Publications

2015

• Development of antibacterials directed against the thymidylate synthase ThyX in Mycobacterium tuberculosis : mechanism kinetic and in silico inhibitor optimisation
  
  • Djaout Kamel
  , 2015. Tuberculosis is a global health issue as the recent data from World Health Organization indicates that one third of the human population is infected with a dormant form of tuberculosis. Consequently, 9 million new cases and 1.5 million deaths caused by Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, were reported in 2014. The treatment for tuberculosis lasts at least 6 months and excessive or misuse of currently available anti-tuberculosis agents has led to the rise of multi and extensively resistant forms of this important human pathogen. The dissemination of Mtb resistant strains is alarming and the need for new therapeutic agents and targets to fight these Mtb strains is urgent. During my thesis, I have investigated reaction and inhibition mechanism of Mtb ThyX. ThyX catalyses the synthesis of deoxythymidine monophosphate (dTMP) by the methylation of carbone 5 of deoxyuridine monophosphate (dUMP), using N5,N10-methylenetetrahydrofolate and NADPH as carbon and hydride donors respectively. As ThyX is absent in humans and they are structurally and mechanistically distinct from analogous human enzyme, these proteins are a promising therapeutic target in Mtb and many other pathogenic bacteria. Moreover, recent genome wide studies of clinical isolates have revealed overexpression of Mtb thyX in some multi-resistant isolates of Mtb suggesting an involvement in a compensatory pathway in the mechanism of acquisition of drug resistance. I have studied the kinetics of Mtb ThyX mechanism under steady-state conditions and by using time resolved spectroscopy. I showed the selectivity of the enzyme for NADPH, and studied the dynamics of dUMP and NADPH binding in the Mtb ThyX active site as well as the reactivity of the flavin cofactor with molecular oxygen. These studies have explained how Mtb, a strict aerobe, can synthetize thymidylate even in the presence of molecular oxygen, despite the reduced flavin-factor (obligatory intermediate of the reaction) is highly oxygen reactive. A second part of my work focused on identifying Mtb ThyX inhibitors with antimycobacterial activity. Two approaches were used: i) selecting naphthoquinone derivatives, by similarity searching, or by picking previously reported compounds with anti-Mtb activity but of unknown target(s), and testing them for Mtb ThyX inhibition ; ii) Developing a pharmacophore model that can be used for screening chemical libraries in silico and/or prioritizing further compounds for additional experimental testing. These approaches have led to the identification of several new Mtb ThyX inhibitors with low micromolar inhibition constants while no significant inhibition was observed on human thymidylate synthase. The inhibition modes of this new set of compounds suggest a novel binding site on Mtb ThyX than previously observed on other ThyX proteins. The most potent compounds like idebenone or 2-[(4-methoxyphenyl)methyl]-1,4-dihydronaphthalene-1,4-dione also exhibit moderate activity against clinical strains of Mtb. The precise binding site of these compounds remains unknown and will require further investigation to guide SAR studies to optimize these inhibitors. Updating the pharmacophore model will allow to select new compounds to be tested in vitro. Idebenone has already passed phase 1 and 2 clinical trials for duchenne muscle dystrophy, exhibiting good ADME-tox properties. Repurposing idebenone derivatives to target ThyX in Mtb will be the focus of future work.
• Thioglycerol-functionalized CdSe quantum dots detecting cadmium ions
  
