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11-23-2020
Interactions of the NLRP3 Inflammasome Complex
Nyasha Makoni University of Missouri-St. Louis, [email protected]
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Interactions of the NLRP3 Inflammasome Complex
Nyasha J. Makoni
M.S., Chemistry, University of Missouri-St. Louis, 2017 B.S., Chemistry, LeMoyne-Owen College, Memphis TN, 2014
A Dissertation Submitted to The Graduate School at the University of Missouri-St. Louis in partial fulfillment of the requirements for the degree Doctor of Philosophy in Chemistry with an emphasis in Biochemistry
December 2020
Advisory Committee
Michael R. Nichols, Ph.D. Chairperson
Cynthia M. Dupureur, Ph.D.
Chung F. Wong, Ph.D.
Wendy M. Olivas, Ph.D.
ACKNOWLEDGEMENTS
I would like to express my gratitude to the UMSL Department of Chemistry
& Biochemistry for accepting me into the Chemistry Ph.D. program. Thank you,
Dr Nichols, for taking me under your wing and mentoring me all these years I have been in your lab. You were kind enough to share your valuable expertise, support, and guidance throughout. It was not an easy road and yes there were challenging times, but you still encouraged me to keep pushing especially when I was getting frustrated with my project. Forever I will be grateful!
Drs. Dupureur, Wong, and Olivas: What an experience to have amazing scientists like you on my dissertation committee. Thanks for sharing your knowledge with me and for all the feedback you gave me. I had the opportunity to take classes with all of you and those were some of my greatest moments at
UMSL. Dr Dupureur, it was a pleasure teaching Biochemistry lab with you. Great moments indeed! Thanks for being a great counselor and helping me maintain my sanity.
Dr. Sanjib Karki thanks for taking your precious time to train me during your last few months in the lab. I hated western blots then, but now I bet I can do a dozen of them in my sleep. Dr. Lisa Picard-Gouwens oh what wonderful times we had. I am lucky I had the opportunity to work with you and you kept us grounded in the lab. I appreciate the great relationship we have.
Adela, Nathan, and Sweta; I appreciate all your help with BCA assays, running gels, and getting milliQ water. Fatima, thanks for all the snacks and
i
NOSH fries. Victoria, you kept me on my toes when we both took Advanced
Biochemistry and Protein Polymers. Evan, thanks for establishing the caspase activity assay and all those countless hours you spent running columns with me.
Kapur, I cannot thank you enough for assisting me with my experiments and your sarcasm never gets old. Always remember the cuvette-washing technique.
Shikha, I appreciate your help, positive vibes, and endless questions. Anna and
Thao, I admire your dedication and you two undoubtedly make the best cloning team ever. Helena, you are one of the smartest and dedicated people I have ever worked with, the best TA buddy ever! Cristina, Marina, Tyler, Antanisha, and
Nick; I enjoyed working with all of you. Catherine and Deva; I cherish the great moments we have shared at UMSL. All the best!
To all my teachers, surely it takes a village to raise a child. Dr Chinn-
Jointer, thanks for helping me get that scholarship at LeMoyne-Owen College. Dr
Hamada, I am grateful that you recommended the UMSL Chemistry program to me. To Dr. Shajilla Siricilla, thanks for training me during my rookie days in the
Kurosu lab. Special thanks to my loved ones for making this journey much easier. To my mother Locadia, I can never sum enough words to express my appreciation for all the sacrifices you have made and your unwavering support.
You are my hero. Molly, Tendai, and Prince; you are the best siblings one could ever ask for. Mr. Ray Jackson, what an honor to have met you. You helped me realize my American dream. Mama Jackie, Muzvare Betty Makoni; I will always be indebted to you all. To the Sherwoods, Mafanas, Davisons; thanks for taking care of me all these years.
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TABLE OF CONTENTS ACKNOWLEDGEMENTS ...... i LIST OF TABLES ...... vii LIST OF FIGURES ...... viii LIST OF ABBREVIATIONS ...... x ABSTRACT ...... xvi CHAPTER 1: INTRODUCTION 1.1. Innate immunity ...... 1 1.2. Amyloid beta (Aβ) and Alzheimer’s disease (AD) ...... 4 1.3. Neuroinflammation and AD ...... 9 1.4. Structure and assembly of the NLRP3 inflammasome ...... 13 1.5. Structural studies of NLRP3 inflammasome proteins (and domains) .... 15 1.6. Apoptosis-associated speck-like protein (ASC) speck formation ...... 19 1.7. Activation of the inflammasome ...... 22 1.8. Regulation of the inflammasome ...... 28 1.8.1. Protein kinases A, C, D (PKA, PKC, PKD) ...... 28 1.8.2. Syk kinase and JNK signaling ...... 29 1.8.3. Bruton’s tyrosine kinase (BTK) ...... 31 1.8.4. Interleukin-receptor-associated kinase1 (IRAK-1) ...... 31 1.8.5. IκB kinase alpha (IKKα) ...... 31 1.8.6. Ubiquitination ...... 32 1.8.7. Sumoylation ...... 33 1.8.8. NIMA-related kinase-7 (NEK7) ...... 33 1.8.9. NOD-like receptor family CARD domain containing proteins (NLRC) ...... 34 1.8.10. Heat shock proteins ...... 34 1.8.11. NLPR3 inflammasome inhibitors ...... 35 1.9. Caspase-1 1.9.1. Initial characterization of caspase-1 ...... 36 1.9.2. Caspase-1 structural studies ...... 37 1.9.3. Caspase-1 functionalization assays ...... 39 1.10. Aβ-NLRP3 inflammasome interactions ...... 40 1.11. Recombinant expression of inflammasome proteins from the literature 40 1.11.1. NLRP3 and ASC proteins ...... 40 1.11.2. Caspase-1 ...... 47 1.12. Overall summary and research goals ...... 50
CHAPTER 2: METHODS 2.1. Gene design and synthesis ...... 51 2.2. Plasmid amplification ...... 54 2.3. ExpiCHO-S Cell Culture ...... 54 2.4. Transfection and expression ...... 55
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2.5. Protein harvesting ...... 56 2.6. Nickel affinity chromatography ...... 57 2.7. Size-exclusion chromatography (SEC) ...... 58 2.8. SEC-multi-angle light scattering (MALS)...... 59 2.9. Dynamic light scattering (DLS) ...... 59 2.10. Circular dichroism (CD) ...... 60 2.11. Transmission Electron Microscopy (TEM) ...... 61 2.12. Protein concentration determination ...... 61 2.13. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) and western blotting...... 62 2.14. In vitro caspase-1 reconstitution assays ...... 63 2.15. Caspase-1 activity assays ...... 64 2.16. Tobacco-etch virus (TEV) cleavage reactions ...... 67 2.17. Interleukin 1-beta (IL-1β) cleavage assays ...... 67 2.18. Aβ protofibril prep and purification ...... 68 2.19. Aβ-NLRP3 inflammasome interactions ...... 69 2.20. Cell culture 2.20.1. THP-1 monocytes ...... 70 2.20.2. Primary microglia ...... 70 2.20.3. BV2 microglia ...... 71 2.21. Cell stimulation assays 2.21.1. Suspension cells ...... 72 2.21.2. Adherent cells ...... 73 2.22. Co-immunoprecipitation (Co-IP) assays ...... 74 2.23. Bis(sulfosuccinimidyl)suberate (BS3) crosslinking ...... 75 2.24. Trichloroacetic acid (TCA) precipitation ...... 76 2.25. Cytokine enzyme-linked immunosorbent assays (ELISA) ...... 77 2.26. Aβ-ASC aggregation assays ...... 78
CHAPTER 3: EXPRESSION AND PURIFICATION OF NLRP3 INFLAMMASOME PROTEINS IN A MAMMALIAN EXPICHO SYSTEM 3.1. The pcDNA3.4-TOPO mammalian vector is ideal for recombinant expression of inflammasome proteins ...... 80 3.2. ExpiCHO-S cells maintain great cell viability at high densities and are ideal for recombinant expression of proteins in suspension ...... 83 3.3. ExpiCHO-S cells maintained good cell viability during the expression period and proteins were detected within 4 days post-transfection ...... 86 3.4. Incorporating detergents, reducing agents, and protein stabilizers in the buffers used during harvesting and lysate preparation maximized protein yields ...... 88 3.5. The three inflammasome proteins were successfully co-expressed in ExpiCHO-S cells ...... 90 3.6. Affinity purification of His-tagged proteins yielded high amounts of expressed proteins ...... 93 3.6.1 Affinity purifications of NLRP3, ASC, and caspase-1 p10 ...... 93 3.6.2 Fusion proteins enhance solubility of expressed proteins ...... 100
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CHAPTER 4: OLIGOMERIZATION STATE DETERMINATION OF EXPICHO- EXPRESSED INFLAMMASOME PROTEINS 4.1. Analysis of the oligomerization state of the recombinant proteins under denaturing conditions ...... 103 4.1.1. Proteins mainly appeared as monomers under denaturing conditions ...... 104 4.1.2. Expressed proteins showed some degree of oligomerization ..... 106 4.1.3. Inclusion of the secretion sequence resulted in differences in oligomerization profiles upon analyzing medium samples ...... 107 4.1.4. Oligomerization profiles of NLRP3 lysates and inclusion bodies are quite distinct ...... 109 4.2. Analysis of inflammasome proteins oligomerization state in solution .. 110 4.2.1. ASC showed some degree of oligomerization ...... 111 4.2.2. NLRP3 existed as a mixture of small and high molecular weight species ...... 113 4.2.3. Optimizing various SEC buffers using bovine serum albumin (BSA) ...... 115 4.2.4. A large portion of ASC fusion proteins appeared in the aggregated form...... 116 4.2.5. SEC-MALS analysis confirmed the existence of protein aggregates ...... 119 4.3. Analysis of protein oligomer structure and morphology 4.3.1. DLS analysis of caspase-1 proteins revealed the presence of monomers and dimers ...... 121 4.3.2. Recombinant ASC exhibits some degree of helicity that is unaffected by changes in pH ...... 123 4.3.3. ASC ball-like aggregates were observed by TEM ...... 125
CHAPTER 5: ISOLATION OF THE ELUSIVE CASPASE-1 ENZYME 5.1 What is the actual structure of the active enzyme? ...... 128 5.2 Recombinant caspase-1 p20p10 reconstitution experiments 5.2.1. Caspase-1 p20p10 reconstitution studies from the literature ...... 132 5.2.2. Caspase-1 stability ...... 133 5.2.3. Caspase-1 p20p10 reconstitution assays +/- inhibitors were unsuccessful ...... 134 5.3 Caspase-1 activity assays 5.3.1. The enzyme assay was optimized using commercial active caspase-1 and inhibitors ...... 137 5.3.2. Recombinant full-length (FL) procaspase-1 exhibited transient enzyme activity ...... 139
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5.4 Various conditions were optimized in order to retain a stable, active enzyme ...... 142 5.5 Recombinant procaspase-1 was unable to cleave the pro form of interleukin-1-beta (IL-1β) ...... 144 5.6 Tobacco-etch virus (TEV) protease cleavage reactions to remove fusion tags ...... 146 5.6.1. The cleavage experiments showed half maximal cleavage efficiency ...... 146 5.6.2. Analysis of cleaved MBP-p10 ...... 149
CHAPTER 6: INTERACTION OF INFLAMMASOME PROTEINS WITH THE ALZHEIMER’S AMYLOID-β PROTEIN 6.1 There is a direct interaction between Aβ PF and NLRP3 protein ...... 152 6.1.1 In vitro interaction assays ...... 154 6.1.2 Cell stimulation assays ...... 155 6.2 Glutathione-S-transferase (GST) fusion tag improves protein solubility ...... 160 6.2.1 Optimizing protein purification conditions using GST-ASC PYD K21A mutant ...... 161 6.2.2 The fusion proteins exhibited concentration-dependent aggregation ...... 164 6.2.3 The soluble and insoluble fractions of GST-ASC CARD showed distinct elution profiles ...... 165 6.2.4 SEC-purified GST-ASC proteins were fairly stable over time ...... 167 6.2.5 The oligomerization state of SEC-purified GST-ASC fusion proteins affects detection by immunoblotting methods ...... 169 6.3 Aβ-ASC aggregation assays ...... 172 6.3.1 GST-ASC domains did not promote aggregation of Aβ monomer ...... 172
CHAPTER 7: OVERALL DISCUSSIONS, CONCLUSIONS, & FUTURE DIRECTIONS 7.1. Conclusions ...... 175 BIBLIOGRAPHY ...... 180 VITA ...... 200
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LIST OF TABLES
CHAPTER 2: METHODS 2.1. All expressed NLRP3 inflammasome proteins ...... 53 2.2. Various protein treatment strategies employed prior to size-exclusion chromatography (SEC) ...... 58 2.3. Reaction vessel components for caspase-1 activity assays ...... 66
CHAPTER 3: EXPRESSION AND PURIFICATION OF NLRP3 INFLAMMASOME PROTEINS IN A MAMMALIAN EXPICHO SYSTEM 3.1. Buffers (and additives) used in the harvesting of inflammasome proteins ...... 89
CHAPTER 4: OLIGOMERIZATION STATE DETERMINATION OF EXPICHO- EXPRESSED INFLAMMASOME PROTEINS 4.1. Various sample treatment conditions employed in order to obtain stable, soluble proteins ...... 110 4.2. DLS experimental data ...... 121
CHAPTER 5: ISOLATION OF THE ELUSIVE CASPASE-1 ENZYME 5.1. Recombinant expression of inflammasome proteins from the literature ...... 132 5.2. UV determined concentrations of refolded caspase-1 p20p10 ...... 136
CHAPTER 6: INTERACTION OF INFLAMMASOME PROTEINS WITH THE ALZHEIMER’S AMYLOID-β PROTEIN 6.1. In vitro inflammasome reconstitution assay set up ...... 154 6.2. hTNFα ELISA and BCA protein assay data using treated cell supernatants and lysates ...... 157 6.3. BCA assays of treated cell lysates before and after TCA precipitation 159 6.4. DLS data of SEC-purified GST-ASC fusion proteins ...... 169
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LIST OF FIGURES
CHAPTER 1: INTRODUCTION 1.1 AD pathology ...... 5 1.2 Generation of Aβ from APP...... 6 1.3 Aβ aggregation pathway ...... 7 1.4 Aβ PF prime and activate the NLRP3 inflammasome ...... 11 1.5 Structure of the NLRP3 inflammasome ...... 13 1.6 Structure of ASC ...... 17 1.7 Activation of the NLRP3 inflammasome ...... 23 1.8 Structure of caspase-1 and its substrate ...... 38
CHAPTER 2: METHODS 2.1. Structure of AMC ...... 65 2.2. Six-well plate experimental set up for LPS Nigericin cell treatment ...... 72 2.3. Aβ-ASC aggregation assays ...... 79
CHAPTER 3: DEVELOPMENT OF A MAMMALIAN EXPICHO CELL SYSTEM FOR EXPRESSION OF NLRP3 INFLAMMASOME PROTEINS 3.1. Plasmid vector with human NLRP3 gene inserted ...... 82 3.2. ExpiCHO-S cell culture, transfection, and protein expression ...... 85 3.3. Monitoring ExpiCHO-S cell viability and secretion of expressed proteins into the medium ...... 87 3.4. Immunoblots showing that the three inflammasome proteins were successfully co-expressed ...... 91 3.5. Schematic showing constructs of all expressed full-length and truncated inflammasome proteins ...... 93 3.6. Initial affinity purification of His-tagged secreted ASC medium using a linear gradient ...... 95 3.7. Affinity purification of His-tagged NLRP3 protein ...... 96 3.8. Affinity purification of His-tagged ASC protein lysate and IB ...... 98 3.9. Affinity purification of His-tagged caspase-1 p10 subunit ...... 99 3.10. Fusion tags enhance the solubility of the ExpiCHO-S-expressed ASC protein ...... 101
CHAPTER 4: OLIGOMERIZATION STATE DETERMINATION OF EXPICHO- EXPRESSED INFLAMMASOME PROTEINS 4.1. Immunoblot analysis confirming identity of affinity-purified inflammasome proteins ...... 105 4.2. Immunoblots of expressed inflammasome proteins showing the presence of SDS-resistant oligomers ...... 106
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4.3. Expressed ASC proteins were present in the ExpiCHO-S medium whether or not they contained a secretion sequence ...... 108 4.4. Oligomerization patterns of NLRP3 pyrin lysates and inclusion bodies are quite distinct ...... 109 4.5. Recombinant ASC exists as oligomers of various molecular weights . 112 4.6. Two rounds of SEC purification yielded monomeric NLRP3 pyrin domain ...... 114 4.7. BSA has different elution profiles in different buffers ...... 115 4.8. Sup 75 purifications of ASC fusion proteins in PBS/ DTT buffer ...... 117 4.9. Superdex 75 elutions of MBP-ASC fusion proteins in various buffers . 118 4.10. SEC-MALS analysis with recombinant MBP-ASC protein ...... 120 4.11. CD studies revealed that recombinant ASC had some structure ...... 124 4.12. TEM images of recombinant ASC ...... 126
CHAPTER 5: ISOLATION OF THE ELUSIVE CASPASE-1 ENZYME 5.1. Schematic showing possible active confomers of caspase-1 ...... 129 5.2. Possible scenarios to explain caspase-1 activation mechanisms ...... 131 5.3. Analysis of refolded caspase-1 p20p10 +/- inhibitors ...... 135 5.4. Optimizing the caspase-1 activity assay with commercial fluorogenic tetrapeptide substrates and inhibitors ...... 138 5.5. Caspase activity assays with refolded p20p10 ...... 139 5.6. FL procaspase-1 possesses enzymatic activity as shown by the fluorogenic peptide assay ...... 141 5.7. Optimizing IL-1β cleavage assay with active commercial caspase-1 .. 145 5.8. TEV cleavage reactions with MBP-p10 ...... 147 5.9. Analysis of cleaved MBP-p10 by affinity purification and immunoblotting ...... 150
CHAPTER 6: INTERACTION OF INFLAMMASOME PROTEINS WITH THE ALZHEIMER’S AMYLOID-β PROTEIN 6.1. Superdex 75 purification of Aβ-42 ...... 153 6.2. Aβ PF show direct interactions with NLRP3 protein ...... 156 6.3. Glutathione s-transferase (GST) fusion tags enhance solubility of ASC proteins ...... 161 6.4. Optimizing sample treatment and purification conditions using affinity- purified GST-ASC PYD K21A mut ...... 162 6.5. Superdex 200 elutions of GST-ASC proteins show concentration- dependent aggregation ...... 165 6.6. Elution profiles of GST-ASC CARD pellet and supernatant samples are distinct ...... 166 6.7. Stability of GST-ASC fusion proteins ...... 168 6.8. Western blot of SEC-purified GST-ASC fusion proteins...... 170 6.9. ASC domains had no effect on Aβ aggregation ...... 173
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LIST OF ABBREVIATIONS
AD, Alzheimer’s disease Aβ, amyloid beta Ac-YVAD-AMC/AFC, acetyl-tyrosine-valine-alanine-aspartic acid- 7-Amino-4- methylcoumarin/ 7-amino-4-trifluoromethylcoumarin Ac-YVAD-CHO, acetyl-tyrosine-valine-alanine-aspartic acid-aldehyde caspase-1 inhibitor Ac-WEHD-AMC, acetyl-tryptophan-glutamic acid-histidine-aspartic acid- 7- Amino-4-methylcoumarin ADDLs, amyloid β-derived diffusible ligands ADP, adenosine diphosphate AFM, atomic force microscopy AIM2, absent in melanoma 2 AKTA-FPLC, integrated system for fast protein liquid chromatography apAbSL, affinity-purified Antibody St. Louis (generated in the Nichols Lab) APDC, (2R,4R)-4aminopyrrolidine-2,4-dicarboxylate APOE4, apolipoprotein E4 APPswe, amyloid precursor protein containing a Swedish mutation (K595N/M596L) ASC, apoptosis-associated speck-like protein containing a CARD (also known as PYCARD) ASC2, a pyrin domain only protein (also known as POP1) ATP, adenosine triphosphate ATPase, enzyme that hydrolyzes ATP to ADP BCA, bicinchoninic acid assay BMDMs, bone marrow derived macrophages BMH, water-insoluble maleimide crosslinker targeting sulfhydryl groups BSA, bovine serum albumin BRCC3, a deubiquitinase BS3, bis(sulfosuccinimidyl)suberate BTK, bruton tyrosine kinase
