NLR Proteins: Methods and Protocols

269 Pages·2016·7.945 MB·English

by  Francesco Di Virgilio, Pablo Pelegrín

Methods in
Molecular Biology 1417
Francesco Di Virgilio
Pablo Pelegrín Editors
NLR Proteins
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
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John M. Walker
University of Hertfordshire
School of Life and Medical Sciences
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NLR Proteins
Methods and Protocols
Edited by
Francesco Di Virgilio
Department of Morphology, Surgery and Experimental Medicine,
University of Ferrara, Ferrara, Italy
Pablo Pelegrín
Molecular Inflammation Group, Murcia Biomedical Research Institute (IMIB-Arrixaca),
Hospital Virgen de la Arrixaca, Murcia, Spain
Editors
Francesco D i Virgilio Pablo P elegrín
Department of Morphology Molecular Inflammation Group
Surgery and Experimental Medicine Murcia Biomedical Research Institute (IMIB-
University of Ferrara Arrixaca), Hospital Virgen de la Arrixaca
Ferrara, Italy Murcia, S pain
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-3564-2 ISBN 978-1-4939-3566-6 (eBook)
DOI 10.1007/978-1-4939-3566-6
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Prefa ce
Infl ammation is the most fundamental defense mechanism developed by multicellular organ-
isms [1]. Central to infl ammation is the ability to discriminate noxious from non-n oxious
agents and to detect signs of tissue damage or cellular distress that might signal an impend-
ing danger. Given the multiplicity of foreign microorganisms which our body gets in contact
with throughout its life, the ability to tell “dangerous” from “non-dangerous” is crucial
because we do not want to rouse a potentially destructive response, such as infl ammation, if
not absolutely necessary. The immune system selected the capability to cause cell or tissue
damage as an unequivocal proof of a given microorganism “dangerousness.” Furthermore,
as it is well known to clinicians, harmful agents are very often of endogenous origin (e.g.,
misfolded proteins or products of abnormal metabolic pathways); thus, an effi cient defense
mechanism should also be able to detect the presence of these agents. Thus, as pointed out
by Carl Nathan, in order to start infl ammation in response to a foreign microorganism, our
body needs to detect the guest (the pathogen) and have evidence of its “dangerousness,”
i.e., detect the damage [2]. This “two-hit” system is based on the ability to identify on one
hand molecular signs of the presence of the pathogen, i.e., pathogen-a ssociated molecular
patterns, PAMPs, and on the other molecular signs of possible cell damage or distress, i.e.,
damage-associated molecular patterns, DAMPs [3]. Very interestingly, DAMPs released as a
consequence of sterile tissue damage, as, for example, in the case of closed trauma, autoim-
mune diseases, or metabolic stress, are themselves suffi cient to trigger infl ammation (sterile
infl ammation), in the absence of PAMPs. This self-suffi ciency of endogenous factors may tell
us something of the evolutionary driving forces behind the infl ammatory response.
Immune cells have developed a sophisticated array of receptors to monitor the extracel-
lular and intracellular environment for the presence of PAMPs and DAMPs. At least four
different families of receptors are known: (1) C-type lectin receptors (CLRs), (2) retinoid-
acid inducible gene (RIG)-I-like receptors (RLRs), (3) toll-like receptors (TLRs), and (4)
nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). TLRs are spe-
cialized to detect pathogens in the extracellular space or in the endosome lumen that can
be equated to the extracellular space. NLRs are specialized to sense pathogen presence in
the cytoplasm; thus, they can be considered the prototypic intracellular pathogen/danger-
sensing receptors. Over the last 10 years, our knowledge of NLRs, as well as the number of
diseases in which these key defense molecules are involved, has increased exponentially. We
now know that some NLRs are intracellularly assembled together with other proteins (i.e.,
ASC and caspase-1) in a macromolecular complex named the infl ammasome and that this
complex undergoes a complex molecular rearrangement during immune cell activation [4].
Such changes (translocation to different intracellular compartments, shift in the affi nity for
intracellular nucleotides, conformational changes) are intimately linked to immune cell
effector responses.
