Publications by authors named "David Pastré"

43 Publications

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain.

Commun Biol 2021 Mar 19;4(1):359. Epub 2021 Mar 19.

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

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved β-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.
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http://dx.doi.org/10.1038/s42003-021-01862-3DOI Listing
March 2021

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

Fused in Sarcoma (FUS) in DNA Repair: Tango with Poly(ADP-ribose) Polymerase 1 and Compartmentalisation of Damaged DNA.

Int J Mol Sci 2020 Sep 24;21(19). Epub 2020 Sep 24.

Institute of Chemical Biology and Fundamental Medicine, SB RAS, 630090 Novosibirsk, Russia.

The fused in sarcoma (FUS) protein combines prion-like properties with a multifunctional DNA/RNA-binding domain and has functions spanning the regulation of RNA metabolism, including transcription, pre-mRNA splicing, mRNA transport and translation. In addition to its roles in RNA metabolism, FUS is implicated in the maintenance of DNA integrity. In this review, we examine the participation of FUS in major DNA repair pathways, focusing on DNA repair associated with poly(ADP-ribosyl)ation events and on how the interaction of FUS with poly(ADP-ribose) may orchestrate transient compartmentalisation of DNA strand breaks. Unravelling how prion-like RNA-binding proteins control DNA repair pathways will deepen our understanding of the pathogenesis of some neurological diseases and cancer as well as provide the basis for the development of relevant innovative therapeutic technologies. This knowledge may also extend the range of applications of poly(ADP-ribose) polymerase inhibitors to the treatment of neurodegenerative diseases related to RNA-binding proteins in the cell, e.g., amyotrophic lateral sclerosis and frontotemporal lobar degeneration.
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http://dx.doi.org/10.3390/ijms21197020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582374PMC
September 2020

Regulation of Poly(ADP-Ribose) Polymerase 1 Activity by Y-Box-Binding Protein 1.

Biomolecules 2020 09 16;10(9). Epub 2020 Sep 16.

Institute of Chemical Biology and Fundamental Medicine, SB RAS, 630090 Novosibirsk, Russia.

Y-box-binding protein 1 (YB-1) is a multifunctional positively charged protein that interacts with DNA or RNA and poly(ADP-ribose) (PAR). YB-1 is poly(ADP-ribosyl)ated and stimulates poly(ADP-ribose) polymerase 1 (PARP1) activity. Here, we studied the mechanism of YB-1-dependent PAR synthesis by PARP1 in vitro using biochemical and atomic force microscopy assays. PAR synthesis activity of PARP1 is known to be facilitated by co-factors such as Mg. However, in contrast to an Mg-dependent reaction, the activation of PARP1 by YB-1 is accompanied by overall up-regulation of protein PARylation and shortening of the PAR polymer. Therefore, YB-1 and cation co-factors stimulated PAR synthesis in divergent ways. PARP1 autoPARylation in the presence of YB-1 as well as trans-PARylation of YB-1 are greatly affected by the type of damaged DNA, suggesting that PARP1 activation depends on the formation of a PARP1-YB-1-DNA ternary complex. An unstructured C-terminal part of YB-1 involved in an interaction with PAR behaves similarly to full-length YB-1, indicating that both DNA and PAR binding are involved in the stimulation of PARP1 activity by YB-1. Thus, YB-1 is likely linked to the regulation of PARylation events in cells via an interaction with PAR and damaged DNA.
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http://dx.doi.org/10.3390/biom10091325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565162PMC
September 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

U2AF assemblies drive sequence-specific splice site recognition.

EMBO Rep 2019 08 4;20(8):e47604. Epub 2019 Jul 4.

SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, Evry, France.

The essential splicing factor U2AF is known to help anchoring U2 snRNP at the branch site. Its C-terminal UHM domain interacts with ULM motifs of SF3b155, an U2 snRNP protein. Here, we report a cooperative binding of U2AF and the related protein CAPERα to the multi-ULM domain of SF3b155. In addition, we show that the RS domain of U2AF drives a liquid-liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF or CAPERα improves the inclusion of cassette exons that are preceded by such repeated pyrimidine-rich motifs. These results support a model in which liquid-like assemblies of U2AF and CAPERα on repetitive pyrimidine-rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi-ULM domain of SF3b155. We anticipate that posttranslational modifications and proteins recruited in dynamical U2AF and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.
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http://dx.doi.org/10.15252/embr.201847604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681011PMC
August 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

PARP-1 Activation Directs FUS to DNA Damage Sites to Form PARG-Reversible Compartments Enriched in Damaged DNA.