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BV-2, immortalized murine neonatal microglia b-VAD-cmk/fmk, biotin valyl-alaninyl-aspartyl chloro/fluoro-methylketone cAMP, cyclic adenosine mononophosphate CARD, caspase recruitment domain CARDINAL, an adaptor protein CASR, calcium-sensing receptor CDL, CARD-domain linker CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (zwitterionic detergent) CLIC 1/4, chloride intracellular channel protein 1/4 CLRs, C-type lectin receptor Co-IP, co-immunoprecipitation COP, card-only protein CryoEM, cryogenic electron microscopy CSF, cerebrospinal fluid C57BL/6J, inbred mouse strain DAMP, danger-associated molecular patterns DD, death domain DLS, dynamic light scattering DMEM, Dulbecco's modified Eagle's medium DTT, dithiothreitol (reducing agent) DUB, deubiquitinating enzyme ECL, enhanced chemiluminescence (western blot substrate) E. coli, Escherichia coli EDTA, ethylenediaminetetraacetic acid EK, enterokinase ELISA, enzyme-linked immunosorbent assay EM, expression medium ExpiCHO-S, mammalian chinese hamster ovary cell line that grows in suspension
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FBS, fetal bovine serum FL, full-length FLICA, fluorescent-labeled inhibitor of caspases FRET, fluorescence resonance energy transfer FT, flow-through GFP, green fluorescent protein GM-CSF, granulocyte-macrophage colony-stimulating factor GPCR, G protein-coupled receptors GST, glutathione-S-transferase GuHCl, guanidine hydrochloride HA, hyaluronan HD2, helical domain 2 HFIP, hexafluoroisopropanol HRP, horse radish peroxidase HPLC, high performance liquid chromatography Hsp, heat shock protein IB, inclusion bodies ICE, interleukin-1 converting enzyme (also known as caspase-1) ICEBERG, CARD-only protein IDL, interdomain linker IFI200, interferon inducible-200 IL-1β, interleukin 1-beta IL-18, interleukin 18 IKKα/β, IκB kinase α/β INCA, CARD-only protein IRAK-1, interleukin-1 receptor-associated kinase 1 JNK, c-Jun N-terminal kinase LB, luria broth LPS, lipopolysaccharide S (found on the outer membrane of Gram – bacteria) LRR, leucine-rich repeat
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LUBAC, linear ubiquitin chain assembly complex (ubiquitin ligase) Lys, lysate (sample) MALS, multi-angle light scattering MBP, maltose-binding protein MCI, mild cognitive impairment MSU, monosodium urate MTOC, microtubule-organizing center
Mw, molecular weight MWCO, molecular weight cut-off MyD88, myeloid differentiation primary response 88 NALP1/3, NACHT, LRR and PYD domains-containing protein 1/3 (same as NLRP1/3) NAC, N-acetyl-L-cysteine (inhibitor of reactive oxygen species) NACHT, central nucleotide-binding and oligomerization domain of NLRP3 containing an ATPase and Walker A, B motifs NADPH, nicotinamide adenine dinucleotide phosphate NBD, nucleotide binding domain NEK7, NIMA (never in mitosis gene a)-related kinase 7 Nf-κB, nuclear factor kappa B (transcription factor) NLR, NOD-like receptor NLRC3/4, NOD-like receptor CARD domain 3/4 NLRP1/3, nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain (PYD)-containing 1/3 NMR, nuclear magnetic resonance NOD, nucleotide-binding oligomerization domain NP-40, nonyl phenoxypolyethoxylethanol-40 OPI, oxaloacetate-pyruvate-insulin PAMP, pathogen-associated molecular patterns PBMCs, peripheral blood mononuclear cell
PBS, phosphate-buffered saline (pH 7.4), [10 mM Na2HPO4 and 150 mM NaCl] Pellino2, E3 ubiquitin ligase
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PF, protofibrils PKA/C/D, protein kinases A/C/D PMA, phorbol 12-myristate 13-acetate POP1, pyrin only protein 1 PP2A, phosphatase 2A PRR, pattern recognition receptor PS1/2, presenilin 1/ 2 PTMs, post-translational modifications PTPN22, protein tyrosine phosphatase, non-receptor type 22 PVDF, polyvinylidene fluoride PYD, pyrin domain Pyk2, proline-rich tyrosine kinase 2 p22phox, a subunit of NADPH oxidase P2X7, ATP-gated ion channel RIPA, radioimmunoprecipitation assay (buffer) RLRs, retinoic acid-inducible gene I (RIG-I)-like receptors RNA, ribonucleic acid ROS, reactive oxygen species RPMI, Roswell Park Memorial Institute (cell culture medium) RVD, regulatory release volume SDA, succinimidyl-ester diazirine SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEC- size exclusion chromatography SEM, scanning electron microscopy SENP3, SUMO Specific Peptidase 3 SFM, serum-free complexation medium siRNA, small interfering ribonucleic acid SGT1, suppressor of the G2 allele of Skp1 (co-chaperone of hsp90) SOC, super optimal broth with catabolite repression medium SR, scavenger receptor
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SUMO, small-ubiquitin-related modifier Syk(I), spleen associated tyrosine kinase (inhibitor) TCA, trichloroacetic acid
TCEP, Tris (2-carboxyethyl) phosphine hydrochloride TEM, transmission electron microscopy TEV, tobacco etch virus THP-1, human monocytic cell line obtained from an acute monocytic leukemia patient ThT, thioflavin T (fluorescent dye) TLR, toll-like receptor TMB, tetramethylbenzidine (ELISA substrate) TNFα, tumor necrosis factor alpha UBC9, SUMO-conjugating enzyme USP 7/47/50, ubiquitin-specific-processing protease 7/47/50 WT, wildtype YFP, yellow fluorescent protein z-YVAD-AMC/AFC, benzyloxycarbonyltyrosyl-valyl-alanyl-aspartic acid 7-Amino- 4-methylcoumarin/ 7-amino-4-trifluoromethylcoumarin z-YVAD-cmk/fmk, benzyloxycarbonyltyrosyl-valyl-alanyl-aspartic acid chloro/fluoromethylketone (irreversible caspase-1 inhibitor)
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ABSTRACT
Makoni, Nyasha J., University of Missouri-St. Louis, December 2020.
Interactions of the NLRP3 Inflammasome Complex. Major Professor: Dr. Michael
R. Nichols.
The innate immune system is the first line of defense in response to danger signals or invasion by pathogens. One of the major pathways in the innate immune system involves a three-protein complex known as the NLRP3 inflammasome. This complex, mainly found in immune cells, is comprised of
NLRP3, ASC, and procaspase-1. The individual proteins are separate under normal conditions, and only assemble in response to external stimuli to form a caspase-1-activating complex. Ultimately, activated caspase-1 facilitates production of interleukin-1β (IL-1β), an inflammatory cytokine. The NLRP3 inflammasome has been implicated in a variety of inflammatory disorders including Alzheimer’s disease (AD). Amyloid beta (Aβ) is the protein that causes
AD and Aβ deposits in the brain activate microglia. Continuous activation of microglia results in a chronic inflammatory state hence neuroinflammation is a characteristic feature of AD pathology.
However, the actual events by which Aβ activates the inflammasome are still unknown. In addition, the actual structure of the inflammatory complex and how that relates to caspase-1 activation is yet to be determined. Hence, my research focused on determining the interactions between Aβ and the NLRP3 inflammasome, and how they lead to caspase-1 activation. To conduct these studies, I expressed full-length, mutants, and truncated versions of the
xvi inflammasome proteins using a mammalian ExpiCHO-S system. This was the first time anyone had ever attempted to use this cell line to express these proteins. Proteins were successfully isolated from the lysates and inclusion bodies. Fusion tags were added to improve solubility. After purification by affinity and size-exclusion chromatography, recombinant proteins were characterized by
SDS-PAGE and immunoblotting. Although monomers were observed, high degree of oligomerization revealed the propensity of the proteins to aggregate.
Biophysical techniques also confirmed the presence of protein aggregates.
Efforts to break the pre-formed aggregates involved applying stringent conditions such as thermal and chemical denaturation, detergents, and reducing agents.
The next aspect of the studies involved performing functional assays with the recombinant caspase-1 proteins in order to determine which exact domains are required for enzyme activity. Using a fluorescence assay, I observed enzymatic activity with procaspase-1 although it was transient. This discovery was surprising and exciting since the proenzyme had been previously dubbed inactive in the published literature. On the other hand, the reconstituted p20p10 and CARDp20 domains had no activity under similar conditions. Some publications, however, have observed enzyme activity with reconstituted p20p10 fragments.
I also investigated the nature of the interactions between Aβ and the
NLRP3 inflammasome using two approaches: cell stimulation assays, and solution interactions with recombinant proteins. In agreement with previous literature, I observed direct interactions between Aβ protofibrils and NLRP3
xvii protein. Thus, the studies I conducted provide some insightful findings regarding the nature of the interactions between Aβ and the inflammasome. In addition, they offer new perspectives relating to the actual structure of active caspase-1 and suggest the possibility that there could be multiple forms of the active enzyme.
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CHAPTER 1: INTRODUCTION
1.1. Innate immunity
Innate immune system is the first line of defense against pathogens
(bacteria, viruses, fungi) [24-28]. It has evolved to be a quite sophisticated system for sensing “danger” whilst remaining unresponsive to the molecules and particles regarded as posing no threat to the general well-being of cells [29]. This system senses pathogens and other microbes via pattern recognition receptors
(PRRs) [30, 31]. PRRs are mainly found in immune and inflammatory cells such as monocytes, microglia, macrophages, neutrophils, and dendritic cells [18, 24,
28]. The main classes of PRRs are Toll-like receptors (TLRs), nucleotide oligomerization and binding domain (NOD)-like receptors (NLR), C-type lectin receptors (CLRs), RIG-I (retinoic acid inducible gene-I)-like receptors (RLRs), and IFI200 family member absent in melanoma 2 (AIM2) [24-27, 29, 32-35].
PRRs can be extracellular/ transmembrane (TLRs) or intracellular (NLRs, RLRs,
AIM2) [36]. TLRs, located in plasma and endosomal membranes, detect pathogen associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) in the extracellular space [18, 24, 27, 36]. PAMPs are unique to microbials and are critical in microbial function [bacterial lipopolysaccharide S (LPS), flagellin, viral RNA etc] while DAMPs are basically endogenous or modified host molecules such as adenosine triphosphate (ATP), amyloid-beta (Aβ), uric acid, hyaluronan as well as heat shock proteins (hsp) -70 and -90 [30, 32, 37]. LPS, a well-known TLR4 ligand [38], is found on the outer wall of gram-negative bacteria. PRRs can sense a variety of stimuli such as
1 microbial cell wall components and nucleic acids, uric acid crystals, ATP, and Aβ
[29, 30]. After sensing stimuli, PRRs promote the assembly of intracellular, multi- protein complexes called “inflammasomes” that activate inflammatory caspases thus resulting in the production of cytokines and chemokines [25, 26, 39].
Inflammasomes are cytosolic protein complexes (about 700 kD in size) that mediate immune responses (via activation of caspases) in the event of microbial infection or cellular damage [25, 26, 30, 39-42]. The inflammasome complex was initially isolated from extracts of human THP-1 monocytic cells by
Tschopp and co-workers and was described as a caspase-activating complex
[39]. In other reports, inflammasomes have been described as “…molecular platforms activated upon cellular infection or stress that trigger the maturation of proinflammatory cytokines such as interleukin-1β to engage innate immune defenses” [29]. Many reviews have been written on inflammasomes [29, 33, 35,
43-46].
Inflammasomes are made up of a sensor (NLR family protein), an adaptor
(ASC) [apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)], and an effector (a caspase) [35]. To date, five PRRs
[nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain (PYD)-containing 1/3 (NLRP1/3), NOD-like receptor CARD domain 4
(NLRC4), Pyrin and AIM2] have been found to form inflammasomes. However, four key inflammasomes have been studied so far (NLRP1, NLRP3, NLRC4,
AIM2) [33, 35, 42, 47].
2
The human NLR family of intracellular receptors consists of 22-23 members [33, 36, 42]. These receptors not only sense microbials but are also triggered by non-microbial particles leading to the activation of caspases 1- and -
5. Subsequently, the proinflammatory cytokines interleukin- 1-beta (IL-1β) and interleukin 18 (IL-18) are released and may cause pyroptosis which is a proinflammatory cell death process [39, 48]. NLR family of proteins utilize ASC as the adaptor protein although other ASC-independent inflammasome complexes exist [25, 29, 43, 45].
The nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasome is part of a highly regulated innate immune system and is an emerging area of research attracting the most interest of any of the inflammasomes. Prolonged and excessive production of IL-
1β causes tissue damage and this is the characteristic of many inflammatory and autoimmune disorders as well as numerous cancers, cardiovascular, and neurodegenerative diseases such as Alzheimer’s disease (AD) [33, 49].
Activation of the NLRP3 inflammasome has been implicated in the occurrence of
AD [27, 49-53]. The interplay between TLR signaling, AD, and the NLRP3 inflammasome has been reviewed here [34].
3
1.2. Amyloid beta (Aβ) and Alzheimer’s disease (AD)
Alzheimer’s disease (AD) is a neurodegenerative disease which is the most common cause of dementia and accounts for 60-70% of the cases [34, 37,
49, 54-56]. AD can be familial or sporadic as in most cases. Although aging is the main risk factor for developing AD, genetic factors which increase overall production of amyloid beta (Aβ) above a critical threshold as well as environmental factors (diet, sedentary lifestyle) contribute towards developing the disease [34, 57, 58]. Genetic factors leading to early onset AD include mutations in the amyloid precursor protein (APP) [58, 59], presenilin genes (PS1/2) [58, 60,
61], and presence of the apolipoprotein E4 (APOE4) gene (late onset) [62, 63].
Although extensive research has been conducted to find drug therapies against
Aβ [49, 64-73], currently there is no effective cure for this debilitating disease. AD pathology usually starts 20-30 years prior to the manifestation of clinical symptoms. It is characterized by extracellular Aβ deposits (senile plaques), intracellular tau tangles, neuronal loss, synaptic damage, and ultimately cognitive decline leading to loss of memory as shown in Fig 1.1 [28, 56, 61, 74-76].
4
Fig 1.1 The Alzheimer’s disease (AD) brain is characterized by extracellular senile (amyloid beta) plaques, intracellular tau tangles, enlarged ventricles, and cortical shrinkage compared to the healthy brain. Severe loss of brain tissue ultimately leads to dementia in AD patients.
Image obtained from: http://www.totalhealthinstitute.com/tips-on-preventing-alzheimers- disease-naturally/
5
Amyloid beta (Aβ), a 38-43 amino acid peptide, is responsible for causing
AD [77]. This protein is processed from its parent protein APP [58, 60, 78] which is an integral membrane protein. As shown in Fig 1.2 [1], cleavage by two aspartyl proteases (β and γ secretases) generates Aβ-42 and Aβ-40 fragments.
Aβ-42 is neurotoxic and more relevant to AD pathology.
Fig 1.2 Processing of amyloid precursor protein (APP), an integral membrane protein, occurs
via amyloidogenic and non-amyloidogenic pathways. β- and –γ secretases facilitate the
generation of amyloid beta fragment via the amyloidogenic pathway [1].
6
Due to the two the extra hydrophobic residues (Ile, Ala) at the C-terminus,
Aβ-42 has a greater propensity to aggregate into β-sheet structures [64, 79, 80] and is predominant in senile plaques [81]. The Aβ aggregation pathway is shown in Fig 1.3 [20].
Fig 1.3 Aβ aggregation pathway. Unstructured Aβ monomers undergo aggregation to form
soluble oligomers (ranging from dimers to high order protofibrils) and ultimately, insoluble
fibrillar species. An alternative route involving the annulus, dodecamer, and amylospheroid is
distinct from the protofibrillar-fibrillar pathway [20].
Aβ42 aggregates were found to be stable and resistant to denaturants
[82]. Many AD-related genetic mutations result in increased amounts of Aβ-42 vs
Aβ-40 [58]. A critical AD biomarker is reduced cerebrospinal fluid (CSF) levels of
Aβ-42 [57, 83-85] while the Aβ-40 levels remain the same [84, 86] resulting in a higher Aβ-40/ Aβ-42 ratio. Under normal conditions, Aβ clearance is facilitated by microglia [49, 87-89] and via translocation to the blood stream and the cerebrospinal fluid [56]. Various microglial receptors including scavenger receptors (SR), TLRs, CD36, and NLRs are involved in the uptake and clearance of Aβ as reviewed by Doens et al [90]. In the case of AD, dysregulation and
7 subsequent elevation of Aβ levels results in the accumulation of insoluble protein aggregates in the brain [51, 52, 91]. Senile Aβ plaques in the brain activate microglia (immune cells in the brain) resulting in the production of inflammatory factors [51, 52, 91]. Although the natural levels of Aβ40 are much higher than those of Aβ42 [54], the former does not form aggregates compared to the latter
[57]. According to the amyloid hypothesis theory [58, 78], formation of senile plaques in the brain is the main cause of AD pathology [92]. Dysregulation of Aβ metabolism results in the deposition of neurotoxic Aβ plaques that eventually trigger a range of processes leading to neurodegeneration [58, 74, 78]. However, more recent studies have disproved this model and have revealed that plaque formation occurs way after the manifestation of the disease [54].
Different forms of Aβ exist [54], ranging from soluble species (monomers, oligomers, protofibrils, Aβ-derived diffusible ligand ADDLs) [79, 93-98] to insoluble fibrils. Studies have shown that fibril formation and amyloid deposition is not a prerequisite for microglia activation [19, 23, 56, 99, 100]. Contrary to reports that implicate fibrils as the toxic species [78, 101, 102], newer studies support the “oligomer hypothesis” [64, 103] which implicates soluble Aβ species such as oligomers [97, 104-109], protofibrils [19, 23, 100], and ADDLs [98] as the most deleterious. Thus, it has been proposed that a “halo” of soluble Aβ species surrounding the plaques causes the toxicity in the brain.
