Publications

2019

• SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation
  
  • Ros Oriol
  • Zagar Yvrick
  • Couvet Sandrine
  • Aghaie Alain
  • Roche Fiona
  • Baudet Sarah
  • Louail Alice
  • Petit Christine
  • Mechulam Yves
  • Nicol Xavier
  , 2019. (10.2139/ssrn.3470393)
  DOI : 10.2139/ssrn.3470393
• Formation of S. pombe Erh1 homodimer mediates gametogenic gene silencing and meiosis progression
  
  • Hazra Ditipriya
  • Andrić Vedrana
  • Palancade Benoit
  • Rougemaille Mathieu
  • Graille Marc
  , 2019. Timely and accurate expression of the genetic information relies on the integration of environmental cues and the activation of regulatory networks involving transcriptional and post-transcriptional mechanisms. In fission yeast, meiosis-specific transcripts are selectively targeted for degradation during mitosis by the EMC complex, composed of Erh1, the ortholog of human ERH, and the YTH family RNA-binding protein Mmi1. Here, we present the crystal structure of Erh1 and show that it assembles as a homodimer. Mutations of amino acid residues to disrupt Erh1 homodimer formation result in loss-of-function phenotypes, similar to erh1∆ cells: expression of meiotic genes is derepressed in mitotic cells and meiosis progression is severely compromised. Interestingly, formation of Erh1 homodimer is dispensable for interaction with Mmi1, suggesting that only fully assembled EMC complexes consisting of two Mmi1 molecules bridged by an Erh1 dimer are functionally competent. We also show that Erh1 does not contribute to Mmi1-dependent down-regulation of the meiosis regulator Mei2, supporting the notion that Mmi1 performs additional functions beyond EMC. Overall, our results provide a structural basis for the assembly of the EMC complex and highlight its biological relevance in gametogenic gene silencing and meiosis progression. (10.1101/787739)
  DOI : 10.1101/787739
• Incorporation de la beta alanine dans des polypeptides
  
  • Nigro Giuliano
  , 2019. Les cellules vivantes utilisent 20 acides α aminés canoniques lors de la synthèse ribosomale des protéines, même si dans de rares occasions, d’autres acides aminés (selenocysteine ou pyrrolysine) peuvent être utilisés. Le répertoire des acides aminés utilisables est donc assez restreint. Cela limite la possibilité de construire des protéines ayant de nouvelles propriétés. Une tendance forte dans le domaine de l’ingénierie des protéines est la construction de systèmes permettant d’incorporer des acides aminés non canoniques pendant la biosynthèse in vivo. Ces travaux ont connu d’importants succès, mais tous les acides aminés incorporés jusqu'aux années 2010 étaient des acides aminés α. L’incorporation d’acides aminés β à des sites spécifiques créerait une flexibilité supérieure à celle des acides aminés α dans la chaîne principale, ce qui augmenterait les possibilités de repliement de la protéine. Il a été montré récemment en utilisant un système de traduction in vitro de synthèse protéique qu’il était possible d’incorporer des acides aminés β à des positions spécifiques (Katoh and Suga, 2018). Pour parvenir à transposer ce système in vivo, la principale limitation est l’aminoacylation des ARNt avec les acides aminés β. Il a été récemment proposé que certaines aminoacyl-ARNt synthetases étaient capables d’utiliser les acides aminées β, mais cette capacité reste limitée.Le but de cette thèse est d’incorporer la β-méthionine dans des polypeptides in vivo. Pour cela nous avons caractérisé la reconnaissance et l’utilisation de la L-β-homométhionine par la méthionyl-ARNt synthetase (MetRS) d’E. coli. Nous avons en particulier déterminé une structure cristallographique à haute résolution du complexe MetRS: β -Met. En utilisant la spectroscopie de fluorescence ainsi que la spectroscopie de masse, nous avons pu mettre en évidence l’activation de la -Met en adénylate ainsi que son estérification à un ARNtMet. Toutefois, les efficacités mesurées sont très petites par rapport à ce qui avait été publié. Une contamination de la -Met commerciale par de la méthionine pourrait expliquer ces différences. Une étude in silico basée sur la structure du complexe MetRS:β-Met a été menée en collaboration avec l’équipe de Bio-informatique du laboratoire afin de rechercher des enzymes mutantes plus efficaces vis à vis des acides aminés β. Enfin, nous avons amorcé la mise en place d’une méthode d’évolution dirigée destinée à améliorer l’efficacité d’incorporation des acides aminés β in vivo.
• Insights into the control of mRNA decay by YTH proteins during the transition from meiosis to mitosis in yeasts.
  