Cell Rep 2019 05;27(6):1809-1821.e5

SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France. Electronic address:

PARP-1 synthesizes long poly(ADP-ribose) chains (PAR) at DNA damage sites to recruit DNA repair factors. Among proteins relocated on damaged DNA, the RNA-binding protein FUS is one of the most abundant, raising the issue about its involvement in DNA repair. Here, we reconstituted the PARP-1/PAR/DNA system in vitro and analyzed at the single-molecule level the role of FUS. We demonstrate successively the dissociation of FUS from mRNA, its recruitment at DNA damage sites through its binding to PAR, and the assembly of damaged DNA-rich compartments. PARG, an enzyme family that hydrolyzes PAR, is sufficient to dissociate damaged DNA-rich compartments in vitro and initiates the nucleocytoplasmic shuttling of FUS in cells. We anticipate that, consistent with previous models, FUS facilitates DNA repair through the transient compartmentalization of DNA damage sites. The nucleocytoplasmic shuttling of FUS after the PARG-mediated compartment dissociation may participate in the formation of cytoplasmic FUS aggregates.
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http://dx.doi.org/10.1016/j.celrep.2019.04.031DOI Listing
May 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

Relation Between Stress Granules and Cytoplasmic Protein Aggregates Linked to Neurodegenerative Diseases.

Curr Neurol Neurosci Rep 2018 11 8;18(12):107. Epub 2018 Nov 8.

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

ᅟ: A hallmark of neurodegenerative diseases is the accumulation of cytoplasmic protein aggregates in neurons of affected subjects. Among recently identified elements of these aggregates are RNA-binding proteins (RBPs) involved in RNA metabolism and alternative splicing and have in common the presence of low complexity domains (LCD) that are prone to self-assemble and form aggregates. The mechanism of cytoplasmic protein aggregation remains elusive. Stress granules (SGs) that are micrometric RNA-protein assemblies located in the cytoplasm of cells exposed to environmental stress are suspected to play the role of seeds. The review sheds light on the recent experimental results that suggest a link between SGs and cytoplasmic protein aggregates but also propose other routes for the formation of these aggregates. PURPOSE OF REVIEW: To analyze the potential relationship between cytoplasmic protein aggregates in neurons of affected subjects and stress granules. RECENT FINDINGS: Liquid phase separation explains how protein and RNA could assemble in membraneless compartments, notably SGs. These results highlight the importance of RBPs with LCD in the SG assembly. Maturation of SGs and in particular the dense core is a potential source of insoluble protein aggregates. Several lines of evidence linked stress granule dynamics to pathogenic protein aggregates. (i) Proteins that accumulate in cytoplasmic aggregates are also SG components. (ii) Neurons are specifically exposed to stress events due to their high metabolism and long lifespan. (iii) Diseases linked protein mutations affect the SG dynamics. (iv) SG dense core could be a breeding ground for protein aggregates. However, we should also keep in mind that SGs are not the only RNA-protein assembly in the cytoplasm; the RNA transport granules could also play a role in the formation of insoluble protein aggregates.
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http://dx.doi.org/10.1007/s11910-018-0914-7DOI Listing
November 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

Vectofusin-1, a potent peptidic enhancer of viral gene transfer forms pH-dependent α-helical nanofibrils, concentrating viral particles.

Acta Biomater 2017 12 7;64:259-268. Epub 2017 Oct 7.

