a regulatory protein. Here we show that YB-1 has a significant inhibitory effect on the cleavage of AP sites located in single-stranded DNA and in DNA bubble structures. Such interference may be considered as a possible mechanism to prevent single-stranded intermediates of DNA replication, transcription and repair from being converted into highly genotoxic DNA strand breaks, thus allowing the cell to coordinate different DNA processing mechanisms.
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http://dx.doi.org/10.1002/jmr.2435DOI Listing
February 2015

Reply to "Comment on 'Structural properties of POPC monolayers under lateral compression: computer simulations analysis'".

Langmuir 2015 Jan 8;31(2):888-9. Epub 2015 Jan 8.

INSERM, U829, Laboratoire Structure - Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val-d'Essonne , F-91025 Evry, France.

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http://dx.doi.org/10.1021/la504104eDOI Listing
January 2015

Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules.

Nucleic Acids Res 2014 Jul 10;42(13):8678-91. Epub 2014 Jul 10.

Institut National de la Santé et de la Recherche Médicale (INSERM), UMR829; Université Evry-Val d'Essonne, Evry 91025, France

The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.
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http://dx.doi.org/10.1093/nar/gku582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117795PMC
July 2014

Mutations in zinc finger 407 [ZNF407] cause a unique autosomal recessive cognitive impairment syndrome.

Orphanet J Rare Dis 2014 Jun 7;9:80. Epub 2014 Jun 7.

Qatar Biomedical Research Institute, Medical Genetics Center, 69 Lusail Street, West Bay Area, P,O, Box: 33123, Doha, Qatar.

Background: A consanguineous Arab family is affected by an apparently novel autosomal recessive disorder characterized by cognitive impairment, failure-to-thrive, hypotonia and dysmorphic features including bilateral ptosis and epicanthic folds, synophrys, midface hypoplasia, downturned mouth corners, thin upper vermillion border and prominent ears, bilateral 5th finger camptodactyly, bilateral short 4th metatarsal bones, and limited knee mobility bilaterally.

Methods: The family was studied by homozygosity mapping, candidate gene mutation screening and whole Exome Next Generation Sequencing of a single affected member to identify the offending gene and mutation. The mutated gene product was studied by structural bioinformatics methods.

Results: A damaging c.C5054G mutation affecting an evolutionary highly conserved amino acid p.S1685W was identified in the ZNF407 gene at 18q23. The Serine to Tryptophane mutation affects two of the three ZNF407 isoforms and is located in the last third of the protein, in a linker peptide adjoining two zinc-finger domains. Structural analyses of this mutation shows disruption of an H-bond that locks the relative spatial position of the two fingers, leading to a higher flexibility of the linker and thus to a decreased probability of binding to the target DNA sequence essentially eliminating the functionality of downstream domains and interfering with the expression of various genes under ZNF407 control during fetal brain development.

Conclusions: ZNF407 is a transcription factor with an essential role in brain development. When specific and limited in number homozygosity intervals exist that harbor the offending gene in consanguineous families, Whole Exome Sequencing of a single affected individual is an efficient approach to gene mapping and mutation identification.
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http://dx.doi.org/10.1186/1750-1172-9-80DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070100PMC
June 2014

Structural properties of POPC monolayers under lateral compression: computer simulations analysis.

Langmuir 2014 Jan 7;30(2):564-73. Epub 2014 Jan 7.

INSERM, U829, Laboratoire Structure - Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val-d'Essonne , F-91025 Evry, France.

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a lipid comprising a saturated and an unsaturated acyl chain, belongs to the class of glycerophosphatidylcholines, major lipids in eukaryotic cell membranes. To get insight into the structural properties of this lipid within monolayers as membrane models, we performed molecular dynamics (MD) simulations of POPC monolayers under compression at the air/water interface. MD simulations were carried out at 300 K and at different surface pressures using the all-atom general Amber force field (GAFF). A good agreement was found between the simulated data and experimental isotherms. At surface pressures greater than 15 mN/m, two orientations of the head groups clearly appear: one nearly parallel to the monolayer interface and another one pointing toward the water. On the basis of the analysis of headgroup dihedral angles, we propose that the conformational variations around the bonds connecting the phosphorus atom to the adjacent oxygens are involved in these two orientations of the headgroup. The glycerol group orientation is characterized by a large distribution centered around 50° with respect to the monolayer normal. The acyl chains are predominantly in trans configuration from 7.5 to 43 mN/m surface pressures. Moreover, the calculated order parameter profiles of both chains suggest an independent behavior of the saturated and unsaturated chains that could be correlated with the formation of chain-type clusters observed along the simulated trajectories.
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http://dx.doi.org/10.1021/la4043809DOI Listing
January 2014

Structural basis for the association of MAP6 protein with microtubules and its regulation by calmodulin.

J Biol Chem 2013 Aug 6;288(34):24910-22. Epub 2013 Jul 6.

Institut National de la Santé et de la Recherche Médicale (INSERM), UMR829, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université Evry-Val d'Essonne, Evry 91025, France.

