ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:http://www.springer.com/series/7651

Beta-Arrestins

Methods and Protocols

Edited by

Mark G. H. Scott

Institut Cochin, INSERM U1016, Paris, France

CNRS, UMR8104, Paris, France

Université Paris Descartes, Sorbonne Paris Cité, Paris, France

Stéphane A. Laporte

Department of Medicine, Research Institute of the McGill University Health Center (RI-MUHC), McGillUniversity, Montreal, QC, Canada

Department of Pharmacology and Therapeutics, McGill University, Montréal, QC, Canada

Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada

RI-MUHC/Glen Site, Montréal, QC, Canada

EditorsMark G. H. ScottInstitut CochinINSERM U1016Paris, France

CNRS, UMR8104Paris, France

Universite Paris Descartes,Sorbonne Paris CiteParis, France

Stephane A. LaporteDepartment of Medicine, Research Institute of the McGillUniversity Health Center (RI-MUHC)McGill UniversityMontreal, QC, Canada

Department of Pharmacology and TherapeuticsMcGill UniversityMontreal, QC, Canada

Department of Anatomy and Cell BiologyMcGill UniversityMontreal, QC, Canada

RI-MUHC/Glen SiteMontreal, QC, Canada

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-4939-9157-0 ISBN 978-1-4939-9158-7 (eBook)https://doi.org/10.1007/978-1-4939-9158-7

Library of Congress Control Number: 2019935574

© Springer Science+Business Media, LLC, part of Springer Nature 2019This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulationsand therefore free for general use.The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed tobe true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cover Caption: This image represents the homology model of AT1R/β-arrestin complex based on the Rhodopsin-arrestin crystal structure (5W0P). This model was graciously provided by Dr. Laporte’s student Aaron Cho.

This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part ofSpringer Nature.The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Preface

The arrestin family is composed of four members: visual arrestin (arrestin-1) and conearrestin (arrestin-4), which are expressed in the eye, and the ubiquitously expressedβ-arrestins (β-arrs), β-arr1 and β-arr2 (also known as arrestin-2 and arrestin-3, respectively).β-arrs were initially appreciated for the roles they play in the desensitization and endocytosisof G protein-coupled receptors (GPCRs), but they are now also known to act as multifunc-tional adaptor proteins binding many non-receptor protein partners to control a plethora ofsignalling pathways. β-arrs are therefore key regulators at the crossroads of external cellinputs and functional outputs in cellular processes ranging from gene transcription to cellgrowth, survival, cytoskeletal regulation, and migration. An increasing number of studieshave also highlighted the scaffolding roles β-arrs play in vivo in both physiological andpathological conditions, which open up therapeutic avenues to explore. As our understand-ing of β-arrs has progressed, multiple new techniques and approaches have been developedto probe β-arr function.

This volume entitled Beta-Arrestins: Methods and Protocols provides approaches to studythe pleiotropic roles of β-arrs in the control of signal transduction and the subsequentcellular and in vivo consequences that arise. Contributions cover established and cutting-edge techniques to investigate different facets of β-arr function. The chapters are provided inan easy-to-follow format with background information, lists of materials and reagents, step-by-step protocols, and a notes section describing tips and variations on techniques that canbe used, as well as potential problems that can arise and how they can be circumvented. Afirst series of chapters cover various methods, including bioluminescence resonance energytransfer (BRET)-based techniques, to investigate the canonical roles of β-arrs in GPCRregulation and trafficking (Chapters 3–6). A spectrum of methods is next provided to studythe scaffolding functions of β-arrs with non-receptor protein partners that influence MAPKactivation, cAMP hydrolysis, and cytoskeletal dynamics (Chapters 7–11). In addition,methods to validate β-arr-partner binding interfaces predicted by a docking algorithm(Chapter 12) and methods describing a mathematical framework to dynamically modelβ-arr-signalling networks (Chapter 13) are included. Methods to perform global proteomicanalysis of the β-arr interactome using mass spectrometry are also detailed (Chapter 14).While GPCRs can signal through both G proteins and β-arrs, in some situations, agonistscan skew the receptor to preferentially activate one effector in what is termed "ligand bias."A stepwise protocol is provided to analyze bias (Chapter 15). β-arrs have also been shown toplay non-canonical roles in the nucleus and in ciliogenesis; methods are furnished toinvestigate the nucleocytoplasmic trafficking functions of β-arrs (Chapter 16) and to moni-tor targeting of β-arr2 to the centrosome, basal body, and primary cilium (Chapter 17). Theability of β-arrs to perform such a broad range of functions within cells depends on theircapacity to adopt multiple conformations; chapters detailing methods to monitor β-arrconformational rearrangement using a mass spectrometry-based structural assay(Chapter 18) and FlAsH-BRET intramolecular biosensors are provided (Chapter 19). Thefinal chapters cover methods to study the roles of β-arrs in physiopathology, using cellularand in vivo models, in the contexts of host–pathogen interactions (Chapter 20), allergicinflammatory airway disease (Chapter 21), Type 2 diabetes (Chapters 22 and 23), Parkin-son’s disease (Chapter 24), and cancer (Chapter 25).