CNRS, Univ. of Strasbourg, Institut de Chimie UMR_7177, Strasbourg, France. Electronic address:

Gene transfer using lentiviral vectors has therapeutic applications spanning from monogenic and infectious diseases to cancer. Such gene therapy has to be improved by enhancing the levels of viral infection of target cells and/or reducing the amount of lentivirus for greater safety and reduced costs. Vectofusin-1, a recently developed cationic amphipathic peptide with a pronounced capacity to enhance such viral transduction, strongly promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium. To clarify the molecular basis of its action the peptide was investigated on a molecular and a supramolecular level by a variety of biophysical approaches. We show that in culture medium vectofusin-1 rapidly forms complexes in the 10 nm range that further assemble into annular and extended nanofibrils. These associate with viral particles allowing them to be easily pelleted for optimal virus-cell interaction. Thioflavin T fluorescence, circular dichroism and infrared spectroscopies indicate that these fibrils have a unique α-helical structure whereas most other viral transduction enhancers form β-amyloid fibrils. A vectofusin-1 derivative (LAH2-A4) is inefficient in biological assays and does not form nanofibrils, suggesting that supramolecular assembly is essential for transduction enhancement. Our observations define vectofusin-1 as a member of a new class of α-helical enhancers of lentiviral infection. Its fibril formation is reversible which bears considerable advantages in handling the peptide in conditions well-adapted to Good Manufacturing Practices and scalable gene therapy protocols.
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http://dx.doi.org/10.1016/j.actbio.2017.10.009DOI Listing
December 2017

Nanoscale Analysis Reveals the Maturation of Neurodegeneration-Associated Protein Aggregates: Grown in mRNA Granules then Released by Stress Granule Proteins.

ACS Nano 2017 07 5;11(7):7189-7200. Epub 2017 Jul 5.

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

TDP-43 and FUS are two mRNA-binding proteins associated with neurodegenerative diseases that form cytoplasmic inclusions with prion-like properties in affected neurons. Documenting the early stages of the formation of TDP-43 or FUS protein aggregates and the role of mRNA stress granules that are considered as critical intermediates for protein aggregation is therefore of interest to understand disease propagation. Here, we developed a single molecule approach via atomic force microscopy (AFM), which provides structural information out of reach by fluorescence microscopy. In addition, the aggregation process can be probed in the test tube without separating the interacting partners, which would affect the thermodynamic equilibrium. The results demonstrate that isolated mRNA molecules serve as crucibles to promote TDP-43 and FUS multimerization. Their subsequent merging results in the formation of mRNA granules containing TDP-43 and FUS aggregates. Interestingly, TDP-43 or FUS protein aggregates can be released from mRNA granules by either YB-1 or G3BP1, two stress granule proteins that compete for the binding to mRNA with TDP-43 and FUS. Altogether, the results indicate that age-related successive assembly/disassembly of stress granules in neurons, regulated by mRNA-binding proteins such as YB-1 and G3BP1, could be a source of protein aggregation.
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http://dx.doi.org/10.1021/acsnano.7b03071DOI Listing
July 2017

Polyamine signal through gap junctions: A key regulator of proliferation and gap-junction organization in mammalian tissues?

Bioessays 2016 06 29;38(6):498-507. Epub 2016 Apr 29.

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

We propose that interaction rules derived from polyamine exchange in connected cells may explain the spatio-temporal organization of gap junctions observed during tissue regeneration and tumorigenesis. We also hypothesize that polyamine exchange can be considered as signal that allows cells to sense the proliferation status of their neighbors. Polyamines (putrescine, spermidine, and spermine) are indeed small aliphatic polycations that serve as fuels to sustain elevated proliferation rates of the order observed in cancer cells. Based on recent reports, we consider here that polyamines can be exchanged through gap junction channels between mammalian cells. Such intercellular exchange of polyamines has critical consequences on the local control of growth. In line with this hypothesis, the complex protein network that keeps polyamine levels finely tuned in mammalian cells can translate polyamine efflux or influx into integrated signals controlling transcription, translation, and cell communications.
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http://dx.doi.org/10.1002/bies.201500195DOI Listing
June 2016

Role of tau in the spatial organization of axonal microtubules: keeping parallel microtubules evenly distributed despite macromolecular crowding.

Cell Mol Life Sci 2016 10 13;73(19):3745-60. Epub 2016 Apr 13.

Institut National de la Santé et de la Recherche Médicale (INSERM), UMR1204, Université Evry-Val d'Essonne, Evry, 91025, France.

