Papers by Jean-Jacques Lacapère
<p>In the upper diagram, before 24 hours of treatment, TNF activates the production of IL-8... more <p>In the upper diagram, before 24 hours of treatment, TNF activates the production of IL-8 through NFκB signaling, as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152919#pone.0152919.ref063" target="_blank">63</a>]. Under this condition, ROS are produced and the antioxidant defenses (MnSOD) are activated simultaneously to TSPO transcription by the JNK pathway [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152919#pone.0152919.ref052" target="_blank">52</a>]. ROS induce the formation of TSPO polymers acting as ROS scavengers, and they induce an increase in TSPO turnover. This mechanism maintains the integrity of the mitochondria (inner ultrastructure membrane complexes) and favors regulation of the ROS production. In the lower diagram, after 24 hours, treatment with TNF causes an overflow of antioxidant defenses together with sustained TSPO turnover, leading to the dysfunction of mitochondrial membrane complexes responsible of the disruption of inner mitochondrial integrity; the apoptotic cascade is then activated, leading to cell death.</p
<p>(A), Representative images of HT-29 cells expressing green fluorescent protein (GFP) tar... more <p>(A), Representative images of HT-29 cells expressing green fluorescent protein (GFP) targeted to mitochondria in control and treated cells. (B and C) Quantitative analysis of mitochondrial structure using morphological parameters (Aspect Ratio and Form Factor) following Koopman et al. treatment. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152919#pone.0152919.ref023" target="_blank">23</a>] At least 500–600 mitochondria were analyzed per condition and normalized to control conditions. The time course of the Aspect Ratio (B) and Form Factor (C) changes in TNF-treated cells (black bars), 1 μM of PK 11195-treated cells (grey bars), and cells treated simultaneously with 10 ng/mL of TNF and 1 μM of PK 11195 (dashed bars). mRNA expression of mitofusins (<i>Mfn1</i> and <i>Mfn2</i> in panels D and E, respectively) relative to <i>GAPDH</i> as a function of time of treatment with 10 ng/mL of TNF (open square), the simultaneous presence of TNF and PK 11195 (open diamonds), and controls without treatment (closed circle). (F) Western blot analysis of mitofusins (1 and 2) in DDM and Digit-solubilized mitochondrial membranes after 72 hours of treatment with TNF [T], PK 11195 [P], both TNF and PK 11195 [T+P], and controls [C]. (G) Densitometry evaluation of western blot of mitofusins. The results are expressed as the means ± standard error of the mean.</p
<p>(A) Electron micrographs of different types of encountered mitochondria [<a href=&quo... more <p>(A) Electron micrographs of different types of encountered mitochondria [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152919#pone.0152919.ref062" target="_blank">62</a>] and their distribution in control cells (open bars), in TNF-treated cells (black bars), in 1 μM PK 11195-treated cells (grey bars), and in cells treated simultaneously with 10 ng/mL of TNF and 1 μM PK 11195 (dashed bars). (B) <i>OPA1</i> (optic atrophy 1) mRNA expression relative to <i>GAPDH</i> as a function of time of treatment with 10 ng/mL of TNF (open square), the simultaneous presence of TNF and PK 11195 (open diamonds), and controls without treatment (closed circle). (C) Western blot analysis of OPA1 protein in 0.1% dodecyl maltoside (DDM) and 0.1% digitonin (Digit)-solubilized mitochondrial membranes after 24 hours and 72 hours of treatment with 10 ng/mL of TNF [T], 1 μM of PK 11195 [P], and both 10 ng/mL of TNF and 1 μM PK 11195 [T+P] and controls [C]. (D) Densitometry evaluation of western blot of OPA1 protein in DDM (open and grey bars for long and short forms, respectively) and digitonin (dashed and black bars for long and short forms, respectively). (E) Electron micrograph of the mitochondria of HT-29 cells treated for 72 hours with 10 ng/mL of TNF. (F) Western blot analysis of the SLP-2 protein (stomatin-like protein-2) in DDM and Digit-solubilized mitochondrial membranes after 24 hours and 72 hours of treatment with TNF [T], PK 11195 [P], both TNF and PK 11195 [T+P], and controls [C], as in panel C. (G) Densitometry evaluation of western blot of SLP-2 in DDM-solubilized mitochondrial membranes. The results are expressed as the means ± standard error of the mean.</p
Biochimica et Biophysica Acta (BBA) - Biomembranes
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Methods in molecular biology (Clifton, N.J.), 2017
Structural studies of membrane proteins (MP) in a native or native-like environment remain a chal... more Structural studies of membrane proteins (MP) in a native or native-like environment remain a challenge. X-ray crystallography of three-dimensional crystals of MP in lipids and cryo-electron microscopy of two-dimensional crystals also in lipids have given atomic structures of several MP. Recent developments of solid-state NMR (ssNMR) provided structural data of MP in lipids and should give access to the dynamic behavior of MP's in a native-like environment. Preparation of samples for ssNMR is not trivial with overexpressed proteins since purified recombinant MP have to be reincorporated in proteoliposomes and concentrated in the small volume of the rotor used for ssNMR studies. We present here the protocol that we have used to study the recombinant mouse TSPO1, an integral membrane protein of 20 kDa mostly found in the outer membrane of mitochondria and overexpressed in E. coli bacteria.