Many in vitro cell stimulation studies with Aβ resulted in the secretion of inflammatory cytokines such as tumor necrosis factor α (TNFα) and IL-1β [56,
110-112]. Our lab has been extremely thorough in characterizing the various
8 forms of Aβ [100, 113]. Using size-exclusion chromatography (SEC), we can isolate pure monomers and protofibrils (PF). Pre-fibrillar forms of Aβ were found to induce TNFα production in THP1 cells [114]. Later experiments would identify
PF as the most toxic species compared to both monomers and fibrils [19, 100].
Cell-uptake experiments conducted with primary murine microglia cells indicated that the cells preferentially take up PF compared to monomers [22]. Similarly, a previous study had reported that primary microglia cells internalized soluble and fibrillar Aβ differently. However, the cells only partially degraded both forms of Aβ and released most of the protein still intact thus promoting accumulation of aggregated protein [87]. All these findings clearly show the varying toxicity of Aβ-
42 species and their involvement in the inflammatory response processes facilitated by immune cells.
1.3. Neuroinflammation and AD
Neuroinflammation is characteristic of AD pathology [28, 51-53]. Data has shown that both soluble and insoluble Aβ species are neurotoxic and are involved in activating the NLRP3 inflammasome [19, 23, 37, 53, 56, 100, 115].
Heneka and colleagues showed cross-seeding between microglia-derived ASC specks and Aβ monomers thus promoting aggregation of the latter [2]. In addition, they showed that Aβ-ASC composites augment NLRP3 inflammasome activity and subsequent cell death [116]. Aβ deposits/ senile plaques in the brains of AD patients cause continuous activation of microglia [49]. Ultimately, this results in the prolonged release of inflammatory cytokines, IL-1β and TNFα,
9 leading to a chronic inflammatory state, neuronal loss, and ultimately dementia
[57]. Overproduction of IL-1β, due to dysregulation of the innate immune system, is the main cause of inflammation [49, 89].
Various reports have implicated the NLRP3 inflammasome in AD-related neuroinflammation [37, 49, 51, 53, 56, 115, 117]. Elevated amounts of IL-1β were found in AD brains [118]. A later study also reported the discovery of elevated IL-
1β and IL-6 in the cerebrospinal fluid of Alzheimer’s patients [117]. Stimulation of primary mouse and immortalized microglia with Aβ fibrils (coupled with LPS priming) also resulted in lysosomal damage and the release of mature IL-1β.
Control experiments with NLRP3/ ASC-deficient macrophages failed to activate
IL-1β maturation [37].
In other studies, in vivo multiphoton imaging technique was used to study
APPswe/PS1d9xYFP (B6C3-YFP) transgenic mice bearing mutations associated with early-onset AD. These AD-model experiments were conducted to show the effect of plaque deposition on microglial activation. They discovered that microglia surrounded the areas of plaque formation and morphological changes were detected in the neurites surrounding the plaques [119]. These findings were in agreement with the amyloid hypothesis theory [64]. Moore and colleagues showed the involvement of the CD36 receptor in facilitating uptake of soluble Aβ species by macrophages resulting in NLRP3 activation [115]. Further studies by
Heneka and colleagues showed increased caspase-1 activation in brains of mild cognitive impairment (MCI) and AD patients [53]. In vitro studies conducted by the Nichols group [23] and others [56, 116] confirmed the secretion of IL-1β by
10
Aβ-stimulated microglia. Fig 1.4 summarizes the studies by the Nichols group leading to the NLRP3 inflammasome studies.
A. B.
C.
11
Fig 1.4 summarizes findings from 3 previous publications from the Nichols lab. Aβ PF were
identified as the most toxic species [19]. In addition, primary murine microglia were found to
internalize PF more than monomers [22]. Panel A. Size-exclusion chromatography (SEC)
elution profiles of Aβ-42 purification in aCSF buffer on a Superdex 75 column. Protofibrils
and monomers elute in two distinct peaks. Panel B. Data from ELISA experiments shows
that TNFα and IL-1β mRNA production (left and right panel respectively) is reduced in the
MyD88−/− microglia in response to Aβ PF stimulation Panel C. Schematic shows the
involvement of TLRs in Aβ PF uptake. Experiments with MyD88 knockout mice confirmed
the involvement of this cytosolic adaptor protein in the TLR pathway. Thus, the findings
confirmed that PF prime and activate the NLRP3 inflammasome resulting in IL-1β secretion
[23].
The involvement of the NLRP3 inflammasome in this inflammatory response ignited our interest hence we decided to get a better understanding of this inflammatory complex. Recent reviews explain in depth the roles of Aβ and microglia in neuroinflammatory processes and subsequent neurodegeneration
[28, 120]. It is important to note that apart from Aβ, tau protein and other amyloidogenic proteins also activate the NLRP3 inflammasome via similar mechanisms [76, 97].
12
1.4. Structure and assembly of the NLRP3 inflammasome
The nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain (PYD)-containing 3 (NLRP3) inflammasome also known as cryopyrin/ NALP3 is a cytoplasmic receptor which is normally expressed in immune cells (predominantly in the microglia) [28]. NLRP3, a member of the NLR family, is the most characterized inflammasome to date [44, 45, 121, 122]. This protein complex is made up of three proteins namely NLRP3 (sensor protein),
ASC (an adaptor protein also known as PYCARD), and the cysteine protease caspase-1 as shown in Fig 1.5. NLRP3 binds with ASC via homotypic PYD-PYD interactions. Likewise, ASC utilizes its CARD domain to recruit caspase-1 to the inflammasome.
13
Fig 1.5 The NLRP3 inflammasome complex is made up of NLRP3 (senses stimuli via the
leucine-rich region LRR), ASC (adaptor protein), and caspase-1 (effector protein) which is
a cysteine protease. The domains making up each individual protein are highlighted. The
dashed lines show the homotypic interactions, facilitated by hydrophobic and charged
residues, that keep the three proteins together. In the presence of stimuli, the NLRP3
inflammasome assembles resulting in the activation of caspase-1 which subsequently
cleaves proIL-1β to mature IL-1β. *The actual stoichiometric components of each
individual protein in the complex are still unknown.
Various studies have identified NLRP3 as a vital component of the inflammasome complex. In addition, NLRP3 oligomerization is a key process in the inflammasome assembly process [39, 121, 123]. The NACHT domain of
NLRP3 (protein) is common to all NLR members and it oligomerizes in the presence of ATP thus promoting activation of the protein complex [18, 31, 36, 40,
47]. Ting and co-workers showed that cryopyrin (NLRP3) exhibited ATPase activity and co-immunoprecipitation studies verified that it interacted with ASC only in the presence of ATP. Further studies revealed that the Walker A motif
(located in the NACHT domain) is responsible for ATP binding. Mutation of the critical residues in this region abolished interactions with ATP [124]. On the other hand, the leucine-rich region (LRR) senses PAMPs and DAMPs and is involved in autoregulation of the inflammasome [125, 126]. However, Jerala and colleagues provided evidence disputing the previous claims regarding the functions of the LRR. Truncated NLRP3 proteins lacking the LRR were
14 responsive to stimuli in a manner similar to that of the full-length NLRP3. Thus, the group proposed that the PYD may have some autoinhibitory effect and keeps the inflammasome inactive in the absence of stimuli [127].
Studies identified ASC as an inflammasome adaptor protein [128, 129].
Gumucio and colleagues reported direct interactions between pyrin and ASC
[125]. Dowds et al showed that cryopyrin (NLRP3) co-immunoprecipitated with
ASC. In addition, siRNAs targeting endogenous ASC prevented IL-1β secretion thus indicating the importance of ASC in inflammasome assembly [130]. Thus, the three proteins that constitute the NLRP3 inflammasome all play vital roles in the assembly of the protein complex. Upon sensing danger signals, NLRP3 inflammasome proteins assemble to form a caspase-1-activating facilitating platform resulting in IL-1β maturation and cytokine release coupled with pyroptosis.
1.5. Structural studies of the NLRP3 inflammasome proteins
Many studies have been conducted on the pyrin domains of both NLRP3 and ASC and are summarized in a report compiled by Stehlik and colleagues
[131]. ASC2, a pyrin-only protein (POP) was used as a model to study the pyrin domain. Nuclear magnetic resonance (NMR) studies on recombinant ASC2 conducted by Hill and colleagues showed six parallel helices, characteristic of the death-domain (DD) proteins. Sequence alignment of ASC2 showed some degree of similarity with the pyrin domains of ASC and NALP1 (NLRP1), particularly the
15 presence of conserved hydrophobic residues [132]. Do and Park performed crystallization studies using recombinantly-expressed POP1 at 3.6 Å [133].
Although many studies have been done to determine the significance of the residues in the PYD domains of both NLRP3 and ASC [4, 5, 134, 135], very few studies have been done on the CARD domain. Using multiple point mutations to study the pyrin domain of ASC, various groups discovered the role of charged & hydrophobic residues in facilitating oligomerization [5, 134, 136].
Sagara and co-workers discovered that these mutations abolished the formation of ASC filaments. In addition, C-terminal deletion experiments showed that a fragment containing a minimum of 90 amino acids was necessary for filament formation [134]. Two binding surfaces on ASC PYD were found to be involved in its self-association and interactions with NLRP3 PYD [5, 135, 136]. Interestingly,
ASC PYD self-association binding affinities were found to be much stronger than those with NLRP3 PYD [135].
On the other hand, there is quite a few biophysical studies that have been conducted with some domains of the inflammasome proteins. Initial NMR studies on ASC PYD were done by Otting and co-workers [137]. The de Alba group also conducted some ASC structural studies. In their earlier NMR studies with full- length human ASC expressed in E.coli, they were able to determine the residues involved in the CARD domain interactions [138]. In later studies they utilized atomic force microscopy (AFM), NMR relaxation techniques, and circular dichroism (CD) to determine ASC structure and solubility at neutral and acidic pH
(4.0). Their data revealed that ASC is made up of two independent 6-helix
16 bundles separated by a flexible linker as shown in Fig 1.6. In addition, the structure was not perturbed by changes in pH. The size of ASC oligomers remained unchanged at both pH conditions [17]. The protein was more soluble under acidic conditions as physiological pH promoted oligomerization and filament formation [17]. Studies by another group also revealed the CARD domain of ASC to be six-helix bundle [139].
Figure 1.6 Structure of ASC. A ribbon structure of full-length ASC based on the solution
structure solved by de Alba and colleagues. It shows the pyrin and CARD domains as
6-helix bundles connected by a flexible linker (23 residues long) [17].
17
Wu and colleagues utilized cryogenic electron microscopy (cryo-EM) to study ASC PYD filaments at near atomic resolution for the first time and their data confirmed the importance of opposite surface charges in the formation of filaments. They also showed that both PYD and CARD domains are involved in filament formation [140, 141]. Cryo-EM and solid-state NMR structural studies conducted by Hiller and colleagues using mouse ASC suggested the PYD facilitated filament formation while the flexible CARD domain remained exposed outside the filament [4]. Physiological pH conditions were found to promote formation of ASC filaments just like the previous findings by de Alba and colleagues [17].
By modeling the ASC-CARD dimer, de Alba and co-workers used a 7- member ring model for ASC oligomerization [142]. Further analysis of the individual PYD and CARD domains by TEM indicated that the two domains formed structurally distinct filaments. Moreover, full-length ASC filaments were noticeably different from those formed by ASC PYD only. Contrary to the previous cryo-EM reports that only the PYD is involved in the formation of ASC filaments [4], TEM and NMR studies by the de Alba group showed that CARD-
CARD interactions are also essential in filament formation [143]. Fluorescence studies revealed that both PYD and CARD domains were essential in ASC foci formation during inflammasome assembly [144]. Single molecule fluorescence resonance energy transfer (FRET) experiments would also prove that the two domains were in very close proximity during oligomerization [145]. These findings
18 reveal that ASC CARD is not only involved in the recruitment of caspase-1 but also participates in ASC filament formation with PYD. It is important to also note that the filament formation experiments were mostly conducted under non- physiological conditions and may not reflect the actual cellular processes.
Nonetheless, all these findings emphasize the importance oligomerization during inflammasome assembly and the directionality of domain-domain interactions.
In other studies, Bae and Park reported the crystal structure of NLRP3
PYD at 1.7Å resolution. Just like the ASC PYD, the surface of the NLRP3 PYD is made up of charged and hydrophobic residues that are crucial in promoting self- dimerization [146]. Thus, these various structural studies provided a better insight on the key residues and interactions involved in keeping the inflammasome complex intact.
1.6. ASC speck formation
ASC-dependent inflammasomes undergo nucleation-dependent polymerization. Many groups have studied ASC oligomerization during inflammasome activation and assembly. ASC has been described as a
“molecular glue” which brings the inflammasome proteins together. When overexpressed separately, the PYD and CARD domains of ASC formed filaments
[128]. Structural studies have shown that after activation, the inflammasome proteins oligomerize and recruit ASC which forms fiber-like structures that ultimately assemble into large particles known as “specks”. Numerous reports on
ASC speck formation have been published [3, 141, 147-151].
19
Initial studies by Alnemri and colleagues reported that ASC dimers were involved in speck formation ultimately resulting in the formation of a supramolecular complex termed “pyroptosome.” In addition, they revealed that only the pyrin domains of ASC were involved in the assembly while the CARD domains participated in the recruitment of caspase-1 [147]. The notion that ASC dimers are the basis of filament formation was further investigated by Egelman and colleagues [141]. Later studies would prove the involvement of the CARD domain during the formation of the inflammasome complex [143, 144]. The kinetics involved in speck formation were studied by Gumucio and co-workers.
Confocal laser scanning microscopy time-lapse studies conducted using ASC-
YFP (yellow fluorescent protein)-transfected HeLa cells revealed ASC aggregation to be an extremely fast process i.e at 20 µM, the collision rate of two
ASC dimers is 6x1011 sec-1 per cell. Diffusion was the main process involved in speck formation. The specks formed in both cytoplasmic and nuclear compartments. Moreover, the rate of depletion of ASC from the cytoplasm was found to be constant. The authors proposed a two-step process for speck formation involving rapid diffusion of ASC dimers followed by their (dimers) binding to the growing speck [148]. Follow-up experiments by Sester and colleagues later would utilize a flow cytometry-based assay to assess inflammasome assembly [152].
Using recombinantly-expressed proteins, Wu and colleagues showed that the pyrin domain of NLRP3 is required for polymerization with ASC-PYD. While
NLRP3-PYD alone did not induce ASC-PYD polymerization, addition of the
20 nucleotide binding domain NBD (NLRP3-PYD-NBD) resulted in nucleation of
ASC filaments. Fluorescence polarization assays were done to assess ASC-PYD filament formation. Cryo-EM studies of ASC-PYD filament at near atomic resolution also revealed the role of opposing charges in filament assembly [141].
The same group also performed studies with the absent-in-melanoma 2 (AIM2) inflammasome. The studies revealed that ASC-dependent inflammasomes (both
NLRP3 and AIM2) assemble via a unified mechanism involving nucleation- induced polymerization [140, 141].
Recombinant ASC-PYD was shown to form fibers that facilitated prion-like polymerization by converting inactive ASC into an active prion form. However, mutations of charged and hydrophobic residues (R41A, D48A, and P40A) resulted in the loss of prion-forming capabilities [149]. In vitro and in vivo studies by Latz and co-workers also illustrated the prion-like nature of ASC [150].
Pelegrin and colleagues suggested that the extracellular NLRP3 inflammasome exists as oligomeric particles. Their immunoblot analysis indicated the release of inflammasome components when mouse bone-marrow derived macrophages (BMDMs) were stimulated with LPS/ Nigericin. Co- immunoprecipitation assays showed interactions between extracellular ASC and
NLRP3. Time-lapse confocal microscopy with mCherry-tagged ASC and cross- linking experiments with succinimidyl-ester diazirine reagent (SDA) showed formation of ASC specks and oligomers respectively [153]. Another group showed that mutating the key charged residues in the binding surfaces prevented speck formation [154]. Broz and colleagues also studied ASC speck assembly
21 and discovered that the pyrin domain facilitated assembly into helical filaments while the CARD was involved in cross-linking these filaments. Mutating key residues in the CARD domain not only blocked CARD/CARD homotypic interactions, but also resulted in larger and less dense specks. These specks did not promote caspase-1 activation, release of IL-1β or pyroptosis [3].
In conclusion, the formation of ASC specks is a tightly controlled, very rapid, all-or-none process. Initial aggregation of ASC is slow, but nucleation speeds up the process thus promoting speck formation. Detailed reviews have described ASC speck formation as well as the intra- and -extracellular functions of ASC specks [155, 156]. A recent review by de Alba also explored structure, interactions, and dynamics of ASC to a greater depth [157].
1.7. Activation of the inflammasome
The NLRP3 inflammasome is very distinct from other inflammasomes because it is activated by a wide variety of ligands such as pathogens, environmental irritants or endogenous danger particles released during tissue injury or cell stress [25, 26, 29, 45, 122, 158]. Activation of the NLRP3 inflammasome occurs in two distinct steps as shown in Fig 1.7 [18].
22
Figure 1.7 Activation of the NLRP3 inflammasome. The figure shows the two-signal mode of activating the NLRP3 inflammasome as proposed by Liu and colleagues [18]. The priming step which involves the TLR signaling pathways is triggered by DAMPs and
PAMPs. The transcription factor Nf-κB facilitates synthesis of pro-IL-1β and optimal NLRP3.
The activation step is initiated by NLRP3 activators such as ATP and involves three pathways; potassium efflux, generation of reactive oxygen species (ROS), or phalysosomal rupture. Activation promotes assembly of the inflammasome complex which activates caspase-1 and ultimately, IL-1β secretion.
23
The initial step termed “priming” or Signal 1 is induced by toll-like receptor
(TLR) ligands such as bacterial LPS. Basal levels of NLRP3 proteins are very low and are only upregulated in the event of stimuli. The priming step involves myeloid differentiation primary response 88 (MyD88) signaling [23] and LPS- induced protein synthesis of NLRP3 transcripts via the transcription factor Nf-κB.
This results in the assembly of the caspase-1 processing machinery i.e. expression of pro-IL-1β and optimal NLRP3 [159]. It has been suggested that
NLRP3 is produced in an autoinhibited form that is released in the presence of
NLRP3 activators [28]. The activation step or Signal 2 involves NLRP3 activators such as ATP and thus leads to activation of caspase-1 and ultimately, production of mature IL-1β.
Common NLRP3 activators include Aβ, uric acid crystals, ATP, silica, asbestos, aluminum salts, and hyaluronan [37, 53, 123, 160-164]. Silica and asbestos stimulate production of reactive oxygen species (ROS) while crystalline and particulate ligands (alum, silica) result in membrane destabilization [160,
161]. Studies showed that uptake of soluble Aβ species such as oligomers and protofibrils (PF) [23, 56] and insoluble fibrillar Aβ [2, 37, 53, 116, 158] by microglia results in NLR3 inflammasome activation.