Opposing views have been proposed regarding the role of tau, the principal microtubule-associated protein in axons. On the one hand, tau forms cross-bridges at the interface between microtubules and induces microtubule bundling in neurons. On the other hand, tau is also considered a polymer brush which efficiently separates microtubules. In mature axons, microtubules are indeed arranged in parallel arrays and are well separated from each other. To reconcile these views, we developed a mechanistic model based on in vitro and cellular approaches combined to analytical and numerical analyses. The results indicate that tau forms long-range cross-bridges between microtubules under macromolecular crowding conditions. Tau cross-bridges prevent the redistribution of tau away from the interface between microtubules, which would have occurred in the polymer brush model. Consequently, the short-range attractive force between microtubules induced by macromolecular crowding is avoided and thus microtubules remain well separated from each other. Interestingly, in this unified model, tau diffusion on microtubules enables to keep microtubules evenly distributed in axonal sections at low tau levels.
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http://dx.doi.org/10.1007/s00018-016-2216-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002045PMC
October 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

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

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

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

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

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

Gap junctions favor normal rat kidney epithelial cell adaptation to chronic hypertonicity.

Am J Physiol Cell Physiol 2011 Sep 15;301(3):C705-16. Epub 2011 Jun 15.

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

Upon hypertonic stress most often resulting from high salinity, cells need to balance their osmotic pressure by accumulating neutral osmolytes called compatible osmolytes like betaine, myo-inositol, and taurine. However, the massive uptake of compatible osmolytes is a slow process compared with other defense mechanisms related to oxidative or heat stress. This is especially critical for cycling cells as they have to double their volume while keeping a hospitable intracellular environment for the molecular machineries. Here we propose that clustered cells can accelerate the supply of compatible osmolytes to cycling cells via the transit, mediated by gap junctions, of compatible osmolytes from arrested to cycling cells. Both experimental results in epithelial normal rat kidney cells and theoretical estimations show that gap junctions indeed play a key role in cell adaptation to chronic hypertonicity. These results can provide basis for a better understanding of the functions of gap junctions in osmoregulation not only for the kidney but also for many other epithelia. In addition to this, we suggest that cancer cells that do not communicate via gap junctions poorly cope with hypertonic environments thus explaining the rare occurrence of cancer coming from the kidney medulla.
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http://dx.doi.org/10.1152/ajpcell.00128.2011DOI Listing
September 2011

The C terminus of tubulin, a versatile partner for cationic molecules: binding of Tau, polyamines, and calcium.

J Biol Chem 2011 Jan 9;286(4):3065-78. Epub 2010 Nov 9.

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

The C-terminal region of tubulin is involved in multiple aspects of the regulation of microtubule assembly. To elucidate the molecular mechanisms of this regulation, we study here, using different approaches, the interaction of Tau, spermine, and calcium, three representative partners of the tubulin C-terminal region, with a peptide composed of the last 42 residues of α1a-tubulin. The results show that their binding involves overlapping amino acid stretches in the C-terminal tubulin region: amino acid residues 421-441 for Tau, 430-432 and 444-451 for spermine, and 421-443 for calcium. Isothermal titration calorimetry, NMR, and cosedimentation experiments show that Tau and spermine have similar micromolar binding affinities, whereas their binding stoichiometry differs (C-terminal tubulin peptide/spermine stoichiometry 1:2, and C-terminal tubulin peptide/Tau stoichiometry 8:1). Interestingly, calcium, known as a negative regulator of microtubule assembly, can compete with the binding of Tau and spermine with the C-terminal domain of tubulin and with the positive effect of these two partners on microtubule assembly in vitro. This observation opens up the possibility that calcium may participate in the regulation of microtubule assembly in vivo through direct (still unknown) or indirect mechanism (displacement of microtubule partners). The functional importance of this part of tubulin was also underlined by the observation that an α-tubulin mutant deleted from the last 23 amino acid residues does not incorporate properly into the microtubule network of HeLa cells. Together, these results provide a structural basis for a better understanding of the complex interactions and putative competition of tubulin cationic partners with the C-terminal region of tubulin.
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http://dx.doi.org/10.1074/jbc.M110.144089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3024800PMC
January 2011

A central role for polyamines in microtubule assembly in cells.

Biochem J 2010 Aug;430(1):151-9

Institut National de la Santé et de la Recherche Médicale, UMR, Université Evry-Val d'Essonne, Evry, France.