  • Brahim Nassim Ben
  • Mohamed Naim Bel Haj
  • Echabaane Mosaab
  • Haouari Mohamed
  • Chaâbane Rafik Ben
  • Negrerie Michel
  • Ouada Hafedh Ben
  Sensors and Actuators B: Chemical, Elsevier, 2015, 220, pp.1346-1353. Water-soluble CdSe quantum dots (QDs) were synthesized using thioglycerol (TG) as the surface capping agent through a one-step process at low temperature T (100 degrees C). The CdSe quantum dots were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, UV-visible absorption and fluorescence spectroscopies. These measurements revealed that the TG-capped CdSe QDs possess a high crystalline quality with an average diameter in the range 2.5-2.8 nm and exhibit particular optical properties. The UV-visible absorption of CdSe QDs is enhanced by the addition of cadmium ions, with a simultaneous shift of the edge band (400 nm), while seventeen other tested metal cations have no effect on the absorption of QDs. Moreover, the binding of Cd2+ ions induces a quenching of the fluorescence emission of TG-CdSe QDs. At particular absorption wavelengths, the response is linearly proportional to the cadmium ions concentration ranging from 1.0 to 22 mu M with a detection limit of 0.32 mu M (37 mu g L-1). Based on these optical properties, the TG-CdSe QDs could be used as a highly selective probe for the detection of Cd2+ ions in aqueous solutions, a species highly toxic for cells. (C) 2015 Elsevier B.V. All rights reserved. (10.1016/j.snb.2015.07.049)
  DOI : 10.1016/j.snb.2015.07.049
• Conservation and Role of Electrostatics in Thymidylate Synthase
  
  • Garg Divita
  • Skouloubris Stephane
  • Briffotaux Julien
  • Myllykallio Hannu
  • Wade Rebecca C
  Scientific Reports, Nature Publishing Group, 2015. Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome. The electrostatic potential of a protein plays a crucial role in steering ligands to their binding sites, and orienting them correctly for binding 1. In enzymes, the active site electrostatic potential is important for stabilizing the transition state and thereby catalyzing the reaction 2. Therefore, conservation of protein function across a protein family is often accompanied by conservation of the electrostatic potential in the active site region, even though the rest of the protein may lack a conserved electrostatic potential 3,4. Consequently, comparison of protein electrostatic potentials has been employed as a tool to predict protein function and to derive similarities in protein function across protein families 5–7. Optimizing the electrostatic complementarity between a ligand and the binding site of a protein is also an important aspect in drug design 8,9 and may provide a route to gain target selectivity 10 . (10.1038/srep17356)
  DOI : 10.1038/srep17356
• DNA Replication Restart in Archaea
  
  • Lestini Roxane
  • Delpech Floriane
  • Myllykallio Hannu
  , 2015, pp.381-404. One fundamental challenge of cells is to accurately copy their genetic material for cell prolif‐ eration. This task is performed by core machineries considered conserved in all three domains of life: bacteria, archaea and eukaryotes... (10.5772/59994)
  DOI : 10.5772/59994
• DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii
  
  • Lestini Roxane
  • Delpech Floriane
  • Myllykallio Hannu
  Biochimie, Elsevier, 2015, 118, pp.254-263. Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green. Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea. (C) 2015 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). (10.1016/j.biochi.2015.07.022)
  DOI : 10.1016/j.biochi.2015.07.022
• Ultrafast conformational changes in biomolecules studied by time-resolved circular dichroism
  
  • Changenet-Barret Pascale
  • Hache François
  , 2015, 9652. Circular dichroism (CD) is known to be a very sensitive probe of molecular conformation, and it is in particular widely used in biochemistry. Measuring the CD as a function of time is therefore very appealing to access information on the dynamics of conformational changes in molecules or biomolecules. We have implemented such a time-resolved experiments in two complementary configurations: a sub-picosecond pump-probe one and a microsecond detection of CD coupled to a T-jump experiment. We present two experiments based on these techniques: the ultrafast motion of the carbonyl group in the chromophore of the Photoactive Yellow Protein after photoexcitation and the dynamics of thermal denaturation in model peptides. © (2015) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only. (10.1117/12.2216989)
  DOI : 10.1117/12.2216989
• Arbitrary-detuning asynchronous optical sampling with amplified laser systems
  