The tumultuous growth of interest on the NLRs requires a parallel increase in the tech-
nical weaponry for the molecular and biochemical investigation. This is the need that this
book aims to satisfy. In the fi rst chapter (by Edward Lavelle), we provide a succinct albeit
authoritative appraisal of current knowledge of innate immune receptors. In the second
chapter Fayyaz Sutterwala reports on the so-called “atypical” infl ammasomes. Then four
chapters, by Isabelle Couillin, David Brough, Pablo Pelegrin, and Veit Hornung, follow on
v
vi Preface
classical biochemical and novel bioluminescence techniques for the measurement of IL-1β
release as a readout of infl ammasome activation. Francesco Di Virgilio describes a novel
bioluminescent probe for in vivo imaging of extracellular ATP, the prototypic DAMP. The
following fi ve chapters, by Christian Stehlik, Bernardo Franklin, Fatima Martin, Monica
Comalada, and Ming-Zong Lai, reports on different biochemical and microscopy tech-
niques that can be used to monitor NLR oligomerization. The following chapters focus on
the consequences of infl ammasome activation. Virginie Petrilli describes techniques to
measure caspase-1 activation. Fabio Martinon reports on cell-f ree systems for the study of
infl ammasome function. On the other hand, Vincent Compan details the protocols for
NLR reconstitution in a cell model such as HEK293 cells. Gloria Lopez-Castejon describes
the procedure to investigate posttranscriptional NLR modifi cations. Lorenzo Galluzzi
describes the protocols for the study of one of the most unfavorable consequences of
infl ammasome activation, i.e., cell death. F inally, Marco Gattorno and Anna Rubartelli
give an update appraisal of the application of infl ammasome studies to the clinic .
We hope that this book will provide a sound basis for the molecular investigation of
NLR function in health and disease and will sparkle interest in these fascinating molecules
by investigators from many different and faraway disciplines.
Ferrara, Italy Francesco Di Virgilio
Murcia, Spain Pablo Pelegrín
References
1 . Medzhitov R (2008) Origin and physiologi- 3 . S chroder K, Tschopp J (2010) The infl amma-
cal roles of inflammation. Nature 454: somes. Cell 140:821–832
428–435 4 . Gross O, Thomas CJ, Guarda G, Tschopp J
2 . Nathan C (2002) Points of control in infl amma- (2011) The infl ammasome: an integrated view.
tion. Nature 420:846–852 Immunol Rev 243:136–151
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
1 I nnate Immune Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Natalia Muñoz-Wolf and E d C. L avelle
2 A typical Inflammasomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5
Ann M . J anowski and F ayyaz S. Sutterwala
3 A ssessment of Inflammasome Activation by Cytokine
and Danger Signal Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3
Nicolas R iteau , A urélie G ombault , and Isabelle Couillin
4 I nvestigating IL-1β Secretion Using Real-Time Single-Cell Imaging. . . . . . . . . . . . 7 5
Catherine D iamond , James Bagnall , D avid G. Spiller , Michael R. White ,
A lessandra M ortellaro , P awel Paszek , and David Brough
5 M easuring IL-1β Processing by Bioluminescence Sensors I:
Using a Bioluminescence Resonance Energy Transfer Biosensor . . . . . . . . . . . . . . . 8 9
Vincent Compan and P ablo P elegrín
6 Measuring IL-1β Processing by Bioluminescence Sensors II: The iGLuc System . . . 9 7
Eva B artok , Maria Kampes , and Veit Hornung
7 Assessing Extracellular ATP as Danger Signal In Vivo: The pmeLuc System . . . . . . 1 15
Francesco D i Virgilio , P aolo Pinton , and Simonetta F alzoni
8 Measuring NLR Oligomerization I: Size Exclusion Chromatography,
Co-immunoprecipitation, and Cross-Linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 31
Sonal K hare , Alexander D . Radian , Andrea Dorfleutner ,
and C hristian Stehlik
9 M easuring NLR Oligomerization II: Detection of ASC Speck
Formation by Confocal Microscopy and Immunofluorescence . . . . . . . . . . . . . . . . 1 45
Michael B eilharz , Dominic D e Nardo’s , E icke L atz , and Bernardo S. Franklin
10 M easuring NLR Oligomerization III: Detection of NLRP3 Complex
by Bioluminescence Resonance Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 59