v

It was our goal here to provide a helpful resource to those interested in investigating thefunction and regulation of β-arrestins. As editors, we express our sincere gratitude to thescientists that have contributed their time, expertise, and enthusiasm in producing chaptersfor this volume. Clearly, without their valuable input this book would not have beenpossible.

Paris, France Mark G. H. ScottMontreal, QC, Canada Stephane A. Laporte

vi Preface

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I INTRODUCTION

1 A Brief History of the β-Arrestins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Marc G. Caron and Lawrence S. Barak

2 β-Arrestins: Multitask Scaffolds Orchestrating the Whereand When in Cell Signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Stephane A. Laporte and Mark G. H. Scott

PART II METHODS TO STUDY ROLES OF β-ARRESTINS

IN GPCR REGULATION

3 Methods to Monitor the Trafficking of β-Arrestin/G Protein-CoupledReceptor Complexes Using Enhanced Bystander BRET . . . . . . . . . . . . . . . . . . . . . 59Yubo Cao, Yoon Namkung, and Stephane A. Laporte

4 Methods to Investigate β-Arrestin-Mediated Regulation of GPCRFunction in Human Airway Smooth Muscle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Tonio Pera and Raymond B. Penn

5 Measuring Recruitment of β-Arrestin to G Protein-CoupledHeterodimers Using Bioluminescence Resonance Energy Transfer . . . . . . . . . . . . 83Dany Fillion, Dominic Devost, and Terence E. Hebert

6 Detection of β-Arrestin-Mediated G Protein-Coupled ReceptorUbiquitination Using BRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93Karim Nagi and Sudha K. Shenoy

PART III METHODS TO STUDY SCAFFOLDING FUNCTIONS OF β-ARRESTINS

7 Using In Vitro Pull-Down and In-Cell Overexpression Assays to StudyProtein Interactions with Arrestin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107Nicole A. Perry, Xuanzhi Zhan, Eugenia V. Gurevich, T. M. Iverson,and Vsevolod V. Gurevich

8 Methods to Investigate Arrestins in Complex with Phosphodiesterases . . . . . . . . 121Amy J. Tibbo, Tara Busiau, and George S. Baillie

9 Methods to Characterize Protein Interactions with β-Arrestin In Cellulo . . . . . . 139Revu Ann Alexander, Isaure Lot, and Herve Enslen

10 Methods to Investigate the β-Arrestin-Mediated Control of ARF6Activation to Regulate Trafficking and Actin Cytoskeleton Remodeling . . . . . . . 159Ricardo Charles, Mohamed Bourmoum, Shirley Campbell,and Audrey Claing

vii

11 Methods to Investigate the Roles of β-Arrestin-Dependent RalGDSActivation in GPCR-Stimulated Membrane Blebbing . . . . . . . . . . . . . . . . . . . . . . . 169Stephen S. G. Ferguson

12 Methods to Determine Interaction Interfaces Between β-Arrestinsand Their Protein Partners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177Thomas Bourquard, Astrid Musnier, Aurelie Trefier, Flavie Landomiel,Thomas Boulo, Eric Reiter, Pascale Crepieux, and Anne Poupon

13 Workflow Description to Dynamically Model β-Arrestin SignalingNetworks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195Romain Yvinec, Mohammed Akli Ayoub, Francesco De Pascali,Pascale Crepieux, Eric Reiter, and Anne Poupon

14 Proteomic Analysis of the β-Arrestin Interactomes . . . . . . . . . . . . . . . . . . . . . . . . . . 217Yang Zhao and Kunhong Xiao

PART IV METHODS TO ANALYSE LIGAND BIAS

15 Quantitating Ligand Bias Using the Competitive Modelof Ligand Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235Edward L. Stahl, Frederick J. Ehlert, and Laura M. Bohn

PART V METHODS TO STUDY β-ARRESTIN LOCALIZATION

16 Methods to Investigate the Nucleocytoplasmic ShuttlingProperties of β-Arrestins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251Elodie Blondel-Tepaz, Thomas Guilbert, and Mark G. H. Scott