  • Hazra Ditipriya
  , 2019. Insights into the control of mRNA decay by YTH proteinsduring the transition from meiosis to mitosis in yeasts.Keywords: Epitranscriptomics, mRNA decay, meiosis, multi-protein complexes, YTH domainCell cycle is controlled by multi-layered processes. A gene is transcribed in mRNA which is translated in proteins but innumerable regulation processes are working to control every step of this apparently simple process. Among these regulatory check points, post-transcriptional regulation is an important one, where formation of a protein-RNA complex may direct the cellular fate. Among these RNA binding proteins, YTH domain proteins are most novel, discovered in late 90s. YTH domain proteins are abundant in eukaryotes and absent in prokaryotes. YTH domain proteins constitute the majority of reader proteins that can specifically identify m6A modification. Human beings have five YTH domain proteins YTHDF1-3, YTHDC1-2 (Hazra, D., Chapat, C., & Graille, M. (2019). m6A mRNA Destiny: Chained to the rhYTHm by the YTH-Containing Proteins. Genes, 10(1), 49.). Although it is evident that these proteins are controlling cellular fate, the function of each protein and their network is yet to be elucidated. In yeast, there is only one YTH domain protein present: Pho92 in Saccharomyces cerevisiae and Mmi1 in Schizosaccharomyces pombe. Apart from the YTH domain there is no sequence homology between these two proteins but their cellular function is similar.It is well established that Mmi1 is responsible for degradation of meiosis specific transcripts during vegetative growth of the cell. Mmi1 forms a tight complex with a small protein, Erh1 (Erh1-Mmi1 complex or EMC). EMC can physically interact with Not1 of CCR4-Not complex and recruit it for degradation of DSR (determinant of selective removal) containing RNAs. The action of Mmi1 is in turn regulated by an RRM domain protein, Mei2. During meiosis, Mei2, along with a lncRNA meiRNA sequesters Mmi1 in a nuclear dot, rendering it inactive and ensuring smooth continuance of meiosis. These three proteins, Mmi1-Erh1-Mei2 play a key role in mitosis to meiosis switch.In S. cerevisiae, Pho92 is involved in the degradation of PHO4 transcripts contributing to phosphate metabolism pathway, during phosphate starvation and also participates in the degradation of mRNAs containing the N6-methyladenosine (m6A) epitranscriptomics marks. Similarly, to S. pombe Mmi1, Pho92 recruits CCR4-Not complex by physical interaction with Not1.During my PhD, I have tried to elucidate the role of these two YTH domain proteins from two model organisms, S. cerevisiae and S. pombe, in mRNA degradation and cell cycle regulation using biochemical and structural approaches.Pho92 of S. cerevisiae physically interacts with Not1 of CCR4-Not complex, we were able to determine the boundaries of this interaction. The interaction between these two proteins was studied by Fluorescence anisotropy. The protein complex was successfully purified and crystallization trials are ongoing.From S. pombe, structure of Mei2-RRM3 was solved with and without an RNA. RNA binding properties of Mei2-RRM3 was studied by ITC. The structure of Erh1 was also solved and we tried to elucidate its importance for biological function of Mmi1. A co-crystallization trial was performed with Mmi1-Mei2-RNA but it was unsuccessful and we ended up with Mmi1 crystals.
• The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112
  
  • Van tran Nhan
  • Ernst Felix G m
  • Hawley Ben
  • Zorbas Christiane
  • Ulryck Nathalie
  • Hackert Philipp
  • Bohnsack Katherine
  • Bohnsack Markus
  • Jaffrey Samie
  • Graille Marc
  • Lafontaine Denis L j
  Nucleic Acids Research, Oxford University Press, 2019, 47 (15), pp.7719-7733. N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5–TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5–TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid. (10.1093/nar/gkz619)
  DOI : 10.1093/nar/gkz619
• IN SITU PEPTIDE RELEASE KINETICS FROM LIPOSOME FORMULATION MONITORED BY NMR SPECTROSCOPY
  
  • Doyen Camille
  • Larquet Eric
  • Coureux Pierre-Damien
  • Frances Oriane
  • Herman Frédéric
  • Sablé Serge
  • Burnouf Jean-Pierre
  • Sizun Christina
  • Lescop Ewen
  , 2019.
• Suicide and suicide attempts after subthalamic nucleus stimulation in Parkinson disease
  