Though structurally diverse, inflammasome activators somehow share a common mechanism of activation. Despite numerous studies that have been conducted on the NLRP3 inflammasome, it is still quite unclear whether NLRP3 directly binds to any of its activators. Various mechanisms have been proposed regarding to NLRP3 activation. One such mechanism proposes a possible direct
24 interaction between the activating molecules and the LRR domain of NLRP3 after entering the cell. Another possible scenario is the modification of membrane proteins by the activators thus triggering a particular signaling cascade.
Nonetheless, various reports have suggested that activation of the NLRP3 inflammasome occurs via three distinct mechanisms: potassium efflux; the generation of mitochondria-derived reactive oxygen species (ROS); and phagolysosomal destabilization [29, 30, 35, 42, 45-47, 165].
Potassium (K+) efflux acts upstream of NLRP3 inflammasome activation.
Almost all NLRP3 activators, especially pore-forming bacterial toxins and ATP, induce potassium efflux via P2X7, an ATP-gated cation channel ([42, 47, 166].
Dubyak and colleagues showed that activation of caspase-1 by the P2X7 receptor (P2X7R) was dependent on TLR signaling and Nf-κB-facilitated protein synthesis. Their studies showed that LPS/ ATP stimulation facilitated caspase-1 activation and subsequent secretion of mature IL-1β. Although they identified
LPS priming as an important prerequisite step in the activation of the inflammasome by P2X7R, this step alone was not sufficient in activating the receptor hence further stimulation by ATP was required. Nigericin (an ionophore and P2X7R mimic) was also found to induce K+ efflux after LPS priming [163].
Interestingly, K+ efflux regulates Ca2+ signaling to some extend (via promoting Ca2+ influx) [167-169]. Extracellular Ca2+ stimulates G protein-coupled calcium sensing receptors (GPCRs) such as GPCR6A [168] and CASR [167].
Subsequently, the inflammasome is activated upon sensing increased intracellular Ca2+ levels [167, 168] coupled with a decrease in cellular cyclic
25 adenosine monophosphate (cAMP) levels. Interestingly, binding of cAMP to the
NBD region of NLRP3 prevents inflammasome activation [167].
Many studies have proposed that crystalline/ particulate particles such as monosodium urate (MSU) crystals, silica, and asbestos are taken up by cells via endocytosis (phagocytosis) subsequently inducing ROS production and inflammasome activation [160, 161, 170]. Studies by Tschopp and colleagues
[160, 170] showed that MSU, silica and asbestos stimulated the secretion of mature IL-1β by THP-1 cells and human monocyte-derived macrophages.
Treating the cells with cytochalasin D and latrunculin A (which inhibit actin polymerization) prevented mature IL-1β secretion upon stimulation with asbestos and MSU. However, stimulation with nigericin was not affected. Knockdown of p22phox, the common NADPH oxidase subunit, and the use of ROS inhibitors such as N-acetyl-L-cysteine (NAC) and (2R,4R)-4aminopyrrolidine-2,4- dicarboxylate (APDC) severely diminished IL-1β production upon cell stimulation
[160].
Studies by Latz and colleagues demonstrated that pretreatment of human peripheral blood mononuclear cell (PBMCs) with cytochalasin D before stimulation with silica and MSU impaired IL-1β production. As shown previously by Tschopp et al, stimulation by non-crystalline activators (ATP and R848) was not affected by cytochalasin D [161].
Hyaluronan (HA), a glycosaminoglycan released from the extracellular matrix after tissue injury, acts as an endogenous signal for inflammasome activation. HA stimulation of peritoneal macrophages showed dose-dependent
26 release of mature IL-1β which was diminished upon digestion of HA with hyaluronidase or chondroitinase [164].
On the other hand, some studies have proposed that the fluctuations in the levels of other ions (Na+ influx, Cl- efflux) affect the cell membrane potential and overall cell volume resulting in inflammasome activation [171]. Cell swelling and regulatory volume decrease (RVD) processes maintain the overall cellular homeostasis and any changes are sensed by the inflammasome [172]. Pelegrin and co-workers emphasized that although K+ efflux played a vital part in activating the inflammasome, that alone was not sufficient. They proposed the interplay between cell volume and K+/ Cl- efflux and a possible common mechanism utilized by all inflammasome activators [171]. Prior studies showed that high amounts of extracellular chloride were found to have a negative effect on the secretion of IL-1β [173]. Other groups later mentioned Cl- efflux via chloride intracellular channel (CLIC) proteins as a crucial pathway in the activation of the inflammasome [174-176]. Moreover, they discovered that Cl- efflux promotes the interaction between NLRP3 and NIMA-related kinase-7
(NEK7) which promotes NLRP3 oligomerization and subsequent formation of
ASC specks [175]. Thus, the authors proposed Cl- efflux as an additional priming step during inflammasome activation in addition to K+ efflux [176].
In sum, all these studies show the versatility of the NLRP3 inflammasome and its particulate nature. However, there is no single unifying event to describe inflammasome activation. A plethora of diverse ligands activate this protein complex and perhaps these particles perhaps trigger a similar downstream
27 pathway that is sensed by the NLRP3 inflammasome. However, it is still quite unknown whether there is any direct binding between these activators and
NLRP3.
1.8. Regulation of the NLRP3 inflammasome
Various mechanistic pathways are involved in the regulation of the NLRP3 inflammasome activity as reviewed here [35, 47, 122]. These include post- translational modifications (PTMs) (with phosphorylation and ubiquitination being the common ones), chemical and small molecule inhibitors as well as NLRP3 interacting partners. As reviewed by Lamkanfi and Dixit, bacterial factors and viral particles also modulate inflammasome activity and function(s) e.g. inhibition of caspase-1 activity by orthopoxvirus-encoded serpins [26]. Many kinases have been implicated in the activation of the NLRP3 inflammasome and they include protein kinase C (PKC) [177], c-Jun N-terminal kinase (JNK), spleen tyrosine kinase (Syk) [178-182], interleukin-1 receptor-associated kinase1 (IRAK-1) [183],
Bruton’s tyrosine kinase (BTK) [184], and proline-rich tyrosine kinase (Pyk2)
[185].
1.8.1. Protein kinases A, C, D (PKA, PKC, PKD)
Post-translational modifications (PTMs) play a vital role in inflammasome regulation with phosphorylation being one common feature. PKA phosphorylates
Ser 291 (human Ser 295) in the NACHT domain of NLRP3 thus favoring ubiquitination and subsequent degradation of the protein ([186-188].
Phosphorylation of the same residue by PKD however promotes inflammasome
28 activation [189]. Phosphorylation of Ser 533 of NLRC4 by PKC resulted in activation of the inflammasome whilst immuno-depletion of the kinase from cell lysates and the use of rottlerin, a well-known PKC inhibitor, significantly inhibited phosphorylation of the residue and ultimately caspase-1 activation [177].
1.8.2. Syk kinase and JNK signaling
Phosphorylation of NLRP3 (Ser 194) by JNK during priming positively regulates inflammasome activation [190]. Syk, a non-receptor tyrosine kinase normally expressed in immune cells, also plays vital roles in the processes that govern inflammasome activation [191]. A Syk-Card9-controlled signaling pathway
(Card9 being the downstream protein) was implicated in the proinflammatory response by murine bone-marrow-derived macrophages (BMDMs) stimulated with fungi (C. albicans) [178]. Mitsuyama and co-workers provided evidence on the role of the Syk-JNK signaling pathway (independent of Card9) in the phosphorylation of mASC Tyr 144 (Tyr 146 in humans) located in the CARD domain. They revealed that phosphorylation of ASC promotes its oligomerization and subsequently, activation of caspase-1. Although phosphorylation of ASC is vital, that alone is insufficient to induce speck formation in the absence of stimuli
[180]. Mutating the critical residues, deletion of both Syk and Card9 genes, and pre-treatment of cells with Syk inhibitor (R406) all prevented caspase-1 activation and IL-1β secretion upon stimulation [178, 180]. Interestingly though, Syk inhibition and deletion did not affect caspase-1 activation and IL-1β maturation when cells were stimulated by LPS/ ATP/ Nigericin thus suggesting that
29 inflammasome activation by these ligands utilizes a Syk-independent pathway
[178].
Lin and colleagues showed that the kinase domain of Syk directly binds to both ASC and NLRP3 [182]. In contrast to previous studies suggesting that stimulation by ATP/nigericin/alum/MSU utilizes a Syk-independent pathway
[178], employing Syk inhibitors (SykI and BAY61-3606) resulted in decreased amounts of secreted IL-1β by stimulated macrophages [182]. These discrepancies could be attributed to the different inhibitors used or the origin of the cells used in the experiments i.e BMDMs [178] vs murine primary liver fetal macrophages [182]. Nunez and colleagues showed that 3,4-methylenedioxy-β- nitrostyrene (MNS), a well-known kinase inhibitor, interacts directly with the NOD and LRR domains of NLRP3 thus blocking its ATPase activity [181]. Meanwhile, studies by Latz and co-workers revealed that phosphorylation of Ser5 (located in the pyrin domain) negatively regulates inflammasome assembly [192].
Dephosphorylation by phosphatase 2A (PP2A) [192] and protein tyrosine phosphatase non-receptor 22 (PTPN22) [193] reversed the process.
All these findings indicate the importance of phosphorylation in the regulation of the inflammasome. In addition, Syk kinase is activated by various stimuli and depending on the stimuli, takes on various mechanistic pathways.
Another important aspect highlighted is how the cell type used when conducting experiments affects the response to various stimuli and how this can give rise to conflicting findings.
30
1.8.3. Bruton’s tyrosine kinase (BTK)
BTK is a member of the Tec family of tyrosine kinases (TK) that are structurally related to the Syk kinase. The kinase directly interacts with both
NLRP3 and ASC ultimately facilitating oligomerization of ASC. Studies by Morita and co-workers revealed that BTK inhibitors abolished inflammasome activation in stimulated cells resulting in reduced ASC oligomerization and smaller specks.
Additional in vivo experiments proved that ibrutinib (BTK inhibitor) had some neuroprotective effects in mice models of ischemic brain injury [184]. Other studies emphasized the role of BTK and tubulin polymerization during inflammasome activation [194-196]. Shuttling of NLRP3 to the microtubule- organizing center (MTOC) is an essential step in the inflammasome assembly and activation process [195].
1.8.4. Interleukin-1 receptor-associated kinase1 (IRAK-1)
IRAK-1 is a TLR signaling molecule that activates the NLRP3 inflammasome during simultaneous stimulation of TLR (by LPS) and NLRP3 (by
ATP). Upon stimulation, IRAK-1 interacts with ASC but the authors were not able to determine its exact involvement in the activation process [183].
1.8.5. IκB kinase alpha (IKKα)
IKKα facilitates NF-κB signaling and mediates the expression of genes involved in immune responses [197]. The kinase is found in both the cytoplasm and the nucleus and its translocation to the nucleus occurs upon stimulation (by activators such as TNFα) resulting in NF-κB-mediated gene transcription [198].
Martin and colleagues showed that under normal conditions, interactions
31 between IKKα and ASC prevent inflammasome activation. However, in the presence of stimuli, Signal 2 prompts the recruitment of a phosphatase which has an inhibitory effect on the kinase hence allowing free ASC to participate in inflammasome assembly [199]. Loss of IKK kinase activity results in elevated inflammasome activity [200-202].
1.8.6. Ubiquitination
Ubiquitination by ubiquitin ligases has emerged as one of the many significant mechanisms involved in immune regulation [35, 203, 204]. Although ubiquitination generally inhibits activation of the NLRP3 inflammasome, ubiquitination by the ligases Pellino2 (targets NLRP3) or linear ubiquitin chain assembly complex (LUBAC, targets ASC) activates the inflammasome [35].
NLRP3 is activated via deubiquitination during both TLR-priming and ATP- activation. Deubiquitination by deubiquitinating enzymes (DUBs) activates the inflammasome [205]. However, A20 (a DUB located in the cytoplasm) negatively regulates the inflammasome by inhibiting NF- κB [206, 207]. Yuan and colleagues identified BRCC3, a DUB which positively regulates the NLRP3 inflammasome by deubiquitinating the LRR region of NRP3 prior to assembly.
Inhibition of BRCC3 by an isopeptidase suppresses inflammasome activation
[208]. Just like BRCC3, ubiquitin-specific peptidase 50 (USP50) positively regulates NLRP3 activation by targeting Lys 63 of ASC thus promoting ASC oligomerization and speck formation [209]. Other DUBs, USP7 and USP47, also modulate NF-κB activity and their blockage by inhibitors suppresses inflammasome activation [210]. Thus, DUB inhibitors are potential therapeutic
32 agents for inflammatory disorders. A review by Moretti and Blander 2020 explores in depth the various deubiquitination mechanisms involved in inflammasome regulation [35].
1.8.7. Sumoylation
Protein modification via sumoylation is a vital regulatory process and at present three small ubiquitin-like modifier (SUMO) proteins have been identified in mammals [211]. Sumoylation of NLRP3 at multiple lysine residues by SUMO
E-3 ligase (localized in the mitochondria) negatively regulates the NLRP3 inflammasome. Loss of sumoylation occurs upon stimulation with NLR-ligands
(Signal 2) thus SUMO proteases activate the inflammasome [212]. Interactions between NLRP3 and UBC9, a SUMO-conjugating enzyme, allow SUMO1 to modify NLRP3 at Lys 204. This promotes inflammasome activation, oligomerization of ASC, and ultimately maturation of IL-1β. Meanwhile, deSUMOylases such as SUMO-specific protease 3 (SENP3) reverse these processes and seven mammalian SENPs have been identified to date [213].
1.8.8. NIMA-related kinase 7 (NEK7)
NEK7 belongs to the family of mammalian NIMA (never in mitosis gene a)- related kinases and is involved in the progression of the cell cycle by facilitating mitotic spindle formation [194, 214]. It is a multifunctional serine-threonine
(Ser/Thr) kinase which acts downstream of K+ efflux and directly activates the
NLRP3 inflammasome by regulating expression of genes involved in NLRP3 signaling [215, 216]. According to recent studies, expression levels of both NEK7 and NLRP3 showed a positive correlation [215] and interactions between the two
33 proteins have been reported. In the presence of stimuli, NEK7 binds to the LRR and NACHT of NLRP3 and in doing so induces oligomerization of the latter [214,
217, 218]. NLRP3 inhibitors MCC950 [219] and oridonin [220] inhibit interactions between NEK7 and NLRP3. Hence, NEK7 has emerged as a plausible therapeutic target for NLRP3-related disorders [194, 216].
1.8.9. NOD-like receptor family CARD domain containing proteins
(NLRC)
NLRC proteins have been known to have an inhibitory effect on inflammasome activation. NLRC5 [221] and NLRC3 [222-225] interact with both
ASC and caspase-1 thus disrupting the interactions between the two inflammasome proteins and preventing ASC speck formation. The presence of
NLRC3 was found to prohibit inflammasome assembly and ultimately causing a decline in the amounts of secreted IL-1β [225].
1.8.10. Heat shock proteins
Heat shock proteins (Hsp) also play vital roles in inflammasome regulation. Hsp90 is a chaperone that confers a protective effect to NLRP3 while in its inactive state [226]. Previous studies showed that Hsp90 works with a co- chaperone protein, suppressor of the G2 allele of Skp1 (SGT1), which is normally associated with ubiquitin ligase [227]. Binding of Hsp90 to NLRP3 prevents degradation of the latter. After priming, Hsp90 is released thus allowing NLRP3 to participate in inflammasome assembly [226]. In contrast, Hsp70 negatively regulates the inflammasome by binding to NLRP3. However, the interactions are disrupted upon stimulation with NLRP3 activators [228].
34
1.8.11. NLRP3 inflammasome inhibitors
Chemical inhibitors and isolated domains of inflammasome proteins also regulate inflammasome assembly as reviewed here [25]. Pyrin-only proteins such as POP1/ ASC2 not only bind to ASC but also inhibit the kinase activity of IKKs thus preventing subsequent NF-κB activation [229]. Likewise, POP2 negatively regulates the inflammasome by binding to ASC [230] while CARD-only proteins
(COPs) such as INCA and ICEBERG pose the same effect when bound to caspase-1 [231]. One group designed stapled peptides that could inhibit NLRP3 activation by interacting with the pyrin domain of ASC thus preventing formation of specks [232]. A similar strategy was employed by Hafner-Bratkovic and colleagues who designed various small peptide inhibitors with sequences identical to specific regions of NLRP3 inflammasome proteins [233]. On the other hand, synthetic chemical inhibitors such as Bay 11-7082 [234], MCC50 [219,
235-237], CY-09 [238], IFN39 [239], and tranilast (TR) [240] have been reported.
In addition, natural products such as oridonin [220], shikonin (a naphthoquinone)
[241], parthenolide [234], and resveratrol [242] were also successful in suppressing inflammasome activation and assembly. Recent reviews discussed various NLRP3 inflammasome inhibitors and their modes of action [33, 243].
All the above findings prove the versatility and diverse nature of the mechanistic pathways involved in inflammasome regulation. These controlled processes allow assembly of the inflammasome complex only in the presence of stimuli. More so, inflammasome regulation prevents its persistent stimulation that would result in a prolonged inflammatory state. It is also important to note that
35 certain signaling pathways are only triggered by specific ligands and may not be responsive to other activators. Although inflammasome regulators use somehow distinct pathways, they all regulate the immune response in a controlled manner.
1.9. Caspase-1
Caspase-1 belongs to the class I (inflammatory) caspases and is the first caspase that was discovered in mammals [6, 244]. It is known as the “effector” protein of the inflammasome complex [32, 33, 39] and a report mentioned that caspase-1 was released as part of a stable, high molecular weight (HMW) complex [245]. This cysteine protease facilitates the maturation of the pro forms of interleukins 18 (IL-18) and 1-beta (IL-1β). A recent publication, however, identified p62 (autophagy adaptor protein) as a potential caspase-1 substrate
[246]. In their recent review, Julien and Wells describe the use of proteomics as a vital tool in identifying caspase-1 substrates [247]. The many roles of caspases
(including caspase-1) are reviewed here [247-251]. The enzyme is released as an inactive zymogen and its recruitment to the inflammasome platform facilitates its activation via induced proximity dimerization leading to autoproteolysis [16,
252-262]. Inhibitors block the dimerization process [263]. Although caspase-1 is required for IL-1β production, it is not necessarily required for pyroptosis [264].
1.9.1. Initial characterization of caspase-1
The enzyme was originally isolated in 1989 from stimulated THP1 cell lysates [265, 266] and human peripheral blood monocytes [266] and was later named interleukin-1β (IL-1β) converting enzyme (ICE) [6, 244]. Treatment of the
36 enzyme with iodoacetate and N-ethylmaleimide (reagents which modify cysteines) rendered the enzyme inactive hence they identified it as a cysteine protease. Interestingly though, the enzyme was not affected by E64, a well- known inhibitor of cysteine proteases [265]. Similarly, isolation of the enzyme from cell lysates followed by purification, molecular cloning, and transfection yielded an active enzyme as confirmed by IL-1β cleavage [6, 244]. Both reversible aldehyde (Thornberry 1992) and irreversible ketone-based inhibitors
[6, 267, 268] had a negative effect on the activity of the recombinant human enzyme.