Owing to preferential electrostatic adsorption of multivalent cations on highly anionic surfaces, natural multivalent polyamines and especially quadrivalent spermine can be considered as potential regulators of the complex dynamical properties of anionic MTs (microtubules). Indeed, the C-terminal tails of tubulin display many negative residues in a row which should enable the formation of a correlated liquid-like phase of multivalent counterions on its surface. Although it is known that polyamine counterions promote MT assembly in vitro, little is known about the relevance of this interaction in vivo. In the present study, we have explored the relationship between polyamine levels and MT assembly in HeLa and epithelial NRK (normal rat kidney) cells using DFMO (alpha-difluoromethylornithine), an irreversible inhibitor of ornithine decarboxylase, and APCHA [N-(3-aminopropyl)-N-cyclohexylamine], a spermine synthase inhibitor. Under conditions of intracellular polyamine depletion, the MT network is clearly disrupted and the MT mass decreases. Addition of spermine to polyamine-depleted cells reverses this phenotype and rapidly promotes the extensions of the MT network. Finally, we show that polyamine levels modulate the coating of MTs with MAP4 (MT-associated protein 4), an MT-stabilizing protein, and the spatial distribution of EB1 (end-binding protein 1), an MT plus-end-binding protein. In addition, polyamines favour the formation of gap junctions in NRK cells, a process which requires MT extensions at the cell periphery. The present study provides a basis for a better understanding of the role played by polyamines in MT assembly and establishes polyamine metabolism as a potential cellular target for modulating MT functions.
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http://dx.doi.org/10.1042/BJ20091811DOI Listing
August 2010

High-resolution imaging of microtubules and cytoskeleton structures by atomic force microscopy.

Methods Cell Biol 2010 ;95:157-74

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM - UEVE UMR U829, Evry 91025, France.

Atomic force microscopy (AFM), which combines a nanometer-scale resolution and a unique capacity to image biomolecular interactions in liquid environment, is a promising tool for the investigation of biological samples. In contrast with nucleic acids and nucleoprotein complexes, for which AFM is now of common use and participates in the recent advances in the knowledge of DNA-related biomolecular processes, AFM investigations of cytoskeleton structures and especially microtubules remain rare. The most critical step to observe biomolecules using AFM is the spreading of the biological material on a flat surface. This issue is now better documented concerning DNA but a lot remains to be done concerning microtubules. This is a prerequisite to further document this issue for a proper and large use of AFM to study cytoskeleton structures. We present here an overview of the various procedures previously used to spread microtubules on a flat surface and advance an easy-to-use and efficient experimental protocol for microtubule imaging by AFM in air. We show application of this protocol to observe intermediate structures of microtubule assembly without using any stabilizing agent and the observation of more complex systems like proteins or messenger ribonucleoprotein particles in interaction with microtubules.
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http://dx.doi.org/10.1016/S0091-679X(10)95009-7DOI Listing
August 2010

Role of microtubules in stress granule assembly: microtubule dynamical instability favors the formation of micrometric stress granules in cells.

J Biol Chem 2009 Dec 19;284(52):36569-36580. Epub 2009 Oct 19.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM/Université d'Evry-Val d'Essonne, U829, EA3637, Evry 91025, France. Electronic address:

Following exposure to various stresses (arsenite, UV, hyperthermia, and hypoxia), mRNAs are assembled into large cytoplasmic bodies known as "stress granules," in which mRNAs and associated proteins may be processed by specific enzymes for different purposes like transient storing, sorting, silencing, or other still unknown processes. To limit mRNA damage during stress, the assembly of micrometric granules has to be rapid, and, indeed, it takes only approximately 10-20 min in living cells. However, such a rapid assembly breaks the rules of hindered diffusion in the cytoplasm, which states that large cytoplasmic bodies are almost immobile. In the present work, using HeLa cells and YB-1 protein as a stress granule marker, we studied three hypotheses to understand how cells overcome the limitation of hindered diffusion: shuttling of small messenger ribonucleoprotein particles from small to large stress granules, sliding of messenger ribonucleoprotein particles along microtubules, microtubule-mediated stirring of large stress granules. Our data favor the two last hypotheses and underline that microtubule dynamic instability favors the formation of micrometric stress granules.
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http://dx.doi.org/10.1074/jbc.M109.042879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794772PMC
December 2009