  • Giannini Giulia
  • Francois Matthieu
  • Lhommée Eugénie
  • Polosan Mircea
  • Schmitt Emmanuelle
  • Fraix Valérie
  • Castrioto Anna
  • Ardouin Claire
  • Bichon Amélie
  • Pollak Pierre
  • Benabid Alim-Louis
  • Seigneuret Eric
  • Chabardes Stephan
  • Wack Maxime
  • Krack Paul
  • Moro Elena
  Neurology, American Academy of Neurology, 2019, 93 (1). (10.1212/WNL.0000000000007665)
  DOI : 10.1212/WNL.0000000000007665
• Trm112, a toolbox for RNA methyltransferases
  
  • Graille Marc
  , 2019.
• SponGee: A Genetic Tool for Subcellular and Cell-Specific cGMP Manipulation
  
  • Ros Oriol
  • Zagar Yvrick
  • Ribes Solène
  • Baudet Sarah
  • Loulier Karine
  • Couvet Sandrine
  • Ladarre Delphine
  • Aghaie Alain
  • Louail Alice
  • Petit Christine
  • Mechulam Yves
  • Lenkei Zsolt
  • Nicol Xavier
  Cell Reports, Elsevier Inc, 2019, 27 (13), pp.4003-4012.e6. cGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals. We provide proof-of-concept strategies by using this tool to highlight the role of cGMP signaling in vivo and in discrete subcellular domains. SponGee enables the investigation of local cGMP signals in vivo and paves the way for therapeutic strategies that prevent downstream signaling activation. (10.1016/j.celrep.2019.05.102)
  DOI : 10.1016/j.celrep.2019.05.102
• NMR solution and X-ray crystal structures of a DNA molecule containing both right- and left-handed parallel-stranded G-quadruplexes
  
  • Winnerdy Fernaldo Richtia
  • Bakalar Blaž
  • Maity Arijit
  • Vandana J Jeya
  • Mechulam Yves
  • Schmitt Emmanuelle
  • Phan Anh Tuân
  Nucleic Acids Research, Oxford University Press, 2019, 47, pp.gkz349. Analogous to the B- and Z-DNA structures in double-helix DNA, there exist both right- and left-handed quadruple-helix (G-quadruplex) DNA. Numerous conformations of right-handed and a few left-handed G-quadruplexes were previously observed, yet they were always identified separately. Here, we present the NMR solution and X-ray crystal structures of a right- and left-handed hybrid G-quadruplex. The structure reveals a stacking interaction between two G-quadruplex blocks with different helical orientations and displays features of both right- and left-handed G-quadruplexes. An analysis of loop mutations suggests that single-nucleotide loops are preferred or even required for the left-handed G-quadruplex formation. The discovery of a right- and left-handed hybrid G-quadruplex further expands the polymorphism of G-quadruplexes and is potentially useful in designing a left-to-right junction in G-quadruplex engineering. (10.1093/nar/gkz349)
  DOI : 10.1093/nar/gkz349
• The DEAH-box RNA helicase Dhr1 contains a remarkable carboxyl terminal domain essential for small ribosomal subunit biogenesis
  
  • Roychowdhury Amlan
  • Joret Clément
  • Bourgeois Gabrielle
  • Heurgué-Hamard Valérie
  • Lafontaine Denis
  • Graille Marc
  Nucleic Acids Research, Oxford University Press, 2019, pp.gkz529. Ribosome biogenesis is an essential process in all living cells, which entails countless highly sequential and dynamic structural reorganization events. These include formation of dozens RNA helices through Watson-Crick base-pairing within ribosomal RNAs (rRNAs) and between rRNAs and small nucleolar RNAs (snoRNAs), transient association of hundreds of proteinaceous assembly factors to nascent precursor (pre-)ribosomes, and stable assembly of ribosomal proteins. Unsurprisingly, the largest group of ribosome assembly factors are energy-consuming proteins (NTPases) including 25 RNA helicases in budding yeast. Among these, the DEAH-box Dhr1 is essential to displace the box C/D snoRNA U3 from the pre-rRNAs where it is bound in order to prevent premature formation of the central pseudoknot, a dramatic irreversible long-range interaction essential to the overall folding of the small ribosomal subunit. Here, we report the crystal structure of the Dhr1 helicase module, revealing the presence of a remarkable carboxyl-terminal domain essential for Dhr1 function in ribosome biogenesis in vivo and important for its interaction with its coactivator Utp14 in vitro. Furthermore, we report the functional consequences on ribosome biogenesis of DHX37 (human Dhr1) mutations found in patients suffering from microcephaly and other neurological diseases. (10.1093/nar/gkz529)
  DOI : 10.1093/nar/gkz529
• Cortical branched actin determines cell cycle progression
  