Microtubules are highly dynamic αβ-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90-177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90-177) to microtubules with a 1:1 MAP6(90-177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90-177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca(2+)-calmodulin competes with microtubules for MAP6(90-177) binding and that the binding mode of MAP6(90-177) to microtubules and Ca(2+)-calmodulin involves a common stretch of amino acid residues on the MAP6(90-177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca(2+)-calmodulin.
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http://dx.doi.org/10.1074/jbc.M113.457267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750186PMC
August 2013

Detection of single DNA molecule hybridization on a surface by atomic force microscopy.

Small 2013 Nov 15;9(21):3630-8. Epub 2013 May 15.

Inserm U829, Laboratoire Structure-Activité des, Biomolécules Normales et Pathologiques, Université d'Evry-Val d'Essonne, Evry 91025, France.

Improving the detection of DNA hybridization is a critical issue for several challenging applications encountered in microarray and biosensor domains. Herein, it is demonstrated that hybridization between complementary single-stranded DNA (ssDNA) molecules loosely adsorbed on a mica surface can be achieved thanks to fine-tuning of the composition of the hybridization buffer. Single-molecule DNA hybridization occurs in only a few minutes upon encounters of freely diffusing complementary strands on the mica surface. Interestingly, the specific hybridization between complementary ssDNA is not altered in the presence of large amounts of nonrelated DNA. The detection of single-molecule DNA hybridization events is performed by measuring the contour length of DNA in atomic force microscopy images. Besides the advantage provided by facilitated diffusion, which promotes hybridization between probes and targets on mica, the present approach also allows the detection of single isolated DNA duplexes and thus requires a very low amount of both probe and target molecules.
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http://dx.doi.org/10.1002/smll.201300546DOI Listing
November 2013

An intercellular polyamine transfer via gap junctions regulates proliferation and response to stress in epithelial cells.

Mol Biol Cell 2013 May 20;24(10):1529-43. Epub 2013 Mar 20.

Institut National de la Santé et de la Recherche Médicale, UMR829, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université Evry-Val d'Essonne, Evry 91025, France.

In the organism, quiescent epithelial cells have the potential to resume cycling as a result of various stimuli, including wound healing or oxidative stress. Because quiescent cells have a low polyamine level, resuming their growth requires an increase of their intracellular polyamine levels via de novo polyamine synthesis or their uptake from plasma. Another alternative, explored here, is an intercellular exchange with polyamine-rich cycling cells via gap junctions. We show that polyamines promote gap junction communication between proliferating cells by promoting dynamical microtubule plus ends at the cell periphery and thus allow polyamine exchange between cells. In this way, cycling cells favor regrowth in adjacent cells deprived of polyamines. In addition, intercellular interactions mediated by polyamines can coordinate the translational response to oxidative stress through the formation of stress granules. Some putative in vivo consequences of polyamine-mediated intercellular interactions are also discussed regarding cancer invasiveness and tissue regeneration.
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http://dx.doi.org/10.1091/mbc.E12-10-0729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3655814PMC
May 2013

Mapping the conformational stability of maltose binding protein at the residue scale using nuclear magnetic resonance hydrogen exchange experiments.

Biochemistry 2012 Nov 24;51(44):8919-30. Epub 2012 Oct 24.

Centre National de la Recherche Scientifique UMR 8587, Université Evry-Val d'Essonne et Cergy Pontoise, Laboratoire d'Analyse et de modélisation pour la Biologie et l'Environnement, Evry 91025, France.

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to β-cyclodextrin (MBP-βCD) under native conditions. We observed that the last helix of MBP (helix α14) has a lower protection factor than the rest of the protein. Further, HX experiments were performed using guanidine hydrochloride under subdenaturing conditions to discriminate between local fluctuations and global unfolding events and to determine the MBP-βCD energy landscape. The results show that helix α4 and a part of helices α5 and α6 are clearly grouped into a subdenaturing folding unit and represent a partially folded intermediate under native conditions. In addition, we observed that amide protons located in the hinge between the two globular domains share similar ΔG(gu)(app) and m values and should unfold simultaneously. These observations provide new points of view for improving our understanding of the thermodynamic stability and the mechanisms that drive folding-unfolding dynamics of proteins.
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http://dx.doi.org/10.1021/bi3003605DOI Listing
November 2012

Microtubule-targeting drugs rescue axonal swellings in cortical neurons from spastin knockout mice.

Dis Model Mech 2013 Jan 5;6(1):72-83. Epub 2012 Jul 5.

Inserm U798, Laboratoire de Neurogénétique Moléculaire, Université d'Evry-Val d'Essonne et Paris XI, Evry, 91057, France.

Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP), a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.
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http://dx.doi.org/10.1242/dmm.008946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3529340PMC
January 2013

Phosphorylation controls the interaction of the connexin43 C-terminal domain with tubulin and microtubules.

Biochemistry 2012 May 14;51(21):4331-42. Epub 2012 May 14.

Institut National de la Santé et de la Recherche Médicale (INSERM), UMR829, Université Evry-Val d'Essonne, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Evry 91025, France.