17 Monitoring β-Arrestin 2 Targeting to the Centrosome, Basal Body,and Primary Cilium by Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 271Anahi Molla-Herman, Kathryn M. Davis, Kirk Mykytyn,and Alexandre Benmerah

PART VI METHODS TO STUDY CONFORMATIONAL CHANGE

OF β-ARRESTIN

18 A Mass Spectrometry-Based Structural Assay for Activation-DependentConformational Changes in β-Arrestins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293Yang Zhao and Kunhong Xiao

19 Probing Arrestin Function Using Intramolecular FlAsH-BRET Biosensors . . . . 309Erik G. Strungs, Louis M. Luttrell, and Mi-Hye Lee

PART VII METHODS TO STUDY ROLES OF β-ARRESTINS

IN PHYSIOPATHOLOGY

20 Methods to Study the Roles of β-Arrestins in Meningococcal Signaling. . . . . . . . 325Zoe Virion, Stefano Marullo, and Mathieu Coureuil

21 Methods to Investigate the Roles for β-Arrestin-2 in AllergicInflammatory Airway Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335Akhil Hegde and Julia K. L. Walker

viii Contents

22 Methods to Study Roles of β-Arrestins in the Regulation of Pancreaticβ-Cell Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345Stephane Dalle, Safia Costes, Gyslaine Bertrand, and Magalie A. Ravier

23 Methods to Investigate β-Arrestin Function in Metabolic Regulation . . . . . . . . . 365Bing Luan, Jian Zhao, and Gang Pei

24 Methods to Investigate the Role of β-Arrestin Signalingin Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385Nikhil M. Urs

25 Methods to Investigate β-Arrestin-1/β-Catenin Signalingin Ovarian Cancer Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393Laura Rosano, Roberta Cianfrocca, and Anna Bagnato

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

Contents ix

Contributors

REVU ANN ALEXANDER � Institut Cochin, INSERM U1016, CNRS UMR8104, UniversiteParis Descartes, Sorbonne Paris Cite, Paris, France

MOHAMMED AKLI AYOUB � PRC, INRA, CNRS, IFCE, Universite de Tours, Nouzilly,France; Biology Department, College of Science, United Arab Emirates University, Al Ain,United Arab Emirates

ANNA BAGNATO � Preclinical Models and New Therapeutic Agents Unit, IRCCS-ReginaElena National Cancer Institute, Rome, Italy

GEORGE S. BAILLIE � Institute of Cardiovascular and Medical Sciences, College of Veterinary,Medical and Life Sciences, University of Glasgow, Glasgow, UK

LAWRENCE S. BARAK � Department of Cell Biology, Duke University Medical Center,Durham, NC, USA

ALEXANDRE BENMERAH � Laboratoire des Maladies Renales Hereditaires, INSERM U1163,Paris, France; Institut Imagine, Universite Paris Descartes, Sorbonne Paris Cite, Paris,France

GYSLAINE BERTRAND � Institut de Genomique Fonctionnelle, CNRS UMR5203, INSERMU1191, Montpellier University, Montpellier, France

ELODIE BLONDEL-TEPAZ � Institut Cochin, INSERM U1016, Paris, France; CNRS,UMR8104, Paris, France; Universite Paris Descartes, Sorbonne Paris Cite, Paris, France

LAURA M. BOHN � Department of Molecular Medicine, The Scripps Research Institute,Jupiter, FL, USA; Department of Neuroscience, The Scripps Research Institute, Jupiter, FL,USA

THOMAS BOULO � PRC, INRA, CNRS, Universite Francois Rabelais-Tours, Nouzilly,France

MOHAMED BOURMOUM � Department of Pharmacology and Physiology, Faculty of Medicine,Universite de Montreal, Montreal, QC, Canada

THOMAS BOURQUARD � PRC, INRA, CNRS, Universite Francois Rabelais-Tours, Nouzilly,France; Department of Human and Molecular Genetics, Baylor College of Medicine,Houston, TX, USA

TARA BUSIAU � Institute of Cardiovascular and Medical Sciences, College of Veterinary,Medical and Life Sciences, University of Glasgow, Glasgow, UK

SHIRLEY CAMPBELL � Department of Pharmacology and Physiology, Faculty of Medicine,Universite de Montreal, Montreal, QC, Canada

YUBO CAO � Department of Pharmacology and Therapeutics, McGill University, Montreal,QC, Canada

MARC G. CARON � Department of Cell Biology, Duke University Medical Center, Durham,NC, USA; Department of Neurobiology, Duke University Medical Center, Durham, NC,USA; Department of Medicine, Duke University Medical Center, Durham, NC, USA