  • Antonucci Laura
  • Bonvalet Adeline
  • Solinas Xavier
  • Daniault Louis
  • Joffre Manuel
  Optics Express, Optical Society of America - OSA Publishing, 2015, 23 (21), pp.27931-27940. We demonstrate that Arbitrary-Detuning ASynchronous OPtical Sampling (AD-ASOPS) makes possible multiscale pump-probe spectroscopy with time delays spanning from picosecond to millisecond. The implementation on pre-existing femtosecond amplifiers seeded by independent free-running oscillators is shown to be straightforward. The accuracy of the method is determined by comparison with spectral interferometry, providing a distribution with a standard deviation ranging from 0.31 to 1.7 ps depending on experimental conditions and on the method used to compute the AD-ASOPS delays. (C) 2015 Optical Society of America (10.1364/OE.23.027931)
  DOI : 10.1364/OE.23.027931
• Large-scale live imaging of adult neural stem cells in their endogenous niche
  
  • Dray Nicolas
  • Bedu Sébastien
  • Vuillemin Nelly
  • Alunni Alessandro
  • Coolen Marion
  • Krecsmarik Monika
  • Supatto Willy
  • Beaurepaire Emmanuel
  • Bally-Cuif Laure
  Development (Cambridge, England), Company of Biologists, 2015, 142 (20), pp.3592-3600. Live imaging of adult neural stem cells (aNSCs) in vivo is a technical challenge in the vertebrate brain. Here, we achieve long-term imaging of the adult zebrafish telencephalic neurogenic niche and track a population of >1000 aNSCs over weeks, by taking advantage of fish transparency at near-infrared wavelengths and of intrinsic multiphoton landmarks. This methodology enables us to describe the frequency, distribution and modes of aNSCs divisions across the entire germinal zone of the adult pallium, and to highlight regional differences in these parameters. (10.1242/dev.123018)
  DOI : 10.1242/dev.123018
• Synthesis of an Artificial Cornea Allowing In Vitro Colonization of the Host Cells
  
  • Tidu Aurelien
  • Ghoubay-Benallaoua D.
  • Teulon C.
  • Lynch Barbara
  • Haye Bernard
  • Illoul Corinne
  • Allain Jean-Marc
  • Borderie V.
  • Schanne-Klein Marie-Claire
  • Mosser G.
  , 2015, 21, pp.S142-S142.
• Quantitative measurement of permeabilization in living cells by terahertz attenuated total reflection
  
  • Grognot M.
  • Gallot Guilhem
  , 2015, pp.1-2. (10.1109/IRMMW-THZ.2015.7327838)
  DOI : 10.1109/IRMMW-THZ.2015.7327838
• High-power, widely tunable, room-temperature picosecond optical parametric oscillator based on cylindrical 5%MgO:PPLN
  
  • Chaitanya Kumar S.
  • Wei Junxiong
  • Debray J.
  • Kemlin Vincent
  • Boulanger B.
  • Ishizuki Hideki
  • Taira T.
  • Ebrahim-Zadeh M.
  Optics Letters, Optical Society of America - OSA Publishing, 2015, 40 (16), pp.3897-3900. We report a high-power picosecond optical parametric oscillator (OPO) based on cylindrical MgO:PPLN synchronously pumped by an Yb-fiber laser. The singly resonant OPO is tunable in the near-infrared signal across 1413–1900 nm and mid-infrared idler over 2418–4307 nm by angle tuning of the crystal at room temperature. With non-optimized output coupling of ∼10%, the OPO simultaneously delivers 2.4 W of signal at 1664 nm and 1.7 W of idler at 2950 nm at an overall extraction efficiency of ∼45% with high beam-pointing stability <30 μrad and <14 μrad for the signal and idler, respectively. The generated signal and idler exhibit passive power stability better than 1% rms and 0.8% rms over 15 h, respectively, in high beam quality with TEM00 profile. The extracted signal pulses from the OPO have duration of 15.2 ps with a spectral bandwidth of 0.7 nm, corresponding to a time-bandwidth product of ΔvΔtau∼1.2. (10.1364/OL.40.003897)
  DOI : 10.1364/OL.40.003897
• Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity
  