Connexins are structurally related transmembrane proteins that assemble to form gap junction channels involved in the mediation of intercellular communication. It has been shown that the intracellular tail of connexin43 (Cx43) interacts with tubulin and microtubules with putative impacts on its own intracellular trafficking, its activity in channel communication, and its interference with specific growth factor signal transduction cascades. We demonstrate here that the microtubule binding of Cx43 is mainly driven by a short region of 26 amino acid residues located within the intracellular tail of Cx43. The nuclear magnetic resonance structural analysis of a peptide (K26D) corresponding to this region shows that this peptide is unstructured when free in solution and adopts a helix conformation upon binding with tubulin. In addition, the resulting K26D-tubulin molecular complex defines a new structural organization that could be shared by other microtubule partners. Interestingly, the K26D-tubulin interaction is prevented by the phosphorylation of K26D at a src kinase specific site. Altogether, the results elucidate the mechanism of the interaction of Cx43 with the microtubule cytoskeleton and propose a pathway for understanding the microtubule-dependent regulation of Cx43 gap junctional communications and the involvement of Cx43 in TGF-β signal transduction.
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http://dx.doi.org/10.1021/bi201806jDOI Listing
May 2012

The state of the guanosine nucleotide allosterically affects the interfaces of tubulin in protofilament.

J Comput Aided Mol Des 2012 Apr 19;26(4):397-407. Epub 2012 Apr 19.

Institut National de la Santé et de la Recherche Médicale (INSERM), UMRS 829, Evry, France.

The dynamics of microtubules is essential for many microtubule-dependent cellular functions such as the mitosis. It has been recognized for a long time that GTP hydrolysis in αβ-tubulin polymers plays a critical role in this dynamics. However, the effects of the changes in the nature of the guanosine nucleotide at the E-site in β-tubulin on microtubule structure and stability are still not well understood. In the present work, we performed all-atom molecular dynamics simulations of a αβα-tubulin heterotrimer harboring a guanosine nucleotide in three different states at the E-site: GTP, GDP-Pi and GDP. We found that changes in the nucleotide state is associated with significant conformational variations at the α-tubulin N- and β-tubulin M-loops which impact the interactions between tubulin protofilaments. The results also show that GTP hydrolysis reduces αβ-tubulin interdimer contacts in favor of intradimer interface. From an atomistic point view, we propose a role for α-tubulin glutamate residue 254 in catalytic magnesium coordination and identified a water molecule in the nucleotide binding pocket which is most probably required for nucleotide hydrolysis. Finally, the results are discussed with reference to the role of taxol in microtubule stability and the recent tubulin-sT2R crystal structures.
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http://dx.doi.org/10.1007/s10822-012-9566-xDOI Listing
April 2012

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1.

J Mol Recognit 2012 Apr;25(4):224-33

Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Prospect Lavrentieva 8, Novosibirsk, 630090, Russia.

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.
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http://dx.doi.org/10.1002/jmr.2182DOI Listing
April 2012

Macromolecular crowding regulates assembly of mRNA stress granules after osmotic stress: new role for compatible osmolytes.

J Biol Chem 2012 Jan 6;287(4):2446-58. Epub 2011 Dec 6.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM U829 and Université Evry-Val d'Essonne, Evry 91025, France.

The massive uptake of compatible osmolytes such as betaine, taurine, and myo-inositol is a protective response shared by all eukaryotes exposed to hypertonic stress. Their accumulation results mostly from the expression of specific transporters triggered by the transcriptional factor NFAT5/TonEBP. This allows the recovery of the cell volume without increasing intracellular ionic strength. In this study we consider the assembly and dissociation of mRNA stress granules (SGs) in hypertonic-stressed cells and the role of compatible osmolytes. In agreement with in vitro results obtained on isolated mRNAs, both macromolecular crowding and a high ionic strength favor the assembly of SGs in normal rat kidney epithelial cells. However, after hours of constant hypertonicity, the slow accumulation in the cytoplasm of compatible osmolytes via specific transporters both reduces macromolecular crowding and ionic strength, thus leading to the progressive dissociation of SGs. In line with this, when cells are exposed to hypertonicity to accumulate a large amount of compatible osmolytes, the formation of SGs is severely impaired, and cells increase their chances of survival to another hypertonic episode. Altogether, these results indicate that the impact of compatible osmolytes on the mRNA-associated machineries and especially that associated with SGs may play an important role in cell resistance and adaption to hyperosmolarity in many tissues like kidney and liver.
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http://dx.doi.org/10.1074/jbc.M111.292748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3268405PMC
January 2012

Rapid assembly and collective behavior of microtubule bundles in the presence of polyamines.

Biophys J 2011 Jul;101(1):205-16

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Institut National de la Santé et de la Recherche Médicale, U829, and Université Evry-Val d'Essonne, EA3637, Evry, France.

Microtubules (MTs) are cylindrical cytoskeleton polymers composed of α-β tubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons.
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http://dx.doi.org/10.1016/j.bpj.2011.05.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3127188PMC
July 2011