RICARDO CHARLES � Department of Pharmacology and Physiology, Faculty of Medicine,Universite de Montreal, Montreal, QC, Canada

ROBERTA CIANFROCCA � Preclinical Models and New Therapeutic Agents Unit, IRCCS-Regina Elena National Cancer Institute, Rome, Italy

AUDREY CLAING � Department of Pharmacology and Physiology, Faculty of Medicine,Universite de Montreal, Montreal, QC, Canada

xi

SAFIA COSTES � Institut de Genomique Fonctionnelle, CNRS UMR5203, INSERM U1191,Montpellier University, Montpellier, France

MATHIEU COUREUIL � Institut-Necker-Enfants-Malades, INSERM, Unite U1151, Paris,France; Faculte de Medecine, Universite Paris Descartes, Sorbonne Paris Cite, Paris, France

PASCALE CREPIEUX � PRC, INRA, CNRS, Universite Francois Rabelais-Tours, Nouzilly,France

STEPHANE DALLE � Institut de Genomique Fonctionnelle, CNRS UMR5203, INSERMU1191, Montpellier University, Montpellier, France

KATHRYN M. DAVIS � Department of Biological Chemistry and Pharmacology, The Ohio StateUniversity, Columbus, OH, USA

FRANCESCO DE PASCALI � PRC, INRA, CNRS, IFCE, Universite de Tours, Nouzilly, FranceDOMINIC DEVOST � Department of Pharmacology and Therapeutics, McGill University,

Montreal, QC, CanadaFREDERICK J. EHLERT � Department of Pharmacology, School of Medicine, University of

California, Irvine, Irvine, CA, USAHERVE ENSLEN � Institut Cochin, INSERM U1016, CNRS UMR8104, Universite Paris

Descartes, Sorbonne Paris Cite, Paris, FranceSTEPHEN S. G. FERGUSON � Department of Cellular and Molecular Medicine, University of

Ottawa Brain and Mind Institute, University of Ottawa, Ottawa, ON, CanadaDANY FILLION � Department of Pharmacology and Therapeutics, McGill University, Montre

al, QC, CanadaTHOMAS GUILBERT � Institut Cochin, INSERM U1016, Paris, France; CNRS, UMR8104,

Paris, France; Universite Paris Descartes, Sorbonne Paris Cite, Paris, FranceEUGENIAV. GUREVICH � Department of Pharmacology, Vanderbilt University, Nashville, TN,

USAVSEVOLOD V. GUREVICH � Department of Pharmacology, Vanderbilt University, Nashville,

TN, USATERENCE E. HEBERT � Department of Pharmacology and Therapeutics, McGill University,

Montreal, QC, CanadaAKHIL HEGDE � School of Nursing, Duke University Medical Center, Durham, NC, USAT. M. IVERSON � Department of Pharmacology, Vanderbilt University, Nashville, TN, USA;

Department of Biochemistry, Vanderbilt University, Nashville, TN, USA; Center forStructural Biology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Institute ofChemical Biology, Vanderbilt University, Nashville, TN, USA

FLAVIE LANDOMIEL � PRC, INRA, CNRS, Universite Francois Rabelais-Tours, Nouzilly,France

STEPHANE A. LAPORTE � Department of Medicine, Research Institute of the McGill UniversityHealth Center (RI-MUHC), McGill University, Montreal, QC, Canada; Department ofPharmacology and Therapeutics, McGill University, Montreal, QC, Canada; Departmentof Anatomy and Cell Biology, McGill University, Montreal, QC, Canada; RI-MUHC/Glen Site, Montreal, QC, Canada

MI-HYE LEE � Department of Medicine, Medical University of South Carolina, Charleston,SC, USA

ISAURE LOT � Institut Cochin, INSERM U1016, CNRS UMR8104, Universite ParisDescartes, Sorbonne Paris Cite, Paris, France

BING LUAN � Department of Endocrinology, Shanghai Tenth People’s Hospital, School ofMedicine, Tongji University, Shanghai, China

xii Contributors

LOUIS M. LUTTRELL � Department of Medicine, Medical University of South Carolina,Charleston, SC, USA; Department of Biochemistry and Molecular Biology, MedicalUniversity of South Carolina, Charleston, SC, USA

STEFANO MARULLO � Faculte de Medecine, Universite Paris Descartes, Sorbonne Paris Cite,Paris, France; Institut Cochin, INSERM, U1016, CNRS UMR 8104, Paris, France

ANAHI MOLLA-HERMAN � Department of Genetics and Developmental Biology, InstitutCurie, CNRS-UMR3215, INSERM-U934, Paris, France; CIRB, College de France,CNRS-UMR7241, INSERM-U1050, Paris, France