  • Fojtikova Veronika
  • Stranava Martin
  • Vos Marten H.
  • Liebl Ursula
  • Hranicek Jakub
  • Kitanishi Kenichi
  • Shimizu Toru
  • Martinkova Marketa
  Biochemistry, American Chemical Society, 2015, 54 (32), pp.5017–5029. The globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction’s kinetics. The kcat values of the heme Fe(III)-OH–, Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1–1.2 min–1, and their KmATP values were 18.9–35.4 μM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min–1 but a very high KmATP value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and KmATP values of 0.4 min–1 and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and KmATP values of 0.3 min–1 and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme’s catalytic activity because they modulate its ATP binding affinity and thus change its kcat/KmATP value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed (10.1021/acs.biochem.5b00517)
  DOI : 10.1021/acs.biochem.5b00517
• Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy
  
  • Nazac André
  • Bancelin Stéphane
  • Teig Benjamin
  • Haj Ibrahim Bicher
  • Fernandez Hervé
  • Schanne-Klein Marie-Claire
  • de Martino Antonello
  Microscopy Research and Technique, Wiley, 2015, 78 (8), pp.723-730. Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the gold standard for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5min, and sample thicknesses of 5 mu m were sufficient in cervical and vaginal tissues. Microsc. Res. Tech. 78:723-730, 2015. (c) 2015 Wiley Periodicals, Inc. (10.1002/jemt.22530)
  DOI : 10.1002/jemt.22530
• Détection d'oxydants
  
  • Bouzigues Cedric I.
  • Abdesselem C.
  • Alexandrou Antigoni
  • Gacoin Thierry
  • Boilot Jean-Pierre
  , 2015.
• Lensfree video microscopy: high throughput monitoring and cell tracking of 2D cell cultures
  
  • Beaurepaire Emmanuel
  • So Peter
  • Pavone Francesco
  • Hillman Elizabeth
  • Allier C.
  • Vinjimore Kesavan S.
  • Cioni O.
  • Momey F.
  • Bordy T.
  • Hervé L.
  • Morel S.
  • Navarro F.
  • Menneteau M.
  • Chalmond B.
  • Freida D.
  • Sulpice E.
  • Gidrol X.
  • Dinten M.
  , 2015, pp.95360J. (10.1117/12.2183468)
  DOI : 10.1117/12.2183468
• Active site dynamics of flavin-dependent methylases
  
  • Sournia P.
  • Vos Marten H.
  • Liebl Ursula
  • Myllykallio Hannu
  , 2015, 282 (S1), pp.336. (10.1111/febs.13339)
  DOI : 10.1111/febs.13339
• In situ determination of collagen fibrils size via absolute measurements of second harmonic generation signals
  
  • Bancelin Stéphane
  • Aimé Carole
  • Gusachenko Ivan
  • Kowalczuk Laura
  • Latour Gael
  • Coradin Thibaud
  • Schanne-Klein Marie-Claire
  , 2015. We correlated SHG and Electron Microscopies to calibrate SHG signals as a function of collagen fibril diameter, down to 30 nm. We observed a fourth power dependence, in agreement with analytical and numerical calculations. We applied this calibration to abnormal fibrils in the Descemet’s membrane of intact unfixed rat corneas.
• Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease
  
  • Camus S. M.
  • de Moraes J. A.
  • Bonnin P.
  • Abbyad P.
  • Le Jeune S.
  • Lionnet F.
  • Loufrani L.
  • Grimaud L.
  • Lambry J.-C.
  • Charue D.
  • Kiger L.
  • Renard J.-M.
  • Larroque C.
  • Le Clesiau H.
  • Tedgui A.
  • Bruneval P.
  • Barja-Fidalgo C.
  • Alexandrou A.
  • Tharaux P.-L.
  • Boulanger C. M.
  • Blanc-Brude O. P.
  Blood, American Society of Hematology, 2015, 125 (24), pp.3805-3814. Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that hemeladen MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of hemeladen MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD. (10.1182/blood-2014-07-589283)
  DOI : 10.1182/blood-2014-07-589283
• Ultrafast Dynamics of Carboxy-Hemoglobin: Two-Dimensional Infrared Spectroscopy Experiments and Simulations
  