Membrane Proteins Production for Structural Analysis, 2014
ABSTRACT The 18-kDa translocator protein (TSPO) is evolutionarily conserved from bacteria to huma... more ABSTRACT The 18-kDa translocator protein (TSPO) is evolutionarily conserved from bacteria to humans. TSPO expression has been observed in essentially all mammalian tissues, with a preferential localization in the outer mitochondrial membrane. TSPO is involved in various physiological functions. The evidence suggests that TSPO may function in different protein complexes. In mammalian cells, the best-characterized activity of TSPO is the transport of cholesterol from the cytosol to the mitochondrial matrix, where cholesterol is converted to a precursor of steroids or bile salts. No atomic structure of TSPO is currently available. TSPO does not belong to any known membrane protein structural family. It has five transmembrane domains containing α-helices that are involved in the transport of cholesterol. Cytosolic loops are involved in ligand binding and the activation of transport. It has been suggested that TSPO could form homopolymers within heteropolymer complexes. Because of the low native abundance of TSPO, production of recombinant TSPO is a first step for any structural study. In this chapter, we present the current understanding of TSPO overexpression studies in bacteria, purification of functional TSPO, and different approaches to solve TSPO structure.
European Journal of Biochemistry, 1989
Terbium ions and terbium formycin triphosphate have been used to investigate the interactions bet... more Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3 +-binding sites have been found: a first class of low-affinity (Kd = 10 pM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (< 0.1 pM) corresponds to the calcium transport sites, their occupancy by terbium induces the El to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H 2 0 by D 2 0 shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2 + El transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the El-E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site. The molecular mechanism by which plasma-membranetransport ATPases convert chemical energy of ATP into osmotic energy has been a challenging subject to investigation for many years. The state of our knowledge of these ATPases varies from one system to another, the reaction mechanism of the Na+/K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase being by far the best understood. The most precise transport schemes used so far are the mechano-chemical schemes, where a major conformational change is thought to cause the ion binding site to be alternately exposed to one side and the other of the membrane. This scheme has been adopted by the vast majority of scientists working on the Ca2+-ATPase and Na'/K+-ATPase [1, 21. Another scheme, close to that advocated by Mitchell, invokes a direct interaction between the phosphorylated group and the transported ions [3,4]. Results of deMeis et al. [5] and Dupont and Pougeois [6] have: highlightened the role of the water activity in the active site of the Ca2'-ATPase, which may be extremely different from that of standard conditions.
Science, 1998
Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic... more Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic cells. Here, a kinesin-like protein that interacts with guanosine triphosphate (GTP)–bound forms of Rab6 was identified. This protein, termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular transport. Thus, a molecular motor is a potential effector of a Rab protein, and coordinated action between members of these two families of proteins could control membrane dynamics and directional vesicular traffic.
Journal of Molecular Biology, 2006
Glycoprotein (GP) VI, a key receptor for collagen-induced platelet activation, recently emerged a... more Glycoprotein (GP) VI, a key receptor for collagen-induced platelet activation, recently emerged as a major target for developing new antithrombotics. However, little is known about its functional domains, which is a disadvantage for the rational development of antagonists. Our aim was to identify the structures determining GPVI specificity. GPVI presents homologies with members of the Ig superfamily (in particular with FcαRI) whose extracellular parts present two domains, D1 and D2 linked by a hinge interdomain. To identify the respective role of these domains in GPVI, we have substituted D1 and D2 by their FcαRI homologue in a soluble GPVI fusion protein (GPVI-Fc) and have modified the linker motif by mutagenesis. Proteins were tested for their binding to ligands and antibodies specific for GPVI and FcαRI. We demonstrate for the first time that D2 plays a specific and significant role in GPVI binding to collagen and that the hinge interdomain is critical for the binding to convulxin. Furthermore, binding to CRP requires elements of D1 and of the linker motif. Our results indicate that GPVI is unique amongst the receptors of its family as it uses different structural domains to interact with several agonists and provide evidence that different sites on GPVI constitute targets to develop antagonists of GPVI.
British Journal of Dermatology, 2008
We report the case of an 83-year-old French woman with multiple melanomas showing a severe DNA re... more We report the case of an 83-year-old French woman with multiple melanomas showing a severe DNA repair deficiency, corrected after transfection by XPC cDNA. Two biallelic mutations in the XPC gene are reported: an inactivating frameshift mutation in exon 15 (c.2544delG, p.W848X) and a missense mutation in exon 11 (c.2108 C&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;T, P703L). We demonstrate that these new mutations are involved in the DNA repair deficiency and confirm the diagnosis of xeroderma pigmentosum from complementation group C (XP-C). We speculate that the coexistence of a MC1R variant may be involved in the phenotype of multiple melanomas and that the unusual long-term survival may be related to a lower ultraviolet radiation exposure and to a regular clinical follow-up. This patient appears to be the first French Caucasian XP-C case and one of the oldest living patients with XP reported worldwide.