  • Molinie Nicolas
  • Rubtsova Svetlana N
  • Fokin Artem
  • Visweshwaran Sai
  • Rocques Nathalie
  • Polesskaya Anna
  • Schnitzler Anne
  • Vacher Sophie
  • Denisov Evgeny V
  • Tashireva Lubov
  • Perelmuter Vladimir M
  • Cherdyntseva Nadezhda
  • Bièche Ivan
  • Gautreau Alexis
  Cell Research, Nature Publishing Group, 2019, 29 (6), pp.432-445. (10.1038/s41422-019-0160-9)
  DOI : 10.1038/s41422-019-0160-9
• Towards NMR characterization of apelin encapsulated in liposomes
  
  • Doyen Camille
  • Larquet Eric
  • Coureux Pierre-Damien
  • Frances Oriane
  • Herman Frédéric
  • Sablé Serge
  • Burnouf Jean-Pierre
  • Sizun Christina
  • Lescop Ewen
  , 2019.
• Purification of tubulin with controlled post-translational modifications by polymerization–depolymerization cycles
  
  • Souphron Judith
  • Bodakuntla Satish
  • Jijumon A. S
  • Lakisic Goran
  • Gautreau Alexis
  • Janke Carsten
  • Magiera Maria M.
  Nature Protocols, Nature Publishing Group, 2019, 14 (5), pp.1634-1660. In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes similar to 13 d; the tubulin purification takes 6-7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins. (10.1038/s41596-019-0153-7)
  DOI : 10.1038/s41596-019-0153-7
• Start Codon Recognition in Eukaryotic and Archaeal Translation Initiation: A Common Structural Core
  
  • Schmitt Emmanuelle
  • Coureux Pierre-Damien
  • Monestier Auriane
  • Dubiez Etienne
  • Mechulam Yves
  International Journal of Molecular Sciences, MDPI, 2019, 20 (4), pp.939. Understanding molecular mechanisms of ribosomal translation sheds light on the emergence and evolution of protein synthesis in the three domains of life. Universally, ribosomal translation is described in three steps: initiation, elongation and termination. During initiation, a macromolecular complex assembled around the small ribosomal subunit selects the start codon on the mRNA and defines the open reading frame. In this review, we focus on the comparison of start codon selection mechanisms in eukaryotes and archaea. Eukaryotic translation initiation is a very complicated process, involving many initiation factors. The most widespread mechanism for the discovery of the start codon is the scanning of the mRNA by a pre-initiation complex until the first AUG codon in a correct context is found. In archaea, long-range scanning does not occur because of the presence of Shine-Dalgarno (SD) sequences or of short 5' untranslated regions. However, archaeal and eukaryotic translation initiations have three initiation factors in common: e/aIF1, e/aIF1A and e/aIF2 are directly involved in the selection of the start codon. Therefore, the idea that these archaeal and eukaryotic factors fulfill similar functions within a common structural ribosomal core complex has emerged. A divergence between eukaryotic and archaeal factors allowed for the adaptation to the long-range scanning process versus the SD mediated prepositioning of the ribosome. (10.3390/ijms20040939)
  DOI : 10.3390/ijms20040939
• Real-time, in situ peptide-liposome formulation release kinetics monitored by NMR spectroscopy
  
  • Doyen Camille
  • Larquet Eric
  • Coureux Pierre-Damien
  • Frances Oriane
  • Herman Frédéric
  • Sablé Serge
  • Burnouf Jean-Pierre
  • Sizun Christina
  • Lescop Ewen
  , 2019.
• m6A mRNA Destiny: Chained to the rhYTHm by the YTH-Containing Proteins
  
  • Hazra Ditipriya
  • Chapat Clément
  • Graille Marc
  Genes, MDPI, 2019, 10 (1), pp.49. The control of gene expression is a multi-layered process occurring at the level of DNA, RNA, and proteins. With the emergence of highly sensitive techniques, new aspects of RNA regulation have been uncovered leading to the emerging field of epitranscriptomics dealing with RNA modifications. Among those post-transcriptional modifications, N6-methyladenosine (m 6 A) is the most prevalent in messenger RNAs (mRNAs). This mark can either prevent or stimulate the formation of RNA-protein complexes, thereby influencing mRNA-related mechanisms and cellular processes. This review focuses on proteins containing a YTH domain (for YT521-B Homology), a small building block, that selectively detects the m 6 A nucleotide embedded within a consensus motif. Thereby, it contributes to the recruitment of various effectors involved in the control of mRNA fates through adjacent regions present in the different YTH-containing proteins. (10.3390/genes10010049)
  DOI : 10.3390/genes10010049
• A minimal sequence for ZG4 formation
  