  • Falvo Cyril
  • Daniault Louis
  • Vieille Thibault
  • Kemlin Vincent
  • Lambry Jean-Christophe
  • Meier Christoph
  • Vos Marten H.
  • Bonvalet Adeline
  • Joffre Manuel
  Journal of Physical Chemistry Letters, American Chemical Society, 2015, 6 (12), pp.2216–2222. This Letter presents a comparison between experimental and simulated 2D mid-infrared spectra of carboxy-hemoglobin in the spectral region of the carbon monoxide stretching mode. The simulations rely on a fluctuating potential energy surface that includes both the effect of heme and the protein surroundings computed from molecular dynamics simulations. A very good agreement between theory and experiment is obtained with no adjustable parameters. The simulations show that the effect of the distal histidine through the hydrogen bond is strong and is directly responsible for the slow decay of the frequency–frequency correlation function on a 10 ps time scale. This study confirms that fluctuations in carboxy-hemoglobin are more inhomogeneous than those in the more frequently studied carboxy-myoglobin. The comparison between simulations and experiments brings valuable information on the complex relation between protein structure and spectral diffusion. (10.1021/acs.jpclett.5b00811)
  DOI : 10.1021/acs.jpclett.5b00811
• Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones
  
  • Skouloubris S.
  • Djaout K.
  • Lamarre I.
  • Lambry J.-C.
  • Anger K.
  • Briffotaux J.
  • Liebl U.
  • de Reuse H.
  • Myllykallio H.
  Open Microbiology Journal, Bentham Open, 2015, 5 (6), pp.150015. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2′-deoxythymidine-5′-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation. (10.1098/rsob.150015)
  DOI : 10.1098/rsob.150015
• Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in human thymidylate synthase.
  
  • Salo-Ahen Outi M H
  • Tochowicz Anna
  • Pozzi Cecilia
  • Cardinale Daniela
  • Ferrari Stefania
  • Boum Yap
  • Mangani Stefano
  • Stroud Robert M
  • Saxena Puneet
  • Myllykallio Hannu
  • Costi Maria Paola
  • Ponterini Glauco
  • Wade Rebecca C
  Journal of Medicinal Chemistry, American Chemical Society, 2015, 58 (8), pp.3572-81. Human thymidylate synthase (hTS), a target for antiproliferative drugs, is an obligate homodimer. Single-point mutations to alanine at the monomer-monomer interface may enable the identification of specific residues that delineate sites for drugs aimed at perturbing the protein-protein interactions critical for activity. We computationally identified putative hotspot residues at the interface and designed mutants to perturb the intersubunit interaction. Dimer dissociation constants measured by a FRET-based assay range from 60 nM for wild-type hTS up to about 1 mM for single-point mutants and agree with computational predictions of the effects of these mutations. Mutations that are remote from the active site retain full or partial activity, although the substrate KM values were generally higher and the dimer was less stable. The lower dimer stability of the mutants can facilitate access to the dimer interface by small molecules and thereby aid the design of inhibitors that bind at the dimer interface. (10.1021/acs.jmedchem.5b00137)
  DOI : 10.1021/acs.jmedchem.5b00137
• Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase
  
  • Yoo Byung-Kuk
  • Lamarre Isabelle
  • Martin Jean-Louis
  • Rappaport Fabrice
  • Negrerie Michel
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2015, 112 (14), pp.E1697-704. We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 μs with [NO] = 200 μM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10(8) M(-1)⋅s(-1). In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 μs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 μs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed. (10.1073/pnas.1423098112)
  DOI : 10.1073/pnas.1423098112
• Theoretical, numerical and experimental study of geometrical parameters that affect anisotropy measurements in polarization-resolved SHG microscopy.
  