ASTRID MUSNIER � PRC, INRA, CNRS, Universite Francois Rabelais-Tours, Nouzilly,France

KIRK MYKYTYN � Department of Biological Chemistry and Pharmacology, The Ohio StateUniversity, Columbus, OH, USA; Neuroscience Research Institute, The Ohio StateUniversity, Columbus, OH, USA

KARIM NAGI � Department of Cell Biology, Duke University Medical Center, Durham, NC,USA; College of Medicine, Member of QU Health, Qatar University, Doha, Qatar

YOON NAMKUNG � Department of Medicine, Research Institute of the McGill UniversityHealth Center (RI-MUHC), McGill University, Montreal, QC, Canada

GANG PEI � State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular CellScience, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences,Shanghai, China; Laboratory of Receptor-Based Bio-Medicine, Shanghai Key Laboratory ofSignaling and Disease Research, School of Life Sciences and Technology, Tongji University,Shanghai, China

RAYMOND B. PENN � Division of Pulmonary, Allergy, and Critical Care Medicine,Department of Medicine, Center for Translational Medicine, Jane and Leonard KormanRespiratory Institute, Thomas Jefferson University, Philadelphia, PA, USA

TONIO PERA � Division of Pulmonary, Allergy, and Critical Care Medicine, Department ofMedicine, Center for Translational Medicine, Jane and Leonard Korman RespiratoryInstitute, Thomas Jefferson University, Philadelphia, PA, USA

NICOLE A. PERRY � Department of Pharmacology, Vanderbilt University, Nashville, TN,USA

ANNE POUPON � PRC, INRA, CNRS, Universite Francois Rabelais-Tours, Nouzilly, FranceMAGALIE A. RAVIER � Institut de Genomique Fonctionnelle, CNRS UMR5203, INSERM

U1191, Montpellier University, Montpellier, FranceERIC REITER � PRC, INRA, CNRS, Universite Francois Rabelais-Tours, Nouzilly, FranceLAURA ROSANO � Preclinical Models and New Therapeutic Agents Unit, IRCCS-Regina

Elena National Cancer Institute, Rome, ItalyMARK G. H. SCOTT � Institut Cochin, INSERM U1016, Paris, France; CNRS, UMR 8104,

Paris, France; Universite Paris Descartes, Sorbonne Paris Cite, Paris, FranceSUDHA K. SHENOY � Department of Cell Biology, Duke University Medical Center, Durham,

NC, USA; Department of Medicine (Cardiology), Duke University Medical Center,Durham, NC, USA

EDWARD L. STAHL � Department of Molecular Medicine, The Scripps Research Institute,Jupiter, FL, USA; Department of Neuroscience, The Scripps Research Institute, Jupiter, FL,USA

ERIK G. STRUNGS � Department of Medicine, Medical University of South Carolina,Charleston, SC, USA

AMY J. TIBBO � Institute of Cardiovascular and Medical Sciences, College of Veterinary,Medical and Life Sciences, University of Glasgow, Glasgow, UK

Contributors xiii

AURELIE TREFIER � PRC, INRA, CNRS, Universite Francois Rabelais-Tours, Nouzilly,France

NIKHIL M. URS � Department of Pharmacology and Therapeutics, University of Florida,Gainesville, FL, USA

ZOE VIRION � Institut-Necker-Enfants-Malades, INSERM, Unite U1151, Paris, France;Faculte de Medecine, Universite Paris Descartes, Sorbonne Paris Cite, Paris, France

JULIA K. L. WALKER � School of Nursing, Duke University Medical Center, Durham, NC,USA; Department of Medicine, Duke University Medical Center, Durham, NC, USA

KUNHONG XIAO � Department of Pharmacology and Chemical Biology, University ofPittsburgh, Pittsburgh, PA, USA; Vascular Medicine Institute, University of Pittsburgh,Pittsburgh, PA, USA; Biomedical Mass Spectrometry Center, School of Medicine, Universityof Pittsburgh, Pittsburgh, PA, USA

ROMAIN YVINEC � PRC, INRA, CNRS, IFCE, Universite de Tours, Nouzilly, FranceXUANZHI ZHAN � Department of Chemistry, Tennessee Technological University, Cookeville,

TN, USAJIAN ZHAO � Translational Medical Center for Stem Cell Therapy, Shanghai East Hospital,

School of Medicine, Tongji University, Shanghai, ChinaYANG ZHAO � Department of Pharmacology and Chemical Biology, University of Pittsburgh,

Pittsburgh, PA, USA

xiv Contributors