Fátima M artín-Sánchez , V incent Compan , and Pablo Pelegrín
11 M easuring NLR Oligomerization IV: Using Förster Resonance Energy
Transfer (FRET)-Fluorescence Lifetime Imaging Microscopy (FLIM)
to Determine the Close Proximity of Inflammasome Components . . . . . . . . . . . . . 1 69
Catrin Youssif , B árbara F lix , O livia Belbin , and Mònica C omalada
12 Measuring NLR Oligomerization V: In Situ Proximity Ligation Assay. . . . . . . . . . . 1 85
Yung-Hsuan Wu and M ing-Zong Lai
13 Assessing Caspase-1 Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 97
Baptiste G uey and V irginie P etrilli
14 Cell-Free Assay for Inflammasome Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 07
Yvan J amilloux and F abio Martinon
vii
viii Contents
15 F unctional Reconstruction of NLRs in HEK293 Cells . . . . . . . . . . . . . . . . . . . . . . 2 17
Vincent C ompan and Gloria López-Castejón
16 M ethod to Measure Ubiquitination of NLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 23
Pablo P alazón-Riquelme and Gloria López-Castejón
17 Cytofluorometric Quantification of Cell Death Elicited by NLR Proteins . . . . . . . . 2 31
Valentina Sica , Gwenola M anic , G uido K roemer , Ilio Vitale ,
and L orenzo G alluzzi
18 NLR in Human Diseases: Role and Laboratory Findings. . . . . . . . . . . . . . . . . . . . . 2 47
Sonia C arta , M arco G attorno , and Anna Rubartelli
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Contributors
JAMES BAGNALL • Faculty of Life Sciences, U niversity of Manchester , M anchester, U K
EVA B ARTOK • Institute of Molecular Medicine, University Hospital, U niversity of Bonn ,
Bonn, G ermany; Institute of Clinical Chemistry and Clinical Pharmacology, University
Hospital Bonn, Bonn, Germany
MICHAEL BEILHARZ • Institute of Innate Immunity, University Hospitals,
University of Bonn , B onn , Germany
OLIVIA BELBIN • Memory Unit, Neurology Department, H ospital de la Santa Creu i Sant
Pau , B arcelona, Spain ; C entro de Investigación Biomédica en Red sobre Enfermedades
Neurodegenerativas (CIBERned) , M adrid, Spain
DAVID BROUGH • Faculty of Life Sciences, U niversity of Manchester , Manchester, U K
SONIA CARTA • Cell Biology Unit, I RCCS Azienda Ospedaliera Universitaria San
Martino-IST , G enoa, Italy
MÒNICA COMALADA • Institute for Research in Biomedicine (IRB Barcelona) , B arcelona,
Spain
VINCENT COMPAN • Institut de Génomique Fonctionnelle, Labex ICST, Centre National
de la Recherche Scientifi que, Unité Mixte de Recherche 5203, U niversité Montpellier ,
Montpellier, F rance ; I nstitut National de la Santé et de la Recherche Médicale Unité
1191 , M ontpellier, F rance
ISABELLE COUILLIN • INEM, CNRS, UMR7355, University of Orleans , Orleans, F rance;
Molecular and Experimental Immunology and Neurogenetics, Orleans, France
CATHERINE DIAMOND • Faculty of Life Sciences, U niversity of Manchester , M anchester, U K;
Singapore Immunology Network (SIgN), Agency for Science Technology and Research
(A*STAR) , S ingapore, Singapore
ANDREA DORFLEUTNER • Division of Rheumatology, Department of Medicine, Feinberg
School of Medicine, Northwestern University , C hicago, IL, U SA
SIMONETTA FALZONI • Department of Morphology, Surgery and Experimental Medicine,
University of Ferrara , F errara , I taly
BÁRBARA FLIX • Memory Unit, Neurology Department, H ospital de la Santa Creu i Sant
Pau , B arcelona, Spain
BERNARDO S. FRANKLIN • Institute of Innate Immunity , U niversity Hospitals, University
of Bonn , B onn , G ermany
LORENZO G ALLUZZI • Gustave Roussy Cancer Campus , V illejuif, F rance ; I NSERM, U1138 ,
Paris , F rance; ’ E quipe 11 labellisée par la Ligue Nationale contre le Cancer, C entre de
Recherche des Cordeliers , P aris , France ; U niversité Paris Descartes/Paris V, Sorbonne
Paris Cité , P aris , France; Université Pierre et Marie Curie/Paris VI, Paris, France
MARCO GATTORNO • UO Pediatria 2, Istituto G. Gaslini , G enoa , I taly
AURÉLIE G OMBAULT • INEM, CNRS, UMR7355, U niversity of Orleans , O rleans , F rance
BAPTISTE GUEY • INSERM U1052, Centre de Recherche en Cancérologie de Lyon , Lyon,
France ; C NRS UMR5286, C entre de Recherche en Cancérologie de Lyon , L yon, France ;
Université de Lyon , L yon, France ; Centre Léon Bérard , L yon, France
ix