Publications by authors named "Loic Hamon"

45 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979924PMC
March 2021

[Excimer laser-assisted DALK: a case report from the Homburg Keratoconus Center (HKC)].

Ophthalmologe 2021 Feb 25. Epub 2021 Feb 25.

Klinik für Augenheilkunde, Universitätsklinikum des Saarlandes (UKS), Kirrbergerstr. 100, Geb. 22, 66421, Homburg/Saar, Deutschland.

Indications: The aim of excimer laser-assisted deep anterior lamellar keratoplasty (excimer-DALK) is, as in mechanical DALK, the treatment of keratectasia (keratoconus and pellucid marginal degeneration), stromal scars or stromal corneal dystrophy. A prerequisite for surgery is the absence of (pre‑) Descemet's scars and an intact endothelium.

Surgical Technique: After excimer laser-assisted trephination to 80% of the corneal thickness at the trephination site, intrastromal air injection (so-called big bubble) and lamellar corneal preparation, a lamellar anterior transplantation of the endothelium-free donor tissue is performed. The technique combines the advantages of DALK and excimer laser trephination. We describe the steps of an excimer-DALK from the Homburg Keratoconus Center (HKC).

Conclusion: Excimer-DALK is a viable treatment option for patients with intact endothelium. In cases of intraoperative perforation, conversion to excimer-perforating keratoplasty (PKP) with all the advantages of excimer laser trephination remains feasible.
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http://dx.doi.org/10.1007/s00347-021-01342-3DOI Listing
February 2021

[Purpureocillium lilacinum : Atypical pathogen of mycotic keratitis in an immunocompetent patient].

Ophthalmologe 2021 Feb 13. Epub 2021 Feb 13.

Klinik für Augenheilkunde, Universitätsklinikum des Saarlandes (UKS), Kirrberger Str. 100, Gebäude 22, 66421, Homburg/Saar, Deutschland.

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http://dx.doi.org/10.1007/s00347-021-01325-4DOI Listing
February 2021

Thickness and Curvature Changes of Human Corneal Grafts in Dextran-Containing Organ Culture Medium Before Keratoplasty.

Cornea 2020 Dec 4. Epub 2020 Dec 4.

Department of Ophthalmology, Saarland University Medical Center, Homburg/Saar, Germany.

Purpose: To determine the changes of corneal thickness and curvature of human corneal grafts in organ culture medium II, containing dextran T500 6%, before keratoplasty.

Methods: We examined the tomography of 24 corneas from our eye bank transferred from medium I into medium II. Images were repeated hourly during 24 hours using an anterior segment optical coherence tomography. The central corneal thickness (CCT) was measured with the manual measurement tool of the anterior segment optical coherence tomography. The radii of curvature (anterior flat and steep and posterior flat and steep) were measured with a MATLAB self-programmed software for "sterile donor tomography."

Results: The mean CCT (±SD) at baseline (T0) was 727 ± 156 μm. It reached 581 ± 103, 506 ± 84, 472 ± 79, and 456±7 μm after 6, 12, 18, and 24 hours, respectively. After 12 hours, 83% of the final deswelling was achieved. The radii of curvature (±SD) at baseline (T0) were (posterior flat, posterior steep, anterior flat, and anterior steep) 6.6 ± 0.5, 6.2 ± 0.5, 7.7 ± 0.4, and 7.4 ± 0.4 mm, respectively. After 24 hours, the radii of curvature reached 6.8 ± 0.1, 6.6 ± 0.3, 7.6 ± 0.1, and 7.4 ± 0.2 mm, respectively.

Conclusions: The kinetics of the deswelling process in medium II follow a hyperbolic curve. Considering a CCT of 506 μm at T12, we assume that a time interval of 12 hours in medium II might be enough for clinical purposes. This result might help to keep storage in medium II as short as possible to escape potential toxic effects of dextran in medium II. The radius of curvature does not seem to change within 24 hours for all measured surfaces.
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http://dx.doi.org/10.1097/ICO.0000000000002543DOI Listing
December 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

Tomographically normal partner eye in very asymmetrical corneal ectasia: biomechanical analysis.

J Cataract Refract Surg 2020 Sep 16. Epub 2020 Sep 16.

From the Department of Ophthalmology, Saarland University Medical Center UKS (Fraenkel, Hamon, Daas, Flockerzi, Suffo, Seitz), Homburg/Saar, Institute of Experimental Ophthalmology, Saarland University (Eppig), Homburg/Saar, and AMIPLANT GmbH (Eppig), Schnaittach, Germany.

Purpose: To point out the biomechanical changes of the topographically and tomographically normal partner eye (NPE) in patients with very asymmetrical corneal ectasia.

Setting: Department of Ophthalmology, Saarland University Medical Center in Homburg/Saar, Germany.

Design: Retrospective study.

Methods: The topographical and tomographical results of the NPE using the Pentacam HR as well as the biomechanical corneal properties using the Ocular Response Analyzer (keratoconus match index [KMI], corneal hysteresis [CH], corneal resistance factor [CRF]) and the Corvis ST (topographic biomechanical index [TBI], Corvis biomechanical index) and compared those results with a normal control group (CG).