  • Teulon Claire
  • Gusachenko Ivan
  • Latour Gaël
  • Schanne-Klein Marie-Claire
  Optics Express, Optical Society of America - OSA Publishing, 2015, 23 (7), pp.9313-28. Polarization-resolved second harmonic generation (P-SHG) microscopy is an efficient imaging modality for in situ observation of biopolymers structure in tissues, providing information about their mean in-plane orientation and their molecular structure and 3D distribution. Nevertheless, P-SHG signal build-up in a strongly focused regime is not throroughly understood yet, preventing reliable and reproducible measurements. In this study, theoretical analysis, vectorial numerical simulations and experiments are performed to understand how geometrical parameters, such as excitation and collection numerical apertures and detection direction, affect P-SHG imaging in homogeneous collagen tissues. A good agreement is obtained in tendon and cornea, showing that detection geometry significantly affects the SHG anisotropy measurements, but not the measurements of collagen in-plane orientation. (10.1364/OE.23.009313)
  DOI : 10.1364/OE.23.009313
• Whole-brain functional imaging with two-photon light-sheet microscopy
  
  • Wolf Sébastien
  • Supatto Willy
  • Debrégeas Georges
  • Mahou Pierre
  • Kruglik Sergei G
  • Sintes Jean-Marc
  • Beaurepaire Emmanuel
  • Candelier Raphaël
  Nature Methods, Nature Publishing Group, 2015, 12 (5), pp.379-380. (10.1038/nmeth.3371)
  DOI : 10.1038/nmeth.3371
• Investigation des Interactions Toxine-Cellule et du Confinement des Récepteurs dans la Membrane Cellulaire
  
  • Richly Maximilian U.
  , 2015. La membrane cellulaire est l’interface de communication et d’échange entre la cellule et le monde extérieur. En tant que telle, sa structure et composition ont une importance centrale à la viabilité de la cellule. Les protéines qui résident dans la membrane apportent la fonctionnalité nécessaire pour permettre à la membrane d’accomplir ces tâches. Ces récepteurs se retrouvent dans un environnement de haute hétérogénéité qui renforce leur efficacité. Nous avons étudié cet environnent en suivant des récepteurs uniques dans la membrane grâce aux nanoparticules dopées aux terres rares. Ces nanoparticules produisent des signaux continus, non-interrompus, permettant de suivre des trajectoires pendant plusieurs minutes. Nous avons ensuite utilisé une méthode basée sur l’inférence bayésienne pour analyser et comparer les trajectoires obtenues, et pour extraire le potentiel de confinement de forme arbitraire correspondant à chaque trajectoire. Nous avons d’abord validé l’approche de l’inférence bayésienne en démontrant que cette méthode peut également être utilisée pour la calibration d’un montage de pinces optiques. Par ailleurs, nous avons démontré que cette approche est supérieure aux techniques couramment utilisées pour la calibration des pinces optiques. Puis, nous avons appliqué cette méthode aux trajectoires des récepteurs de la toxine epsilon (de Clostridium perfringens) dans des cellules rénales canines Madin- Darby (MDCK). En particulier, nous avons étudié l’évolution du potentiel de confinement et de la diffusivité à l’intérieur des domaines confinant les récepteurs pendant l’action d’un agent déstabilisant les domaines de confinement, ainsi que les événement de ‘hopping’ pendant lesquels le récepteur change de domaine de confinement, et déterminé les énergies de ‘hopping’ associées. De plus, nous avons observé l’effet d’une force externe appliquée au récepteur, produite par un flux hydrodynamique. L’application d’une force a mis en évidence une dépendance du confinement des récepteurs du cytosquelette d’actine en plus du confinement produit par la distribution des lipides. Pour approfondir notre investigation du confinement des récepteurs de la membrane, nous avons classifié les potentiels de confinement obtenus pour les récepteurs résidant à l’intérieur et à l’extérieur des radeaux lipidiques. Les potentiels ressentis par les récepteurs en dehors des domaines lipidiques sont plus plats au centre du domaine de confinement et plus abrupts vers les bords du domaine par rapport aux potentiels ressentis par les protéines dans les radeaux. Enfin, nous avons étendu la technique de suivi de particules uniques en 3D en utilisant la largeur de la fonction de réponse du signal de la nanoparticule. De cette manière, nous avons observé le mouvement d’internalisation de nanoparticules couplées à un fragment de la chaine lourde de la toxine botulique A de Clostridium botulinum dans des cellules intestinales de souris de la lignée m-ICcl2.