  • Bakalar Blaž
  • Brahim Heddi
  • Schmitt Emmanuelle
  • Mechulam Yves
  • Phan Anh Tuan
  , 2019.
• A Minimal Sequence for Left-Handed G-Quadruplex Formation
  
  • Bakalar Blaž
  • Heddi Brahim
  • Schmitt Emmanuelle
  • Mechulam Yves
  • Phan Anh Tuân
  Angewandte Chemie International Edition, Wiley-VCH Verlag, 2019, 58 (8), pp.2331-2335. Recently, we observed the first example of a left-handed G-quadruplex structure formed by natural DNA, named Z-G4. We analysed the Z-G4 structure and inspected its primary 28-nt sequence in order to identify motifs that convey the unique left-handed twist. Using circular dichroism spectroscopy, NMR spectroscopy, and X-ray crystallography, we revealed a minimal sequence motif of 12 nt (GTGGTGGTGGTG) for formation of the left-handed DNA G-quadruplex, which is found to be highly abundant in the human genome. A systematic analysis of thymine loop mutations revealed a moderate sequence tolerance, which would further broaden the space of sequences prone to left-handed G-quadruplex formation. (10.1002/anie.201812628)
  DOI : 10.1002/anie.201812628
• Variable Neighborhood Search with Cost Function Networks To Solve Large Computational Protein Design Problems
  
  • Charpentier Antoine
  • Mignon David
  • Barbe Sophie
  • Cortés Juan
  • Schiex Thomas
  • Simonson Thomas
  • Allouche David
  Journal of Chemical Information and Modeling, American Chemical Society, 2019, 59 (1), pp.127-136. Computational protein design (CPD) aims to predict amino acid sequences that fold to specific structures and perform desired functions. CPD depends on a rotamer library, an energy function, and an algorithm to search the sequence/conformation space. Variable neighborhood search (VNS) with cost function networks is a powerful framework that can provide tight upper bounds on the global minimum energy. We propose a new CPD heuristic based on VNS in which a subset of the solution space (a “neighborhood”) is explored, whose size is gradually increased with a dedicated probabilistic heuristic. The algorithm was tested on 99 protein designs with fixed backbones involving nine proteins from the SH2, SH3, and PDZ families. The number of mutating positions was 20, 30, or all of the amino acids, while the rest of the protein explored side-chain rotamers. VNS was more successful than Monte Carlo (MC), replica-exchange MC, and a heuristic steepest-descent energy minimization, providing solutions with equal or lower best energies in most cases. For complete protein redesign, it gave solutions that were 2.5 to 11.2 kcal/mol lower in energy than those obtained with the other approaches. VNS is implemented in the toulbar2 software. It could be very helpful for large and/or complex design problems. (10.1021/acs.jcim.8b00510)
  DOI : 10.1021/acs.jcim.8b00510
• Phosphorylation of Merlin by Aurora A kinase appears necessary for mitotic progression
  
  • Mandati Vinay
  • Maestro Laurence Del
  • Dingli Florent
  • Lombard Berangère
  • Loew Damarys
  • Molinie Nicolas
  • Romero Stephane
  • Bouvard Daniel
  • Louvard Daniel
  • Gautreau Alexis
  • Pasmant Eric
  • Lallemand Dominique
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2019, 294 (35), pp.12992-13005. Although Merlin function as a tumor suppressor and regulator of mitogenic signaling networks such as the Ras/rac, Akt or Hippo pathways is well documented,in mammals as well as in insects, its role during cell cycle progression remains unclear. In this study, using a combination of approaches, including FACS analysis, time-lapse imaging, immunofluorescence microscopy and co-immunoprecipitation, we show that Ser-518 of Merlin is a substrate of the Aurora A kinase during mitosis and that its phosphorylation facilitates the phosphorylation of a newly discovered site, Thr-581. We found that the expression in Hela cells of a Merlin variant that is phosphorylation-defective on both sites leads to a defect in centrosomes and mitotic spindles positioning during metaphase and delays the transition from metaphase to anaphase. We also show that the dual mitotic phosphorylation not only reduces Merlin binding to microtubules but also timely modulates Ezrin interaction with the cytoskeleton. Finally, we identify several point mutants of Merlin associated with Neurofibromatosis type 2 that display an aberant phosphorylation profile along with defective α-tubulin binding properties. Altogether, our findings of an Aurora A-mediated interaction of Merlin with α-tubulin and Ezrin suggest a potential role for Merlin in cell cycle progression. (10.1074/jbc.RA118.006937)
  DOI : 10.1074/jbc.RA118.006937