1.9.2. Caspase-1 structural studies
Structural studies such as amino acid sequencing and mass spectrometry
(MS) revealed the primary structure of the proenzyme. These early findings also provided information regarding the active site location and the folding of the full- length protein [7, 8]. The released proenzyme termed p45 (~45 kD) undergoes autoproteolytic cleavage at three aspartic acid (D) residues to produce p20 (~20 kD) and p10 (~10 kD) fragments [6] as shown in Fig 1.8A. Scheer and colleagues later revealed the sequential autoproteolytic cleavage of caspase-1 [14]. The active enzyme was identified as a heterodimer made up of p20 and p10 subunits
[6]. The active site, composed of Cys 285 & His 237, was found in the p20 subunit [7, 8, 269]. However, both the p10 and p20 were found to be essential in rendering the enzyme active [6, 269]. By employing protease digestion of peptides, a four-residue recognition sequence (Ac-YVAD-AMC) was identified as the minimal substrate for caspase-1. The caspase-1 nomenclature identifies the
37 cleavage site D as the P1 position as shown in Fig 1.8 B. The P1’ position
(adjacent to the P1 position) favors a small, nonpolar residue [6, 270].
Fig 1.8 Structure of human caspase-1 Panel A. shows the three aspartic acid cleavage
sites (D) and the active Cys 285 residue located in the p20 domain. The card domain
linker (CDL) separates the CARD and the p20 subunit while the interdomain linker (IDL)
separates the p20 and p10 domains. The prodomain is made up of the CARD, CDL, and
IDL. Panel B. Typical caspase-1 substrate showing key recognition sites. P1 represents
the cleavage site (D). The P1’ position favors a small, hydrophobic residue such as
glycine (G) or alanine (A). Substitutions in the P2 and P2’ positions are much tolerable
and have no significant effect on substrate specificity.
X-ray crystallization studies would later reveal the crystal structure of recombinant human caspase-1 bound to chloromethylketone [8], reversible aldehyde inhibitors [7], or malonate [11, 12]. The inhibitors occupied the S1 position (an extremely basic region which binds to the side chain of aspartic acid)
38 located in the active site thus enabling the enzyme to adopt an open-like conformation which favors substrate binding [11, 12]. These studies revealed the conformation of the active enzyme as a heterotetramer (p202p102) composed of symmetrical p20p10 dimers linked by hydrophobic interactions and hydrogen bonds [7, 8, 11] contrary to previous findings [6]. The outstanding questions regarding actual structure of the active caspase will be explored in detail in chapter 5.
1.9.3. Caspase-1 functionalization assays
Functionalization assays were conducted to study the mechanisms of caspase-1 autoproteolysis and to also determine the role of each caspase-1 domain. Considering the prodomain of caspase-1 (initial 122 amino acids on the
N terminus ~ 11 kDa and includes the CARD), previous findings reported its importance in keeping the pro-caspase-1 in an inactive state thus its removal would permit dimerization and subsequent enzyme activation [6, 271-273].
However, other studies would also prove the importance of the prodomain in stabilizing the enzyme by promoting dimerization and autoproteolysis thus its removal renders the enzyme inactive [21, 262, 274-278]. Interestingly, other groups completely disagreed with these notions and argued that the prodomain is dispensable and has little to no effect on caspase-1 activity [14, 279, 280].
Considering the complexity of the mechanisms governing caspase-1 activation,
Chapter 5 offers a more in-depth look into this elusive enzyme.
39
1.10. Aβ-NLRP3 inflammasome interactions
Interactions between Aβ and the NLRP3 inflammasome have been an area of interest although it is still not quite clear how Aβ stimulates inflammasome activation. Either there is a direct interaction between Aβ and the inflammasome proteins (most likely NLRP3 the sensor protein) or perhaps another indirect pathway involving intermediate proteins and complex signaling pathways. Direct interactions between Aβ and ASC specks were reported by the
Heneka group [2, 116, 158]. Using a cell-free assay, another group demonstrated the direct interactions between Aβ and NLRP3 [281]. These interactions are further explored in Chapter 6.
1.11. Recombinant expression of inflammasome proteins from the literature
1.11.1. NLRP3 and ASC proteins
Many groups have utilized bacterial expression systems to express inflammasome proteins. These systems are ideal for producing large amounts protein in a short period of time. The downfall, however, is that bacterial expression systems do not offer desirable post translational modifications (PTMs)
(such as methylation, phosphorylation, ubiquitination, acetylation, glycosylation among others) that are critical for the overall functioning of the proteins. With this in mind, we opted to use the ExpiCHO-S mammalian recombinant expression system. It is important to note that this is the first time this cell line has been used to express NLRP3 inflammasome proteins.
40
A number of groups utilized recombinant systems to obtain NLRP3 inflammasome individual proteins and/ or domains for functional studies. The earliest ASC studies were done by two different groups [125, 128]. Sagara and colleagues were the first to isolate ASC from human promyelocytic leukemia
(HL60) cells. They characterized the then novel protein and later designed a
DNA construct which was transfected into Cos 7 cells [128]. In their follow-up studies to study ASC filament formation, they transfected Cos 7 cells with either
WT or mutant GFP-tagged ASC PYD then used fluorescence microscopy for visualization. Deletion mutants were used to determine the minimum ASC fragment required for filament formation while the amino-acid substitution mutants provided information on the role of charged and hydrophobic residues in the self-association of ASC PYD. In addition, E. coli-expressed ASC PYD constructs were purified and used for NMR studies [134].
Gumucio and colleagues investigated the role of pyrin in apoptosis. They used a yeast two-hybrid system in which WT pyrin acted as a “bait” to capture and identify its binding partners. Co-expression of pyrin and ASC in 293T and
HeLa cells followed by co-immunoprecipitation and colocalization assays confirmed the interactions between the two proteins. They transfected the same cell lines with CMV-GFP and either myc-pyrin or FLAG-ASC or both to study speck formation [125]. Using YFP-ASC expressed in HeLa cells, they studied the kinetics of ASC aggregation leading to speck formation [148].
Hill and colleagues [5] also utilized both bacteria and mammalian systems.
They expressed GST-ASC PYD (WT and mutants), Pyrin PYD, and PYD only
41 protein (POP1) in E. coli. In vitro translation of full-length ASC, NLRP3, and
POP1 with slightly different constructs was conducted using TNT T7 coupled reticulocyte lysate system. Subsequent in vitro binding assays with GST-tagged
WT and mutant ASC PYD fusion proteins indicated that ASC core mutations
(E13A, K21A, R41A, D48A, D51A) abolished any interactions with NLRP3 and
POP1. Interestingly, binding between the WT ASC and the mutants was reduced but was not completely abolished. A trend similar to the in vitro assays was observed upon conducting co-expression of the proteins in HEK 293T cells followed by co-immunoprecipitation experiments. HEK 293T cells are an excellent choice because they normally do not express inflammasome components. Although the ASC mutants affected binding of ASC and NLRP3, they didn’t affect overall folding of the proteins [5]. Based on these findings, follow-up studies by the same group looked at how the mutations associated with familial Mediterranean fever affected the structure and function of PYD of pyrin
(protein) [136].
Other groups also utilized the HEK 293T mammalian cell line to express
NLRP3 inflammasome proteins. For example, Satoh and colleagues utilized the
HEK cell system to perform transient expression of all three inflammasome proteins to be used as western blot antibody controls while analyzing the expression levels of each protein in human neural cell lines [282]. Another group expressed FLAG-tagged full-length NLRP3 and a variety of NLRP3-deletion mutants lacking the pyrin domain or the LRR region in order to identify the
42 targets of 3,4-methylenedioxy-β-nitrostyrene (MNS), a kinase inhibitor known to inhibit the NLRP3 inflammasome [181].
To study the mechanisms involved during pyroptosis, Fernandes-Alnemri et al utilized THP-1 macrophages stably expressing recombinant GFP-ASC
[147]. Upon stimulation of the cells with LPS and treatment with various activators, pyroptosome assembly was observed by confocal microscopy. In addition, ASC pyroptosomes isolated and purified from stimulated THP-1 cells were found to activate WT procaspase-1 (but not the inactive C285A mutant) thus prompting IL-1β maturation. WT and mutant ASC constructs (K26A) were expressed together with FLAG-tagged procaspase-1 in HEK 293T cells then activated by incubation at 37 oC. The point mutation prevented ASC pyroptosome formation thus emphasizing the importance of the PYD in ASC self- assembly. In vitro experiments with purified recombinant ASC that was expressed in bacteria cells yielded functional pyroptosomes which enabled procaspase-1 activation. Low intracellular K+ triggered ASC oligomerization and ultimately pyroptosis [147].
In order to reconstitute an active inflammasome, Chen and colleagues transfected WT or mutant ASC constructs with pyrin domain mutations (R41A,
D48A, and P40A) in HEK 293T cells stably expressing FLAG-NLRP3, procaspase-1, and pro-IL-1β. Upon treatment with Nigericin, inflammasome activation was observed only in cells containing the WT or P40A mutant thus reinforcing the importance of charged and hydrophobic residues in PYD self-
43 oligomerization and inflammasome assembly. They also utilized bacterial recombinant expression systems [149].
The Pelegrin group also utilized the HEK cell line to express recombinant mcherry-and YFP-tagged ASC proteins for speck-formation studies. Addition of these specks to WT macrophages or cell-free supernatants of stimulated macrophages resulted in an increase in caspase-1 activity and IL-1β release.
Oligomers of YFP-tagged NLRP3 D303N, the mutant associated with cryopyrin- associated periodic syndromes (CAPS) that involve inflammation, caused nucleation of red fluorescent protein (RFP)–tagged ASC upon co-expression in
HEK cells. The mutant NLRP3 specks also co-localized with extracellular ASC in cell-free supernatants [153].
Nunez and colleagues also co-expressed WT NLRP3 and/or inflammatory disease-associated mutants (R260W, D303N, E637G) with ASC in HEK cells.
The mutants resulted in enhanced Nf-κB activation and IL-1β secretion. Similarly, the same trend was observed when THP-1 cells were transfected with WT/ mutant NLRP3 constructs only (since THP-1 cells have endogenous ASC).
Moreover, the mutants exhibited binding to ASC in the absence of stimuli whereas no binding was observed with WT NLRP3 [130].
Apart from mammalian recombinant expression systems, bacterial systems were also extensively used in these studies. Most studies were focused on the formation of filaments by ASC which is an important step in the inflammasome assembly process that leads to speck formation and subsequently recruitment of caspase-1. de Alba utilized E. coli to express full-length hASC for
44
NMR structural studies [17, 138]. In another study to identify the binding surfaces on ASC PYD responsible for self-association as well as binding with NLRP3
PYD, they utilized the same system to express the pyrin domains of both ASC and NLRP3 and the ASC PYD L25A mutant that prevents oligomerization [135].
Broz and colleagues also used E. coli to express recombinant ASC pyrin domain (residues 1-91 with a C-terminal His tag) for conducting studies on ASC filament formation [3]. Likewise, Wu and colleagues studied formation of ASC
PYD and caspase-1 CARD filaments using Alexa488- and-GFP labelled fusion proteins while also employing point mutations that disrupted the oligomerization process. Using cryo-EM, they determined the structure of ASC PYD (at a resolution of 3.8 Å) [141] and AIM PYD filaments [140]. Apart from the GFP fusion tag, the group also introduced MBP and SUMO solubility tags into their gene constructs. In addition, they also studied caspase-1 polymerization in the presence of card-only proteins (COPs) known as INCA and ICEBERG that are known to have an inhibitory effect on inflammasome assembly [231]. In order to conduct these experiments, the group utilized both E. coli and insect cells to express various domains of all three inflammasome proteins (NLRP3, ASC, and caspase-1) [140, 141, 231].
On the other hand, Hiller and colleagues also studied formation of ASC filaments in vitro using recombinant mouse ASC (FL and ASC PYD) expressed in
E. coli cells. They also introduced a GSGSLE linker into their constructs to minimize the effect of the included His-tag [4]. Later they designed a fluorescence polarization assay to test the effect of small molecules on filament
45 formation using bacterial- expressed WT hASC PYD and the K21A mutant incapable of forming filaments. To enable fluorescent labeling of the PYD fragments, they also engineered a single mutation (E97C) [283].
Since previous studies had primarily focused on filament formation by human ASC PYD, de Alba and colleagues opted to use both FL ASC and ASC
CARD expressed in E. coli for their studies. Using FL ASC presented a better model for determining the formation of ASC filaments. By incorporating computational methods, NMR, and TEM, they discovered that the PYD and
CARD domains of ASC formed structurally distinct filaments. In addition, both domains were found to be crucial in the assembly of ASC filaments. TEM images showed a ring-like structure (~ 7 nm diameter), previously reported by Bryant and colleagues [284], which acted as the building block for filament assembly [143].
Apart from the COPs studies by Edelman and colleagues, other groups were interested in hNLRP3 pyrin domain-only proteins containing (POPs) which also prevent assembly of the inflammasome via interactions with ASC PYD.
Reed and colleagues [229] performed in vitro interactions to demonstrate the binding between the PYD domains of ASC and POP1 (also known as ASC2) expressed in E. coli. Co-expression of the protein domains in HEK 293T cells followed by co-immunoprecipitation also yielded similar results. The group’s findings demonstrated the role of POP1 in preventing assembly of the inflammasome via blocking activities of both IKKα and IKKβ, and subsequently
NF-κB activation [229]. Studies by Hill and colleagues would later reveal the structure and dynamics of E. coli- expressed POP1 [132]. Using bacterially
46 expressed proteins, Park and colleagues also determined the crystal structures of NLRP3 PYD (residues 3-110) [146] and residues 1-89 of cellular pyrin domain- only protein 1 (cPOP1) [133] at resolutions of 1.7Å and 3.6 Å respectively.
Narayanan & Park generated the CARD domains of ASC and caspase-1 in E. coli. In vitro binding studies with purified proteins showed that there were no interactions between the CARD domains of the two proteins [139].
1.11.2. Caspase-1
Conversely, some research groups solely focused their studies on caspase-1 due to its interesting function as an enzyme. As far back as the late
80s and early 90s after the enzyme was initially isolated from THP-1 and human peripheral blood monocytes [6, 244, 265, 266], researchers were already taking advantage of recombinant expression systems to express the full-length protein and/ or domains on a preparative scale since endogenous protein amounts were insufficient for functional assays.
Two groups isolated caspase-1 from THP-1 monocytes and obtained the copy DNA (cDNA) via molecular cloning. Transfection of Cos-7 cells with the expression vector containing the DNA insert successfully yielded an active full- length caspase-1 [6, 244]. Later, another group that utilized a bacterial recombinant system was able to express and isolate FL caspase-1 from the inclusion bodies. The refolded protein underwent autoproteolytic cleavage into the active form that facilitated cleavage of pro-IL-1β [9].
47
Brady and colleagues utilized an E.coli expression system to express the individual p20 and p10 domains of human procaspase-1 since initial expressions with eukaryotic systems were unsuccessful. Proteins were isolated from the inclusion bodies, purified then carefully refolded. Subsequently, refolded proteins were used to conduct enzyme activity studies and for structural analysis by x-ray crystallography [8, 10]. Likewise, a few groups that also utilized the same approach were successful in producing a catalytically active enzyme [13, 14, 16].
In addition, two separate groups led by McDowell [11, 12] and Livingstone [7] proceeded to conduct crystallographic studies with recombinant caspase-1 obtained from refolded p20 and p10 fragments.
Other research groups would later establish protocols regarding the expression of caspases using E.coli systems and the subsequent purification, refolding, and characterization procedures. They explored various conditions
(buffers, pH, salt, detergents, denaturants, stabilizing agents, inhibitors) in an effort to establish the optimum conditions ideal for isolating a functionally stable enzyme [285, 286].
Two different groups also utilized E.coli systems to express caspase-1 p30 fragment (made up of p20 and p10 domains). While one group was interested in studying caspase-1 typical behavioral features such as enzymatic activity and stability [15], the other incorporated the GST fusion tag in their proteins in a study focusing on the regulation and expression patterns of caspase-1 subfamily of proteins [287].
48
In a slightly different approach, wild-type and mutant caspase p35 fragments were expressed using the same expression system to study the sequential cleavage at respective aspartate residues thus leading to autoprocessing of the enzyme [14]. In contrast, Wilson et al utilized mammalian
Cos7 cells to conduct transient expression of caspase-1 p30 WT and/or MUT fragments and ultimately monitored IL-1β processing [7].
Vandenabeele and colleagues used a different mammalian expression system (HEK 293T) to express the full-length human caspase-1 and the enzymatically inactive C285A mutant for functionalization studies. In addition, they also expressed other caspase-1 mutants, D27G and R45D, for further functional assays in an effort to determine the importance of these key residues in the interaction with ASC and self-oligomerization of the CARD domain respectively [278]. The Schroder group also used HEK cells to express an engineered, uncleavable caspase-1 construct containing a thrombin cleavage site to study the impact of the CARD-domain linker (CDL) (removal) in processes governing activation of caspase-1 [21].
49
1.12. Overall summary and research goals:
All the afore-mentioned studies in this chapter have demonstrated the interplay between Aβ, Alzheimer’s disease (AD), and the NLPR3 inflammasome.
The NLRP3 inflammasome has been implicated in a variety of inflammatory disorders including AD. The involvement of the innate immune system in AD is well-documented and neuroinflammation is a characteristic feature of AD pathology. Despite its role in the immune response, not much is known about the
NLRP3 inflammasome regarding its exact structure or mechanism of activation.
Moreover, the actual events involved in the activation of the inflammasome by Aβ are still unknown. Previous studies of the three inflammasome proteins by other groups mainly focused on X-ray crystal structure determination, NMR and computational methods to map the binding surfaces, mutation studies to identity key amino acid residues, and peptide truncations to determine the minimal fragment length required for protein-protein interactions. In addition, numerous studies also focused on speck formation by ASC. However, none of these studies determined the actual stoichiometry of the inflammasome proteins
(individually and as a complex) and how that relates to their overall function.
Thus, the goals of this project are: to study each individual inflammasome protein and how these three proteins interact; to determine the nature of the interactions between Aβ protofibrils and the NLRP3 inflammasome proteins; to understand the mechanisms of activation of the elusive caspase-1, and thus determine which exact domains make up the active form of the enzyme.
50
CHAPTER 2: METHODS
Since endogenous amounts of the inflammasome proteins in immune cells are very low, recombinant expression provides an excellent alternative of obtaining these proteins in large amounts for subsequent structural studies and functional assays. In these studies, the NLRP3 inflammasome proteins were overexpressed in a mammalian ExpiCHO-S cell line. Many studies utilize bacterial systems because these are relatively cheap to maintain, easy to culture, and they produce large amounts of protein in a very short time. However, bacterial systems do not offer the post-translational modifications that eukaryotic systems provide hence the reason why a mammalian cell was chosen for these studies. This chapter highlights the experimental methods employed in these studies: recombinant protein expression and harvesting of expressed proteins; purification and characterization of proteins by biochemical and biophysical methods of analyses; functional assays such as caspase-1 activity studies, Aβ-
NLRP3 inflammasome interaction assays, and Aβ-ASC aggregation assays.