Biology of the Cell, 1998
Annals of the New York Academy of Sciences, 2006
Site-directed mutagenesis and molecular modeling demonstrated that the N-terminal ectodomain of t... more Site-directed mutagenesis and molecular modeling demonstrated that the N-terminal ectodomain of the VPAC1 receptor is a major site of vasoactive intestinal peptide (VIP) binding. Previous studies with the [Bpa 6 ]-VIP and [Bpa 22 ]-VIP probes (substitution with the photoactivable Bpa for the residues 6 and 22 in VIP) showed spatial approximation between the amino acids 6 and 22 of VIP and the 104-108 and 109-119 sequences within the N-terminal ectodomain of the receptor, respectively. Here, we characterize the new probe (Bz 2-K 24)-VIP (substitution with the photoreactive Bz 2-K for the residue 24 in VIP). After photolabeling and sequential digestions of the receptor, the 121-133 sequence of the N-terminal ectodomain was identified as the site of interaction. The N-terminal ectodomain of the VPAC1 receptor is therefore an affinity trap for the central part of VIP, at least between residues 6 and 24.
Methods in Molecular Biology, 2010
, except for brief excerpts in connection with reviews or scholarly analysis. Use in connection w... more , except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Membrane protein determination starts from extraction-purification and reaches atomic structure by crystal formation and X-ray diffraction or electron microscopy analysis, or nuclear magnetic resonance studies combined or not with molecular modelling.
Physiological Research, 2009
Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat... more Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca2+ pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent with ...
iScience, 2020
Conserved translocator proteins (TSPOs) mediate cell stress responses possibly in a cell-type-spe... more Conserved translocator proteins (TSPOs) mediate cell stress responses possibly in a cell-type-specific manner. This work reports on the molecular function of plant TSPO and their possible evolutionary divergence. Arabidopsis thaliana TSPO (AtTSPO) is stress induced and has a conserved polybasic, plant-specific N-terminal extension. AtTSPO reduces water loss by depleting aquaporin PIP2;7 in the plasma membrane. Herein, AtTSPO was found to bind phosphoinositides in vitro, but only fulllength AtTSPO or chimeric mouse TSPO with an AtTSPO N-terminus bound PI(4,5)P 2 in vitro and modified PIP2;7 levels in vivo. Expression of AtTSPO but not its N-terminally truncated variant enhanced phospholipase C activity and depleted PI(4,5)P 2 from the plasma membrane and its enrichment in Golgi membranes. Deletion or point mutations within the AtTSPO N-terminus affected PI(4,5)P 2 binding and almost prevented AtTSPO-PIP2;7 interaction in vivo. The findings imply functional divergence of plant TSPOs from bacterial and animal counterparts via evolutionary acquisition of the phospholipid-interacting N-terminus.
Trends in Pharmacological Sciences, 2019
The translocator protein (TSPO), an 18-kDa transmembrane protein primarily found in the outer mit... more The translocator protein (TSPO), an 18-kDa transmembrane protein primarily found in the outer mitochondrial membrane, is evolutionarily conserved and widely distributed across species. In mammals, TSPO has been described as a key member of a multiprotein complex involved in many putative functions and over the years several classes of ligands have been developed to modulate these functions. This review considers the currently available atomic structures of mouse and bacterial TSPO and proposes a rationale for the development of new ligands for the protein. A review of TSPO monomeric and oligomeric states and their conformational flexibility, together with ligand binding site and interaction mechanisms, is provided. These data are expected to help the development of high-affinity ligands for TSPO-based therapies or diagnostics considerably.
During inflammatory response, blood leukocytes adhere to the endothelium. This process involves n... more During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1. We previously found that CX3CL1 clusters in oligomers. How this cluster assembles and whether it has a functional role remain unknown. Using various biochemical and biophysical approaches, we show that CX3CL1 clusters are homo-oligomers with 3 to 7 CX3CL1 molecules. We demonstrate that the transmembrane domain peptide self-associates at a similar level in both cellular and acellular lipid environments while its random counterpart (a scrambled peptide) does not. Hence, oligomerization is mainly driven by the transmembrane domain intrinsic properties. Molecular modeling suggests that transmembrane peptide oligomers are mostly made of monomers linearly assembled side by side. Using a new adherence assay, we demonstrate that, functionally, oligomerization is mandatory for the adhesive potency of CX3CL1. Our results indi...
International Journal of Molecular Sciences, 2019
The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well ... more The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis...
Biochemical Society Transactions, 1981
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Papers by Jean-Jacques Lacapère