Results: The clinical records of 26 patients recruited from the Homburg Keratoconus Center diagnosed with a very asymmetrical corneal ectasia were reviewed. The NPE (8.5±1.5 mm Hg) showed a significantly more pathological CH (p<0.001) compared to the CG. The CRF was also significantly more pathological (p=0.04) for the NPE (8.3±1.5 mmHg) compared with the CG. The NPE (0.62±0.32) showed a nonsignificant (p=0.08) more pathological KMI compared with the CG. Nineteen of 26 NPE (73%) had a KMI < 0.72 and were considered pathological. Compared with the CG, the TBI of the NPE (0.19±0.25) did not differ significantly overall (p=0.57). However, 5 of 26 eyes (19.2%) had a TBI > 0.29 and were considered pathological.

Conclusions: Topographically and tomographically NPEs in very asymmetrical corneal ectasia frequently showed biomechanical changes. This should be considered before planning any type of refractive corneal surgery in such patients.
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http://dx.doi.org/10.1097/j.jcrs.0000000000000435DOI Listing
September 2020

[A 63-year-old male patient with acute visual deterioration after penetrating keratoplasty for keratoconus].

Ophthalmologe 2020 Sep 15. Epub 2020 Sep 15.

Klinik für Augenheilkunde, Universitätsklinikum des Saarlandes (UKS), Kirrberger Str. 100, Gebäude 22, 66421, Homburg/Saar, Deutschland.

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http://dx.doi.org/10.1007/s00347-020-01226-yDOI Listing
September 2020

Keratoconus staging by decades: a baseline ABCD classification of 1000 patients in the Homburg Keratoconus Center.

Br J Ophthalmol 2020 Aug 23. Epub 2020 Aug 23.

Department of Ophthalmology, Saarland University Medical Center, Homburg, Germany.

Background: This retrospective cross-sectional study aims to analyse the keratoconus (KC) stage distribution at different ages within the Homburg Keratoconus Center (HKC).

Methods: 1917 corneae (1000 patients) were allocated to decades of age, classified according to Belin's ABCD KC grading system and the stage distribution was analysed.

Results: 73 per cent (n=728) of the patients were males, 27% (n=272) were females. The highest KC prevalence occurred between 21 and 30 years (n=585 corneae, 294 patients). Regarding anterior (A) and posterior (B) curvature, the frequency of A was significantly higher than B in all age groups for stage 0, 1 and 2 (A0>B0; A1>B1; A2>B2; p<0.03, Wilcoxon matched-pairs test). There was no significant difference between the number of A3 and B3, but significantly more corneae were classified as B4 than A4 in all age groups (p<0.02). The most frequent A|B combinations were A4|B4 (n=451), A0|B0 (n=311), A2|B4 (n=242), A2|B2 (n=189) and A1|B2 (n=154). Concerning thinnest pachymetry (C), most corneae in all age groups were classified as C0>C1>C2>C3>C4 (p<0.04, Wilcoxon matched-pairs test). For the best distance visual acuity (D), a significantly higher number of corneae were classified as D1 compared to D0 (p<0.008; D1>D0>D2>D3>D4).

Conclusion: The stage distributions in all age groups were similar. Early KC rather becomes manifest in the posterior than the anterior corneal curvature whereas advanced stages of posterior corneal curvature coincide with early and advanced stages of anterior corneal curvature. Thus, this study emphasises the necessity of posterior corneal surface assessment in KC as enabled by the ABCD grading system.
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http://dx.doi.org/10.1136/bjophthalmol-2020-316789DOI Listing
August 2020

Corneae from body donors in anatomy department: valuable use for clinical transplantation and experimental research.

BMC Ophthalmol 2020 Jul 13;20(1):284. Epub 2020 Jul 13.

Department of Ophthalmology, Saarland University Medical Center (UKS), Homburg, Germany.

Background: Explanted corneae are highly needed for the surgical management of patients with severe corneal diseases. The aim of this study was to determine whether the body donors from the Institute of Anatomy are a suitable source of donor corneae.

Methods: At the Institute of Anatomy at Saarland University Medical Center in Homburg, corneae are prelevated from body donors who had consented to the removal of tissues for transplantation purposes during their lifetime. Following the report of death, the LIONS Eye Bank is informed and the contraindications of corneal explantation are clarified. Obtaining a blood sample within 24 h postmortem is mandatory.

Results: The Institute of Anatomy had 150 body donors in the time period from January 2018 to June 2019. Out of these, 68 (45.3%) were reported to the Eye Bank. The age of the donors (median 82 years (range: 57-96)) is not critical since the quality of the corneae depends on the number of endothelial cells (mean: 2109 ± 67 cells/mm (range: 511-2944 cells/mm)). Contraindications were present in 19 (12.6%) cases. The corneae were extracted from 49 (32.7%) body donors. Out of these 98 corneae, 46 (46.9%) were successfully transplanted. Of all non-transplanted corneae, 6 (6.1%) were microbiologically contaminated, 10 (10.2%) had a positive serology, 22 (22.5%) had an endothelial cell count < 2000 cells/mm and 6 (6.1%) are at time of this analysis still in culture medium. The non-transplanted tissues were used for research.

Conclusions: Explanted corneae from the Institute of Anatomy are a valuable option in obtaining grafts for corneal transplantation, which is why we are working toward on expanding cooperation with this department.
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http://dx.doi.org/10.1186/s12886-020-01546-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359266PMC
July 2020

[A 27-year-old man with "corneal opacity" after being injured by a branch].

Ophthalmologe 2021 Feb;118(2):186-189

Klinik für Augenheilkunde, Universitätsklinikum des Saarlandes (UKS), Kirrbergerstr. 100, Gebäude 22, 66421, Homburg/Saar, Deutschland.

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http://dx.doi.org/10.1007/s00347-020-01130-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862528PMC
February 2021

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

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

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

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

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

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