2.1. Gene design and synthesis
Protein sequences (with calculated molecular weights, Mw) for human
NLRP3 (AAI43363; 118,163 Da), NLRP3 pyrin domain (residues 3-110; 12,483
Da), human ASC (NP_037390; 21,626 Da), human procaspase-1 (P29466;
45,156 Da), caspase-1 p20 subunit ( residues 120-297; 19,844 Da) and caspase-
51
1 p10 subunit (residues 317-404; 10,244 Da) were obtained from NCBI and entered in the GeneArt Gene Project Configurator (Life Technologies/
ThermoFisher). Several different gene insert configurations were used that contained the following from the 5’ end: Kozak initiation sequence, Ig κ-chain secretion sequence or not, hexa-histidine (His) tag, enterokinase (EK) or tobacco etch virus (TEV) protease cleavage site, gene of interest, and a stop codon.
Fusion proteins were also constructed with yeast small-ubiquitin-related modifier (SUMO) (Q12306; 11,352 Da) with R64T and R71E mutations termed
SumoStar [288, 289], mammalianized bacterial maltose-binding protein (MBP) containing mutations D82A, K83A, E172A, N173A, K239A, A312V, I317V,
E359A, K362A, D363A [290], and glutathione- S transferase (GST) Schistosoma japonicum, (AAC00518; 21,626Da) [291]. A GST C-terminal linker
(SDGSSMGSS; 814 Da) was introduced (Temple and Dong PDB 6N8U). A point mutation, K21A, was incorporated into the GST-ASC PYD fusion protein [2-5].
Gene synthesis and initial plasmid preparation was done by Life
Technologies/ThermoFisher. All the relevant information with regards to the recombinant full-length proteins and /or domains that were expressed and utilized in these studies is summarized in Table 2.1.
52
Table 2.1. Information on all the expressed proteins. ** Early MBP constructs containing
10 mutations that were supposed to improve protein expression. * Later MBP constructs bearing the 2 mutations essential for expression and purification A312V, I317V. Extinction coefficients and pI values were calculated using available online tools.
Ext coefficient calculations: https://web.expasy.org/protparam/ (W 5690, Y 1490, C 120) pI calculations: https://web.expasy.org/compute_pi/
53
2.2. Plasmid amplification
Plasmids (2 μL, 1 μg/μL) were added to 50 μL chemically-competent
TOPSHOT Escherichia coli (E. coli) cells (ThermoFisher) followed by incubation of the vial on ice for 30 min, in a 42 °C water bath for 30 s, and then back on ice.
250 μL of super optimal broth with catabolite repression (SOC) medium was added and the vial was shaken on its side in a 37 °C incubator for 1 h at 225 rpm. Using a sterile pipette tip, 20 μL of the cell transformation solution was spread evenly on pre-warmed in 100 mm petri dishes containing a hardened
1.5% agar/100 μg/mL ampicillin/Luria broth (LB) solution. The plate was inverted and incubated quiescently at 37 °C overnight. The next day individual colonies were inoculated into a sterile 250 mL Erlenmeyer flask containing 200 mL of 100
μg/mL ampicillin/LB solution and the solution was incubated at 37 °C with rotary shaking overnight. DNA was isolated by maxi-prep (PureLink HiPure,
ThermoFisher) according to manufacturer’s guidelines and the yields were typically 0.5 mL of 1.5 mg/mL pure plasmid (A260/A280 = 1.8).
2.3. ExpiCHO-S cell culture
The ExpiCHO-S™ cell line (ThermoFisher) is a mammalian cell system adapted to grow to high densities in serum-free ExpiCHO Expression Medium
(EM). ExpiCHO-S cells in EM were maintained at 37 °C and 8% CO2 in either
250 mL or 500 mL sterile Erlenmeyer shake flasks on a MidSci 19 mm orbit shaker set at 120 rpm. Three days post-thaw, viable cell density was determined using Trypan Blue with a Cellometer cell counting device. Thereafter, cell density
54 and viability were recorded every other day. Log phase cultures typically maintained viability at >90%. Cells were allowed to achieve a density of 4 × 106 cells/mL (~4-5 days), whereupon they were sub-cultured by 20x dilution into pre- warmed EM. Cells were passaged three times before transfection. At higher densities, the cells exhibited some clumping but that was expected, and the clumping did not affect the cell growth and/or viability.
2.4. Transfection and expression
Prior to transfection, ExpiCHO-S cells were grown to a density of 4-6 × 106 cells/mL. On day −1, pre-transfection, the cells were split to 3-4 × 106 cells/mL and allowed to grow overnight. On the day of transfection, day 0, the cell viability was confirmed to be 95-99% and the cells were diluted to 6 × 106 cells/mL with pre-warmed EM (50 mL total) into a 250 mL shaker flask. As per the GIBCO
ExpiCHO™ Expression System Kit manufacturer’s protocol, the plasmid DNAs were diluted into the cold OptiPro Serum-free Complexation Medium SFM (50 µg plasmid in 2 mL SFM. The ExpiFectamine™ CHO Reagent was diluted with
SFM, gently mixed with plasmid DNA, and the ExpiFectamine CHO / plasmid
DNA complexes were incubated at 25 °C for 1-5 minutes. To co-express the three inflammasome proteins, 15 µg of each plasmid was used (combined 45 µg plasmids per 250 mL flask). The mixture was slowly added to the cells while swirling the flask gently during addition. The cells were incubated on an orbital shaker in a humidified 37 °C incubator at 8% CO2. On day 1 after transfection, the ExpiCHO Feed and ExpiFectamine Enhancer were added directly to the
55 flasks. A rabbit IgG-expressing control vector was used as a positive control for transfection and expression in ExpiCHO-S cells.
2.5. Protein harvesting
Harvesting was done 8-13 days post-transfection initially but later modified to only 2 days post-transfection. ExpiCHO-S cells were collected and centrifuged at 1,000g for 10 min. Medium (M) was removed. Cell pellets were washed in phosphate buffered saline pH 7.4 (PBS) containing 10 mM Na2HPO4 and 150 mM NaCl, weighed, and 4 mL of lysis buffer containing a protease inhibitor cocktail was added per gram weight of cells. Various lysis buffer compositions were utilized: Protocol #1 (150 mM NaCl, 20 mM Tris, pH 7.4), Protocol #2 (150 mM NaCl, 20 mM Tris, 1% Triton X-100, 1% deoxycholic acid, and 0.1% SDS, pH 7.4) or Protocol #3 where 1% NP-40 replaced SDS in the buffer used in
Protocol #2. For these experiments, Protocol #3 was mainly used. Cells were then homogenized on ice using a Dounce tissue homogenizer for 5 min. After homogenization, the cell samples were centrifuged at 22,000g for 20 min at 4 °C and the supernatant, or lysate (L), was collected. The pellet was washed with phosphate or HEPES buffers, solubilized in 6-8 M guanidine hydrochloride
(GuHCl) buffers, sonicated for 5 min, and centrifuged at 22,000g for 20 min. In some preparations, either 1 mM or 5 mM dithiothreitol (DTT) was also included in the solubilization buffer. The ensuing supernatant was collected as the inclusion body (IB) fraction. Solubilized inclusion bodies were refolded overnight in refolding buffer (50 mM HEPES pH 8.0, 100 mM NaCl, 10% sucrose, 1 or 5 mM
56
DTT). 4 mL solubilized IB were added to 46 mL refolding buffer. For caspase-1 fragments refolding experiments, 100 mM malonate (Sigma Aldrich Lot# BCBW
2461) or 1 µM caspase-1 inhibitor (Enzo Life Sciences, Ac-YVAD-CHO Cat #
ALX-260-027-M001) were added to the refolding buffers. Briefly, equimolar amounts of solubilized caspase-1 p20 and p10 fragments were combined to 1 mL then added dropwise to refolding buffer to make a total volume of 50 mL.
Refolding was done overnight at 4 oC with rotation. Centrifugation (22,000g for
20 min) was done the next day to remove any floating debris before proceeding with purification.
2.6. Nickel affinity chromatography
Medium samples (approx. 50 mL) were dialyzed overnight in 3.5 L of dialysis buffer (20 mM Na2HPO4, 500 mM NaCl) before purification. Affinity purification of lysates, inclusion bodies, and medium samples was done using a
20 mL Ni SepharoseTM 6 Fast Flow column (GE Healthcare). The lysates and inclusion bodies were centrifuged for 10 min at 17,000g at room temperature to remove loose debris. Samples were then loaded onto the column using a 50 mL
Superloop (GE Healthcare). The Binding Buffer (A) (20 mM Na2HPO4, 500 mM
NaCl, 20 mM Imidazole, pH 7.4) was allowed to flow for 5 column volumes (100 mL) to remove the unbound proteins. Proteins of interest were then eluted in
Elution Buffer (B) (20 mM Na2HPO4, 500 mM NaCl, 500 mM Imidazole, pH 7.4).
No linear gradient was created therefore the proteins eluted when the elution buffer started flowing at 100%. Elution was maintained for 3 column volumes (60
57 mL). 2 mL fractions were collected at a flow rate of 1 mL/min and approximate concentrations were determined from the UV absorbance trace.
2.7. Size exclusion chromatography (SEC)
Inflammasome proteins were separated on Superdex (75 and 200) or
Superose 6 columns using an AKTA fast protein liquid chromatography (FPLC) system (GE Healthcare). Affinity-purified proteins were purified by SEC without prior treatment. In addition, various sample treatment strategies were employed to maximize amount of soluble (stable) species obtained as shown in Table 2.2.
Table 2.2 Various protein treatment strategies employed prior to size-exclusion
chromatography (SEC) to break pre-formed aggregates.
58
After sample treatment, protein samples were centrifuged at 17,000g (10 min), allowed to cool for 5 min then eluted. Various elution buffers used include
10mM phosphate pH 7.4, 20 mM HEPES pH 8.0, and 10mM Acetate pH 3.6 containing 150mM NaCl, reducing agents (5% BME, 2 mM DTT), detergents
(NP40, CHAPS, Tween, SDS), and/or denaturants (2M GuHCl). The flow rate was set to 0.5 mL/min and 0.5 mL fractions were collected. Bovine serum albumin (BSA, 0.5 -1 mL sample volume, 2.5 or 5 mg/mL) was routinely run as both a control protein and a blocking agent to prevent non-specific binding to the
Superdex resin.
2.8. SEC- multi-angle light scattering (SEC-MALS)
MALS data were obtained in line with SEC purifications as previously described [113] using a DAWN DSP instrument with fixed detectors from Wyatt technology. Data analysis was done using the Astra 4.90.08 software. Baseline correction, normalization, and calculation of the delay volume were all based on
BSA data from detector 11. The Debye plot gave a summary of the molecular weight (s) of the proteins relative to BSA monomer (66 kD).
2.9. Dynamic light scattering (DLS)
DLS measurements (to obtain hydrodynamic radii of proteins RH) were conducted with a Wyatt Technology DynaPro instrument (DP-AMB-01,
Manufactured 05-2005). Measurements were taken using a QS 1.50 mm DLS
59 cuvette. 35 µL samples were loaded into a clean cuvette using a gel-loading tip while making sure there were no air bubbles. The cuvette was capped after loading sample and the sides clean were wiped clean with a Kimwipe. Control measurements were done with 2 mg/mL pure BSA sample dissolved in PBS or
SEC-purified BSA peak fraction. Superdex 75/200-purified inflammasome proteins were analyzed. As described previously [113], light scattering intensity was collected at a 90° angle using a 5 s acquisition time. Calculations for diffusion coefficients D of particle were done using the autocorrelated light
퐾퐵푇 intensity data (>25 acquisitions) and the Stokes−Einstein equation 퐷 = 6ƞ휋푅퐻
(where KB is the Boltzmann’ constant, T is temperature in K, ƞ is solvent viscosity, RH is the hydrodynamic radius). The Dynamics 6.7.1 software generated autocorrelation curves (intensity autocorrelation vs time µs), intensity histograms (% intensity vs R nm), and mean RH values for each peak.
2.10. Circular Dichroism (CD)
CD studies were conducted using a Jacso 1500 spectrometer. A Jasco
J/0556 cuvette with a 0.1 cm pathlength was used for the measurements and the sample volume was 200 µL. Affinity-purified ASC medium fractions ~0.4 mg/mL were analyzed under physiological (PBS pH 7.4) and acidic conditions
(phosphoric acid pH 3.8). Twenty continuous scans were done for each sample from 260 nm to 190 nm. Control buffer spectra were obtained, averaged, and subtracted from the protein spectra. Spectra were obtained using a scan speed of 100 nm/min, 2 nm bandwidth, and a 4 sec data integration time. A response
60 factor of 200 mdeg/1.0 OD was applied. The obtained ([θ]obs, deg) values were converted to mean residue ellipticity ([θ], deg cm2 dmol−1) using the equation [θ]
= [θ]obs × (MRW/10lc), where MRW is the mean residue molecular weight of
ASC (21,626 g/mol divided by 195 residues), l is the optical path length (cm), and c is the concentration (g/cm3).
2.11. Transmission electron microscopy (TEM)
For TEM studies, affinity-purified ASC medium sample was used. ASC sample was diluted to 18.5 µM in PBS buffer then 10 µL of the sample solution was placed onto a 200-mesh formvar-coated copper grid (Ted Pella, Inc.). The sample was adsorbed to the grid for 10 minutes and the excess sample was removed by Kimwipe. The grid was then washed three times by placing it face- down in a droplet of water. After washing, staining was done with uranyl acetate
(Electron Microscopy Sciences, Hatfield, PA) for 10 minutes and excess stain was removed. The grid was air dried and then visualized with a JEOL JEM-2000
FX transmission electron microscope operated at 200 keV.
2.12. Protein concentration determination
Protein concentrations were determined in-line during affinity purification or SEC by UV 280 nm absorbance using the following extinction coefficients calculated using Expasy bioinformatics tools: NLRP3 (119,620 M-1 cm-1), NLRP3 pyrin domain (21,270 M-1 cm-1), ASC (24,870 M-1 cm-1), procaspase-1 (27,470 M-
1 cm-1), caspase-1 p20 (14, 230 M-1 cm-1), caspase-1 p10 (8,730 M-1 cm-1).
61
Additionally, concentrations of the eluted affinity fractions were also determined by bicinchoninic acid (BCA) assay (ThermoFisher) in microplate format using colorimetric measurement at 562 nm on a Molecular Devices SpectraMax plate reader. Proteins fractions measured directly after affinity purification were in elution buffer containing 500 mM imidazole, therefore a standard curve was constructed in the same concentration of imidazole. UV absorbance measurements at 280 nm were also made after pooling and overnight dialysis of the protein fractions in PBS, Tris or HEPES buffers.
2.13. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-
PAGE) and western blotting
Protein fractions obtained from the affinity purification (1-5 μg) were mixed
1:1 with Laemmli SDS sample buffer containing 5% β-mercaptoethanol (BME) and heated at 95 °C for 5 min. 20 μL was then loaded onto a 4-20% gel and run under denaturing conditions. HL-60 whole cell lysate (Santa Cruz Biotechnology) was used as a positive control for all inflammasome proteins and typically run in lane 2 with Mw markers (PageRuler Plus pre-stained 10-250 kD, ThermoFisher) in the first and last lanes of each gel. Proteins were then transferred to a PVDF membrane, dried and blocked with a 5% milk solution for 1 h. After blocking the membrane was washed 3x (5 min each) with PBS containing 0.2% Tween 20.
Membranes were then incubated with specific antibodies for each protein diluted in PBS containing 0.1% Tween 20 and 1% milk for 1 h under gentle rotation (70 rpm). Antibodies used were anti-NLRP3 (8N8E9, ThermoFisher, 1:250 dilution;
62
NBP-2-03947, Novus, 1:500; AG-20B-0014, AdipoGen, 1:1000), anti-ASC
(PYCARD, ThermoFisher, 1:1000 dilution and B-3, Santa Cruz, 1:1000 dilution), anti-caspase-1 (MAB6215, R&D Systems, 1:5000 dilution; PA1440, Boster
Biological, 1:1000; EPR16883, Abcam, 1:20,000), anti-His-tag (HIS.H8,
ThermoFisher, 1:1000 dilution), anti-IL-1β (Cat# AF-201-SP, R&D systems,
1:2000), and anti-GST-tag (Cat# 32839-05111T, AssayPro, 1:1,000). A second wash step was followed by incubation with species-specific HRP-conjugated secondary antibodies for 1 h. After washing, the membrane was then treated with
Pierce ECL substrate for 1 min under vigorous rotation (120 rpm). Biomax XAR film was exposed to the membrane and developed. In later cases, Bio-Rad
ChemiDoc MP was used for imaging. Immuno dot blots were conducted in a similar manner. Samples (1.7 µL) were applied to a pre-wetted nitrocellulose membrane, allowed to dry for 5 min and blocked and washed as described above. Membrane was incubated with respectively primary and HRP-conjugated secondary antibodies followed by ECL reagent with wash steps in between, then imaged.
2.14. In vitro caspase-1 reconstitution assays
Caspase-1 reconstitution assays were conducted in order to obtain a stable, refolded active enzyme. For these studies, two approaches were utilized.
The first method involved re-solubilizing affinity-purified p10 and p20 fragments.
Briefly, affinity peak fractions were pooled, went through a series of buffer exchanges with 6M GuHCl/ 0.5 mM DTT at 5,000xg using Amicon
63
Ultracentrifugal filter devices (10,000 MWCO). p10 and p20 domains were concentrated to 1 mL separately, incubated 15 min at room temp then loaded onto Superdex 200 columns using phosphate buffer (10 mM Phosphate, 150 mM
NaCl, 2 mM DTT). SEC-purified p20 and p10 fractions were combined in equimolar amounts, incubated for 1 hour at 4 oC then samples analyzed by further SEC purification, SDS-PAGE, and immunoblotting. The second approach involved overnight solubilization and refolding of combined p10 and p20 IBs +/- inhibitors (1mM Ac-YVAD-CHO and 100 mM malonate) to stabilize the refolded protein. Refolded samples were then purified by affinity and SEC and further analyzed by gel electrophoresis under denaturing conditions followed by immunoblotting.
2.15. Caspase-1 activity assays
A fluorescence assay [6, 14, 269, 285, 292] was developed to assess caspase-1 activity of the recombinant full-length caspase-1, fragments, and reconstituted domains. This assay utilizes a synthetic caspase-1 tetrapeptide substrate that was designed based on the minimum recognition sequence of the enzyme [6]. The synthetic substrate is generally in the form Ac-XXXD-AMC/AFC;
D represents the aspartate cleavage site, 7-Amino-4-methylcoumarin/ 7-amino-4- trifluoromethylcoumarin (AMC/ AFC) are coumarin-based fluorogenic motifs.
Various storage methods were explored in an effort to retain active enzyme.
These include storing the proteins in solution at 4 oC or frozen at -80 oC, and also
64 adding protein stabilizing cocktail (4X solution, Thermofisher, Prod# 89806, Lot#
SLBT4106) to the SEC-purified fractions.
To optimize the assay, 30 nM of Human Caspase-1 (Active) Recombinant
Protein (Millipore Cat # CC126), 30 µM substrate (Ac-YVAD-AMC and Ac-
WEHD-AMC, Enzo Life Sciences, Cat # ALX-260-024-M001 and ALX-260-057-
M001 respectively), and 1 µM reversible and irreversible inhibitors (Enzo Life
Sciences, Ac-YVAD-CHO Cat # ALX-260-027-M001 and z-YVAD-fmk Cat # ALX-
260-020-M001) were used [10, 13-16, 21, 293]. The structure of AMC is shown in
Fig 2.1.
Fig 2.1 Structure of 7-Amino-4-methylcoumarin (AMC) which is fluorogenic. AMC is
released from the synthetic caspase-1 substrate Ac-WEHD-AMC upon cleavage by
caspase-1 enzyme. Fluorescence assays were conducted with a Cary Eclipse fluorometer
and emission scans were recorded using an excitation wavelength of 350nm and
monitoring emission from 400-500 nm. However, emissions at 450 nm were recorded. Fig
obtained from SigmaAldrich website.
https://www.sigmaaldrich.com/catalog/product/sial/08440?lang=en®ion=US
65
For assays conducted with the recombinant protein, a minimum of 700 nM enzyme was used for assaying. The enzyme and substrate plus/ minus inhibitor were mixed in a cuvette in enzyme assay buffer (25 mM HEPES, 1 mM EDTA,
25 mM KCl, 10% Sucrose, 1 mM DTT, pH 7.4) and the total reaction volume was
100 µl as shown in Table 2.3.
Table 2.3 The reaction vessel components for the caspase-1 activity assay. Final
concentration of active enzyme is 1.8 µg (30 nM) and substrate (30µM). As for
recombinant enzyme, a minimum of 700 nM was required to obtain activity. Enzyme
Assay Buffer: (25 mM HEPES, 1 mM EDTA, 25 mM KCl, 10% Sucrose, 1 mM DTT, pH
7.4)
The samples were covered and kept at room temperature in between measurements. Fluorescence emission measurements were done in a quartz cuvette. Emission scans were conducted on a Cary Eclipse fluorometer using
350 nm excitation and emission scan from 400-500 nm, 5/5 slits, 600 V. To observe change in fluorescence over time, emission intensities were recorded at
450 nm. Time-course kinetic measurements were also done by also monitoring
450 nm emission.
66
2.16. Tobacco etch virus (TEV) cleavage reactions
To cleave the MBP and SUMO fusion tags from the recombinant proteins,
AcTEV Protease Kit (Invitrogen Cat # 12575-015) was used. Protein samples were dialyzed overnight using slide-a-lyzer cassettes (ThermoScientific, Prod#
66380, 10 000 MWCO) in Tris buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA) to get rid of the high amount of imidazole. To the reaction tube containing assay buffer, 20 µg of fusion protein was mixed with 1mM DTT, 10 U enzyme and the rest of the volume with water to a total of 150 µL. In some instances, the amount of fusion protein and/or enzyme was scaled up to 10x to maximize cleavage. The reaction tube was incubated at 30 oC for 4-6 hours and aliquots were collected at various time points. The reaction was quenched with SDS sample buffer containing 5% β-mercaptoethanol. The aliquots were stored at -20 oC for future use. Samples were then heated for 5 mins at 95 oC and allowed to cool then proceeded with SDS-PAGE and western blotting. Further analysis of the cleaved samples was done by affinity purification of the reaction vessel contents. The flow through (FT) and the eluted fractions were collected separately then concentrated 10x by Microcon centrifugal filter devices (various MWCO) for dot blot analysis.
2.17. Interleukin 1-beta (IL-1β) cleavage assays
To test the potency of our recombinant pro-caspase-1, commercial human pro-IL-1β (Sino Biological Cat # 10139-H07E) was used as a substrate. To
67 optimize the assay, Human Caspase-1 (Active) Recombinant Protein (Millipore
Cat # CC126) was used as a positive control for caspase activity. Briefly, the reaction was done in a microcentrifuge tube by combining 1.8 μg caspase-1 enzyme with 500 ng/mL IL-1β protein in caspase-assay buffer cocktail (25 mM
HEPES, 1 mM EDTA, 25 mM KCl, 10 % Sucrose, 1 mM DTT, pH 7.4) to a total of 100 µL. A time-course reaction was done for 4 hours at 25 oC then the reaction was quenched by adding SDS sample buffer containing 5% β-mercaptoethanol.
The aliquots were stored at -20 oC until the cleavage assay was complete.
Samples were then heated for 5 mins at 95 oC and allowed to cool then proceeded with SDS-PAGE and western blotting. Detection was done with human IL-1β/ IL-1F2 polyclonal goat antibody (R&D Systems).
2.18. Aβ protofibril preparation and purification
Lyophilized Aβ-42 was purchased from ERI Amyloid Laboratory, LLC
(Oxford, CT) (formerly W. M. Keck Biotechnology Resource Laboratory) and stored at −20 °C. Aβ peptide was resuspended in 100% hexafluoroisopropanol
(HFIP) (Sigma-Aldrich, St. Louis, MO) to yield a 1 mM Aβ solution then aliquoted in sterile microcentrifuge tubes. The tubes were left uncovered at room temperature overnight in a fume hood to allow the HFIP to evaporate completely.
The following day, the aliquots were capped and stored in desiccant at −20 °C.
Aβ−42 protofibrils were prepared as described previously [19]. Lyophilized Aβ aliquot (1 mg) was reconstituted in 100 µL of 50 mM NaOH followed by 900 µL prefiltered (0.22 μm filters) artificial cerebrospinal fluid [aCSF, 15 mM NaHCO3, 1
68 mM Na2HPO4, 130 mM NaCl, and 3 mM KCl, pH 7.8] to yield 250 μM Aβ solution. The mixture was incubated at room temperature for 15-30 minutes then centrifuged at 17,000 x g for 10 min. The supernatant was loaded onto the column and eluted in aCSF (pH 7.8). Fractions (0.5 mL) were collected, and concentrations were determined from the SEC UV absorbance trace.
2.19. Aβ- NLRP3 inflammasome interactions
To study the interactions between Aβ and the inflammasome proteins we used two approaches. The first approach utilized recombinant inflammasome proteins for in vitro binding assays with isolated Aβ protofibrils. For these assays
SEC-purified fractions of recombinant inflammasome proteins were used.
Equimolar amounts (2 µM final concentration) of Aβ PF, ASC, FL NLRP3 or
Pyrin, and FL pro-caspase-1 were combined in PBS buffer and incubated for 1-2 hours at 4 oC and/ or room temperature to allow binding. This assay was designed based on previous studies [139]. In these experiments, the assumption is that the expected binding events between the proteins will occur and that the concentrations used are optimum. After incubation, immunoprecipitation was done with anti-ASC, anti-NLRP3, or anti-caspase-1 antibodies followed by SDS-
PAGE and immunoblotting with antibodies specific to each protein of interest.
An alternative approach involved isolating the endogenous inflammasome from stimulated immune cells. For these studies, THP-1 monocytes/ differentiated macrophages as well as microglia (both primary murine and immortalized BV-2) were utilized. Briefly, cells were stimulated with either Aβ PF
69 or a combination of LPS/nigericin then pull-down assays were used to analyze the proteins present in the lysates as explained in detail in sections below.
2.20. Cell culture
2.20.1. THP-1 monocytes
THP-1 monocytes purchased from American Type Culture Collection
(ATCC, Manassas, VA) were grown in Roswell Park Memorial Institute (RPMI)
1640 (HyClone, Logan, UT) containing 2 mM L-glutamine, 25 mM HEPES, 1.5 g/L sodium bicarbonate, supplemented with 10% FBS (HyClone), 50 U/mL penicillin, 50 µg/mL streptomycin (HyClone), and 50 µM β-mercapto-ethanol.
o 2 Cells were maintained at 37 C and 5% CO2 in 75 cm (10 mL volume) and 150 cm2 (20 mL volume) Corning culture flasks with canted necks. The cells were passaged and replenished with fresh growth medium twice a week.
2.20.2. Primary microglia cells
Primary microglia cells were isolated from wild-type (WT) C57BL/6 mice
(Harlan Laboratories). Microglia were isolated as previously described [19, 294] from 3–4 day old mouse pups. Briefly, brains were isolated under sterile conditions, minced, and trypsinized. The brain tissue was then resuspended in complete Dulbecco’s modified eagle’s medium (DMEM, HyClone) growth medium containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 0.25 µg/mL amphotericin-B, OPI medium supplement (oxaloacetate, pyruvate, insulin) (Sigma-Aldrich), and 0.5 ng/mL
70 recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-
CSF) (Life Technologies). The cell suspension was filtered, centrifuged, resuspended in complete medium and seeded into Corning 150 cm2 culture flasks with canted neck. Mixed glial cultures were maintained at 37 °C in 5% CO2 until confluent (1–2 weeks). For experiments, microglia were detached from the adherent astrocyte layer by shaking the flask at 250 rpm for 6 hours or overnight at 37 °C in 5% CO2 and collecting the medium.
2.20.3. BV-2 microglia
Immortalized BV-2 murine microglia were cultured as previously described
[19] in DMEM [High glucose (4.5 g/L) + Sodium pyruvate (Hyclone) containing 50
U/mL penicillin, 50 µg/mL streptomycin, 50 µM β-mercaptoethanol, and 5% fetal bovine serum (FBS), Hyclone]. Cell-stimulation experiments were performed using serum-free medium or growth medium containing 2% FBS. Cells were grown in 75 cm2 culture flasks and passaged once a week when they reached confluency. The growth medium was removed using a sterile pasteur pipette by vacuum suction. The flask was rinsed with sterile PBS carefully (without disturbing the cells) by gentle swirling 2-3 times. After suctioning the PBS, 1 mL of 0.25% trypsin (HyClone) pre-warmed at room temp was added to the flask.
o Incubation of the flask was done at 37 C in 5% CO2 for 10 mins to allow cell dislodging. Afterwards, 9 mL of growth medium was then added to inactivate trypsin and cells were spun at 400g for 10 mins. The pellet was resuspended in
10 mL growth medium (1:10 dilution normally done but 1:6 dilution could be done
71 if the cells had to reach confluency faster) and transferred to a 75 cm2 culture flask for further growth.
2.21. Cell stimulation assays
2.21.1. Suspension cell lines
For cell stimulation experiments, THP-1 monocytes were centrifuged at
1,000g for 10 mins and the cell pellet was resuspended in fresh warmed assay medium containing 2% serum. Cell concentrations of 1x106 cells/ml were used
(300 µL cells in 48-well plates, 500 µL in 24-well plates, 2-3 mL in 6-well plates).
Cells were primed with 100 ng/mL ultrapure LPS (InvivoGen, E.coli K12, Cat # tlrl-peklps) for 6 hours then stimulated with 5-10 µM nigericin (Sigma SML 1779,
o Lot# 108M 4196V) for 30 minutes at 37 C in 5% CO2. Five experimental conditions were tested as shown in Fig 2.2 below.
Fig 2.2 Six-well plate experimental set-up for the LPS Nigericin cell
treatment using differentiated THP-1 macrophages.
72
Alternatively, stimulation was done with 5-10 µM Aβ protofibrils for 6 hours. After incubation, the entire medium from each well was carefully removed and kept in a labelled Eppendorf tube. Centrifugation was done at 1,000g for 10 mins to remove cell debris and the supernatants were stored at -20 oC for ELISA analysis. The remaining pellets were rinsed once with 50-100 µL sterile PBS.
Lysis was done immediately by resuspending and pipet mixing each pellet in 100
µL radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 20 mM
Tris, 1M EDTA, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS) containing 40x dilution of protease inhibitor cocktail. The tubes were incubated for 15 min at room temp then the lysate went through a second spin to remove debris (if any).
Samples were stored in the fridge at -20 oC until further analysis.
2.21.2. Adherent cells lines
For adherent cells (PMA-differentiated THP-1 macrophages, primary and immortalized BV-2 microglia), cell stimulation assays were done in a slightly different manner. THP-1 monocytes were prepared as noted in above section. To differentiate the monocytes into macrophages, 100 ng/mL phorbol 12-myristate
13-acetate (PMA) was added to the growth medium used to resuspend the cell pellet. Cell concentrations of 1x106 cells/mL were used and cells were added to wells (300 µL in 48-well plates, 500 µL in 24-well plates, 2-3 mL in 6-well plates) and allowed to adhere overnight. Cell adherence to the bottom of the plates was verified by light microscopy. The medium containing PMA was replaced with
73 fresh, warmed growth medium and cells were left to incubate overnight at 37 oC
5 in 5 % CO2. As for the primary and BV2 microglia cells, 5x10 cells/ml were plated overnight in growth medium. On the day of cell treatment, old medium was removed from all wells which were then rinsed with warmed assay medium. Cells were stimulated with LPS/ nigericin or Aβ PF as described in section above. After treatment, the medium samples were collected from each well, kept in labeled
Eppendorf tubes then stored at -20 oC for TNFα ELISA analysis. The wells were rinsed twice with sterile PBS then lysed with RIPA buffer containing protease inhibitor cocktail. Swirling of the plate and pipetting was done several times during the lysis step followed by 15 min incubation step at room temp. Lysates were collected and stored at -20 oC for further analysis.
2.22. Co-immunoprecipitation (Co-IP) assays
For the immunoprecipitation experiments, magnetic Protein G Dynabeads
(Invitrogen, ThermoFisher, Cat # 10003D) were vortexed for about 30 seconds then 50 µL (1.5 mg) transferred to each microcentrifuge tube. The tubes were placed on a dynamagnet (DynaMag-2, Invitrogen, Cat#12321D) to separate the beads from the solution. The supernatants were removed and discarded. About
2-5 µg of antibodies (ASC-B3, NLRP3 8N8E9,) were diluted in wash buffer (1X
PBS with 0.02% Tween 20). To each tube, 200 µL of antibody solution was added then the tubes were incubated for 0.5 -1 hour at room temperature on a rotating shaker. Afterwards, the tubes were placed on a magnet and supernatants discarded. The beads were washed by gentle pipetting in wash
74 buffer then discarding the wash. The antibodies were cross-linked to the beads as outlined in the BS3 cross-linking section. After crosslinking, the washed beads were incubated with 100-500 µL cell lysates at room temperature for 30 mins to an hour with shaking. The tubes were then placed on a magnet and the supernatants were transferred to clean tubes for later analysis. The beads were washed 3x, resuspended in 100 μL wash buffer then the bead suspension was transferred to a clean tube to avoid co-elution of proteins bound to the tube wall.
After removing and discarding the supernatants, elution was done by adding 20
μL of elution buffer (50 mM Glycine pH 2.8) and 10 μL of pre-mixed Laemmli
Sample Buffer/ 5% BME followed by heating for 5 mins at 95 oC. The tubes were left to cool for a few mins, placed on a magnet, and the supernatants were analyzed by SDS-PAGE or stored at -20 oC for later use. Immunoblotting was done with anti-ASC (B-3, Santa Cruz, 1:1000 dilution), anti-NLRP3 (8N8E9,
ThermoFisher, 1:250 dilution), and anticaspase-1 (PA1440, Boster Biological,
1:1000) antibodies. Anti-Aβ antibodies used were affinity-purified apAbSL 79-2
(PF-specific antibody generated in the Nichols Lab) and Ab5 (Mayo clinic).
Loading control probing was done with GapDH (Invitrogen, Cat# MA5-15738) and α-tubulin (Thermofisher, Cat# PA5-39836) antibodies.
2.23. BS3 cross-linking
During immunoprecipitation, cross-linking was done with BS3, a water- soluble homobifunctional N-hydroxysuccinimide ester (NHS ester) amine crosslinker (ThermoScientific, Lot # UE281932) to prevent co-elution of the
75 antibody. After incubating the beads with the antibody (according to the Co-IP protocol), the beads were washed twice with 200 μL fresh Conjugation Buffer (20 mM Sodium Phosphate buffer, 0.15 M NaCl, pH 8.0). The beads were recovered on the magnet and the supernatants were discarded. Right before use, 100 mM
BS3 was prepared in Conjugation Buffer then this stock solution was further diluted to 5 mM with Conjugation Buffer (1:20). For each sample tube, 250 μL of
5 mM BS3 was used. Resuspension of the beads was done in 250 μL of 5 mM
BS3 followed by incubation at room temperature for 30 min with tilting/rotation.
The cross-linking reaction was quenched by adding 12.5 μL of Quenching Buffer
(1M Tris, pH 7.5) then further incubated at room temperature for 15 min with tilting/rotation. The cross-linked beads were washed three times with 200 μL of
PBST (1X PBS with 0.02% Tween 20) then recovered on the magnet.
Supernatants were discarded followed by Co-IP and antigen elution.
2.24. Trichloroacetic acid (TCA) precipitation
TCA-acetone precipitation was used to extract proteins from lysates of cells stimulated in serum-free medium. TCA stock (T0699, Sigma, Lot #
SLBX1977) was diluted in sterile milliQ water to make a 20% solution. Protein samples were then mixed in a 1:1 ratio with diluted TCA then incubated on ice for an hour. The samples were later spun in a microcentrifuge at 15,000g for 10 min at 4 oC. The supernatants were removed leaving protein pellets intact. The pellets were washed with slightly greater volume (~100 μL) ice-cold acetone by swooshing the tube gently without pipet mixing. The samples were spun again for
76
5 mins in a 4 oC microcentrifuge at 15,000g. The acetone was gently removed and discarded. A total of 3 acetone washes were done with centrifugations in between. The pellets were then allowed to air dry at room temp to drive off acetone. When pellets were completely dry, they were re-suspended in SDS sample buffer with 5% BME and slightly sonicated to dissolve the pellets completely. To the samples that still had residual TCA (sample buffer turned yellow), neutralization was done with 1M Tris buffer which was added dropwise until samples turned blue again. Alternatively, re-suspension was done in 4%
SDS only for subsequent BCA-assay analysis. The samples were incubated for 5 mins at room temp, heated at 95 oC for 5 mins, and then allowed to cool down.
20 μL of each sample was loaded onto a suitable gel for electrophoresis, transferred to PVDF membrane, and later analyzed by immunoblotting.
2.25. Cytokine enzyme-linked immunosorbent assay (ELISA)
Supernatants were analyzed by TNFα ELISA to determine amounts of cytokines secreted. Briefly, 100 µL of 2 µg/mL capture antibodies from R& D
Systems (anti-human monoclonal MAB610, anti-mouse polyclonal AF-410-NA) were plated into 96-well plates overnight at room temperature followed by a wash
(PBS with 0.05% Tween 20) the next day. To each well, 300 μL Blocking Buffer
(PBS containing 1% BSA, 5% sucrose, 0.05% NaN3) was added then plates were incubated for at least 1 h at room temperature. Afterwards, the samples were washed 3x. Successive treatments (with 3 washes in between steps) were done with 50 μL samples or standards for 2 h, 100 μL of biotinylated detection
77 antibodies [(R & D systems, BAF210,polyclonal anti-human antibody, 0.1 µg/mL),
(BAF410, 0.2 µg/mL)] diluted in detection antibody diluent (20 mM Tris with 150 mM NaCl and 0.1% BSA) for 2 h, 100 μL of streptavidin-horseradish peroxidase
(HRP) (R&D Systems) diluted 200 fold into conjugate diluent (PBS with 1% BSA) for 20 min, and 100 μL of equal volumes of 3,3’,5,5’ tetramethylbenzidine (TMB) and hydrogen peroxide (KPL, Gaithersburg, MD) for 20-30 min. The reaction was stopped by the adding 50 µL of 1% H2SO4 solution. The optical density of each sample was analyzed at 450 nm with a reference reading at 630 nm using a
SpectraMax 340 absorbance plate reader (Molecular Devices, Union City, CA). A standard curve was constructed by serial dilution of a TNFR standard from 2000 to 15 pg/mL. TNFα concentrations in the samples were calculated from the standard curve. When necessary, samples were diluted 3 to 5-fold to fall within the standard curve.
2.26. Aβ-ASC aggregation assays
To conduct the aggregation assays, freshly purified Aβ monomers and
GST-ASC proteins were used (GST-ASC PYD, GST-ASC PYD K21A, GST-ASC
CARD). GST-ASC proteins were purified with both Superdex 75 and 200 columns using PBS buffer. SEC-purified void and included peak fractions were diluted 1:10 or 1:100 (ASC:Aβ) depending on the amounts of protein available. aCSF buffer (1 mM Na2HPO4 , 1.5 mM NaHCO3, 3 mM KCl, 130 mM NaCl, pH
7.8) was used for the aggregation assays. Each reaction tube contained Aβ monomers and ThT, both at 10 uM final concentrations, ASC protein, and the
78 rest of the volume was made up to 600 ul with aCSF. Three separate microcentrifuge tubes were used, one for each ASC protein. After obtaining fluorescence value for 0h time point, the sample from each tube was split into three tubes (n=3) with 200 ul of solution in each tube. The tubes were incubated quiescently at 37°C as shown in Fig 2.3. Aβ-ASC solutions were assessed by thioflavin T (ThT) fluorescence at different time points. Fluorescence emission scans (460-520 nm) were acquired on a Cary Eclipse fluorescence spectrophotometer using excitation wavelength of 450 nm. Emission scans were integrated from 470-500 nm to provide a numerical value of ThT relative fluorescence values.
Fig 2.3 Aβ-ASC aggregation assays. Briefly, mixtures SEC-purified Aβ monomers
and GST-ASC proteins plus ThT in aCSF (pH 7.8) buffer were incubated
quiescently at 37°C. ThT fluorescence was assessed at various time points. Using
excitation wavelength of 450 nm, fluorescence emission scans (460-520 nm) were
acquired on a Cary Eclipse fluorescence spectrophotometer and integrated from
470-500 nm to provide a numerical value of ThT relative fluorescence values.
Schematic by Dr. Nichols.
79
CHAPTER 3: DEVELOPMENT OF A MAMMALIAN EXPICHO CELL SYSTEM
FOR EXPRESSION OF NLRP3 INFLAMMASOME PROTEINS
The ExpiCHO-S system was chosen for the transient expression of
NLRP3 inflammasome proteins because of these specific features: as a mammalian cell line it allows desirable post-translational modifications; these modified CHO cells grow in suspension thus allowing for easier harvesting of proteins from the expression medium; the cells grow to extremely high densities while maintaining good cell viability therefore maximizing yields.
The classic CHO cells are great for transient expression but are not as efficient as HEK293 cell line which is the most common system for transient protein expression. The ExpiCHO-S cells, however, are designed for stable expression of proteins with extremely high efficiency. The protein yields obtained from ExpiCHO-S expression systems surpass by far the amounts obtained when using standard CHO, HEK 293T, and Expi293F cell lines [295, 296]. In addition, inclusion of a hexa-histidine tag allowed for the selective purification of the desired proteins by Ni2+ affinity chromatography.
3.1. The pcDNA3.4-TOPO mammalian vector is ideal for recombinant
expression of inflammasome proteins
The plasmid vector chosen for these studies was pcDNA3.4-TOPO, which enables constitutive expression of exceptionally high levels of recombinant protein in mammalian ExpiCHO-S cells. As shown in Fig 3.1 top panel, the vector contains a topoisomerase cloning site, the full-length human cytomegalovirus
80
(CMV) immediate-early promoter/enhancer, and both ampicillin and neomycin resistance genes for subcloning. The synthesized gene that was inserted into the plasmids contained a Kozak sequence, hexa-histidine (His) tag for purification purposes, and either an enterokinase (EK) or tobacco etch virus (TEV) cleavage site for removal of the His tag if desired.
In some of the insert sequences, an Ig κ-chain secretion sequence was included [297, 298] to enable secretion of the expressed proteins into the medium as shown in the Fig 3.1 bottom panel A. Otherwise, the proteins were expressed intracellularly (Fig 3.1 panels B and C). DNA sequences for the protein inserts (full-length and subunits of human NLRP3, ASC, and caspase-1 proteins) were optimized using the GeneArt Gene Synthesis platform
(ThermoFisher/Life Technologies). Synthesis and cloning of the designed DNA into the pcDNA3.4 TOPO vector were also done commercially (Thermo
Fisher/Life Technologies).
81
Fig 3.1 Top: Plasmid vector with human NLRP3 gene inserted. Plasmid synthesis, amplification, and sequencing was done by Life Technologies Bottom: The design of the genes showing the Kozak initiation sequence, hexa-histidine tag, and a cleavage site in all constructs. A secretion tag (Ig κ-chain) and an enterokinase (EK) cleavage site were included in the initial gene design Panel A. Modifications were done to remove the secretion sequence and a tobacco-etch virus (TEV) cleavage site was introduced Panel B. MBP and/or SUMO fusion tags were later introduced as shown in
Panel C. Plasmid and schematic designed by Dr. Nichols
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Small ubiquitin-like modifier (SUMO) and maltose-binding protein (MBP) fusion tags were later added to the gene design to enhance solubility of the recombinant proteins. The SUMO protein is relatively small ~11 kD. Yeast SUMO with R64T/R71E mutations was used and it works in mammalian cells. As shown previously, the mutations prevent cleavage during expression [288]. On the other hand, MBP is a relatively large protein ~42 kD. Initially, the mammalianized version of bacterial MBP (mMBP) which contained 10 mutated residues (D82A,
K83A, E172A, N173A, K239A, A312V, I317V, E359A, K362A, D363A) [290] was utilized. These 10 mutations were expected to enhance expression and yields of the MBP-fusion proteins but further literature search revealed that only 2 mutations (A312V, I317V) were necessary for purification and expression. The other 8 mutations assisted in protein crystallization by reducing flexibility of the linker thereby not necessary for our studies. Therefore, later constructs (MBP- p20, MBP-p10) contained only the essential 2 mutations. A tobacco-etch virus
(TEV) protease site was also introduced to allow for removal of the fusion tags from the protein of interest.
3.2. ExpiCHO-S cells maintain great cell viability at high densities and are
ideal for recombinant expression of proteins in suspension
The ExpiCHO-S mammalian cell line was used for the recombinant expression of NLRP3 inflammasome proteins and was chosen over bacterial cell expression systems to ensure that all relevant post-translational modifications
83
(PTMs) were maintained. As discussed in detail in Chapter 1, PTMs play an essential part in the regulation of the NLRP3 inflammasome hence the need to employ a mammalian cell line for our studies. ExpiCHO-S cells have been mostly used in the transient and stable production of secreted antibodies and biotherapeutics (compared to the generic HEK293 cell line), are designed to grow in suspension and can accommodate very high cell concentrations (>107 cells/ml) hence they seemed to be a great model system for our studies [295,
296].
The overall flow of ExpiCHO-S experimental cell work is shown in Fig 3.2 panel A and an image of the cells is shown in panel B. ExpiCHO-S cells were cultured in serum-free EM and subcultured 2-3 times before transfection in order to optimize cell growth and viability. Cell clumping was observed at high densities, but this did not affect the growth of the cells, thus confirming the adaptation of the cell line to a high-density environment. Following transfection as described in the Methods, ExpiFectamine Enhancer and ExpiCHO Feed were added to the flasks on day 1 post-transection to improve transfection efficiency and ensure maximal protein yields.
84
Fig 3.2 ExpiCHO-S cell culture, transfection, and protein expression. Panel A.
Initiation of the process begins with culturing of the suspension ExpiCHO-S cells in sterile Erlenmeyer shake flasks under continuous rotation at 37 °C in 8% CO2 and passaging several times before transfection. Monitoring and confirmation of high cell viability prior to, and during, expression is an important parameter. Application of
ThermoFisher Feed and Enhancer on day 1 post-transfection enhances the protein expression process allowing acquisition of ExpiCHO-S-expressed proteins typically within 2-13 days post-transfection. Schematic by Dr. Nichols. Panel B. Image of
ExpiCHO-S cells taken during routine cell-counting.
85
3.3. ExpiCHO-S cells maintained good cell viability during the expression
period and proteins were detected within 4 days post-transfection
Good cell viability is an important parameter for high-yield protein expression in the ExpiCHO-S cell line. Viability was determined over the course of the expression and although there was a modest decrease in viability, it routinely stayed above 70% for most of the plasmids (Figure 3.3 panel A). Small variations in viability were observed depending on the types of plasmids used
(data not shown).
The efficiency of transfection and expression was evaluated using a positive control antibody-expressing pcDNA vector and assessing the production of secreted rabbit IgG antibody. A time course of rabbit IgG protein expression and secretion was obtained by taking aliquots from the medium of the expression flasks on different days and performing an immuno-dot blot analysis on the samples. The findings demonstrated that expression and secretion of rabbit IgG protein occurred as early as day 4 post-transfection (Fig 3.3B, top panel). Further analysis of the control rabbit IgG control expression after harvest indicated that the protein was secreted and not present in the non-transfected medium, lysate
(L) or inclusion body (IB) fractions (Fig 3.3B, bottom panel).
86
B
Fig 3.3. Monitoring ExpiCHO-S cell viability and secretion of proteins into the medium.
ExpiCHO-S cells maintained good viability during the course of the protein expression. Panel A. Time-dependent measurements of cell viability during the expression period after transfection with control rabbit IgG (circles) or ASC plasmids
(triangles). Cell viability was generally above 70% until the day of harvest. Fig by Dr.
Nichols. Panel B. A dot blot analysis was used to detect the expression and secretion of rabbit IgG in ExpiCHO-S medium samples. The time-dependence of rabbit IgG expression is shown in the upper panel. The lower panel shows the absence of rabbit
IgG in untransfected ExpiCHO-S cells and the presence of rabbit IgG in the transfected ExpiCHO-S medium, but not the lysate (L) or inclusion bodies (IB) fractions.
87
3.4. Incorporating detergents, reducing agents, and protein stabilizers in
the buffers used during harvesting and lysate preparation maximized
protein yields
Expression of inflammasome proteins was done in a similar manner to that of the antibody and the proteins were typically harvested 2-13 days post- transfection. After going through published protocols from various sources in the literature [10, 16, 231, 285, 286, 299], various buffers and harvesting protocols were employed. Modifications were done over the course of time in order to maximize the protein yields as shown in Table 3.1.
The main focus was on the cell lysate preparation, wash steps, solubilization of inclusion bodies, and refolding. Three ExpiCHO-S fractions
(medium, lysate, and inclusion bodies) were collected from each protein expression flask as described in the Methods. Medium fractions (M) consisted of supernatants after low-speed centrifugation of the ExpiCHO-S cells. In-between wash steps with phosphate or HEPES buffers (50 mM HEPES pH 8.0, 300 mM
NaCl, 1 M GuHCl, 5 mM DTT, 1% Triton X-100) were included to get rid of contaminants and cell debris to ensure getting proteins of the highest purity. The ensuing cell pellets were mechanically disrupted with a Dounce homogenizer in the presence of protease inhibitors, centrifuged at 20,000g for 20 min, and the supernatant collected as the lysate (L). Initially, the most basic Lysis Buffer was used (150mM NaCl, 20mM Tris pH 7.4) but over time various additives were
88 added to maximize protein extraction as shown in Table 3.1. The pellet remaining after the lysate spin was solubilized in 8 M GuHCl, sonicated for 5 min, and the solubilized supernatant was obtained as the inclusion body (IB) fraction. To improve the IB solubilization process, later preps utilized modified Solubilizing
Buffer (6 M GuHCl, 25 mM Tris, 5mM DTT pH 7.5) followed by sonication. The samples were incubated overnight at 4 oC with rotation and subjected to centrifugation again (22,000g, for 20 min) to remove debris. Solubilized IB were added dropwise to refolding buffer followed by overnight refolding (50 mM
HEPES pH 8.0, 100 mM NaCl, 10% sucrose, 1 or 5 mM DTT) at 4 oC with rotation. After another centrifugation step, the supernatant was used as the IB.
Table 3.1 The various buffer protocols and modifications introduced during the
harvesting, lysis, solubilization, refolding, and purification processes to maximize
protein yields.
89
3.5. The three inflammasome proteins were successfully co-expressed in
ExpiCHO-S cells
Some groups have successfully co-expressed NLRP3, ASC, and caspase-1 in HEK cells for microscopy assays (to study ASC speck formation) and co-immunoprecipitation studies (to study binding between the proteins) thus indicating successful inflammasome assembly [153, 235, 236, 300, 301]. Co- transfection of Cos7 cells with caspase-1 and pro-IL-1β resulted in secretion of mature IL-1β [244].
The three inflammasome proteins (FL NLRP3, pro-caspase-1, ASC) were co-expressed in ExpiCHO-S cells. The lysates were affinity-purified followed by
SDS-PAGE and immunoblotting with specific antibodies against each of the proteins as shown in Fig 3.4. All three proteins were successfully co-expressed according to the input lanes 4-6 (affinity-purified peak fractions 10-12 respectively) when probed with antibodies against ASC (Panel A), caspase-1
(Panel B), and NLRP3 (Panel C). Interestingly, all three proteins appeared as monomers under denaturing conditions but tend to form SDS-resistant aggregates when expressed separately (shown in Chapter 4).
90
A. 250
130 100 70 55
35
25
15
10
B. 250 130
100 70 55
35
25
15
10
C. 250 130
100 70 55
35
25
15
10
91
Fig 3.4 Immunoblots showed that three inflammasome proteins were successfully co-
expressed. Lysates of co-expressed proteins were affinity-purified and used to
perform IP assays. IP was done with 2 μg ASC-B3 as explained in detail in the
Methods section followed by SDS-PAGE, transfer, and immunoblotting. Immunoblots
were probed with (Panel A) ASC (22 kD), (Panel B) caspase-1 (45 kD), and (Panel
C) NLRP3 (118 kD) antibodies. Lane 2 contained HL-60 positive control lysate. Lanes
3, 7, 9 were empty. Lanes 4- 6 represent affinity-purified co-expressed proteins.
Sample in lane 5 was used as the input for IP. Lane 8 represents the unbound
proteins (sup) and Lane 10 is the IP proteins.
After successfully verifying the co-expression of all three inflammasome proteins, the next step was to do co-immunoprecipitation (Co-IP) assays to test whether all three proteins existed in a complex. Co-IP experiments were conducted with ASC B3 antibody. Affinity-purified fraction 11 (lane 5) was used for the Co-IP experiments. As shown in Fig 3.4 A-C, there were not any differences between the input (lane 5) and the unbound protein (lane 8) indicating that little to no protein was actually bound to the antibody crosslinked to the magnetic beads. Moreover, no proteins were detected in the IP lysates
(lane 10) thus further agreeing with the fact that the ASC protein did not bind to the antibody during the immunoprecipitation process. A possible explanation could be that ASC is in a complex with NLRP3 or caspase-1 (or both) and the binding epitope is obstructed thus preventing its binding to ASC B3.
92
3.6. Affinity purifications of His-tagged proteins yielded high amounts of
expressed proteins
3.6.1. Affinity purifications of NLRP3, ASC, and caspase-1 p10
Figure 3.5 shows the 17 protein constructs that I expressed successfully in
ExpiCHO-S cells. His-tagged proteins in all three fractions (M, L, and IB) were purified by Ni Sepharose 6 Fast Flow affinity chromatography.
93
Fig 3.5. Schematic showing constructs of all full-length and truncated proteins that
were successfully expressed in ExpiCHO-S mammalian cells. A total of 17 proteins
were expressed. The initial FL NLRP3 and ASC constructs had the Ig κ-chain
secretion sequence to allow secretion into the medium during expression. Later
constructs did not feature the secretion tag. GST, MBP, and SUMO fusion tags were
later introduced to improve solubility of proteins. Caspase-1 active site mutants
(C285A) were also expressed. Schematic prepared by Dr. Nichols.
Proteins were detected in-line with UV absorbance at 280 nm. Non-His- tagged proteins passed through the column as flow through in 20 mM Na2HPO4,
500 mM NaCl, 20 mM imidazole pH 7.4. His-tagged proteins were eluted with 20 mM Na2HPO4, 500 mM NaCl, 500 mM imidazole pH 7.4 and produced well- defined elution peaks. BCA protein assays were also conducted on the eluted fractions and used to corroborate the UV-absorbance-determined concentration.
Fig 3.6 shows one of the earliest affinity purifications of secreted ASC. In this case, we were still optimizing the affinity purification protocol using a linear gradient. Since the ASC contained a secretion sequence, presumably all the protein would be in the medium sample. UV280 nm trace indicated two ASC elution peaks that are labelled as peak 1 and peak 2. The peak fractions would be used for circular dichroism studies in Chapter 4.
94
2000 Affinity purification of ExpiCHO-S ASC medium 100
mAU % buffer B 1500 80 fractions
60
1000
mAU Y Axis 3 Axis Y peak 1 40
500 20
peak 2
0 0 0 20 40 60 80 100 120 140 160 elution volume, mL
Fig 3.6 Affinity purification of His-tagged secreted ASC medium. 41 mL of day 10 medium sample was loaded into a superloop and purified using a Ni SepharoseTM 6
Fast Flow column as described in the Methods. The sample was loaded on the column and washed in Buffer A (20 mM imidazole) followed by elution in Buffer B (500 mM imidazole) using a linear gradient (purple line). Protein concentration was monitored in-line by UV absorbance at 280 nm (A280). Elution of His-tagged proteins started at 92 mL as shown by the buffer B line. Two ASC peaks (1 and 2) were eluted and taken for further analysis. BCA assay determined the protein concentrations as: peak 1 (1.5 mg/mL), peak 2 (0.4 mg/mL). Fig by Dr. Nichols.
95
Figure 3.7A depicts the affinity purification of the ExpiCHO-S-expressed
NLRP3 IB fraction. This particular NLRP3 gene was designed with a secretion sequence, but much of the detectable 118 kD NLRP3 protein was found in the IB or L fractions, not the medium. The BCA-determined protein concentration from the collected fractions aligned well with the in-line UV-determined protein concentration. Quantitative integration of the elution peaks for IB and L fractions indicated that a much higher amount of NLRP3 protein was recovered in the IB fraction (6.4 mg) compared to the L fraction (1.4 mg) (Figure 3.7B). These calculated protein values were used to provide a distribution between the two fractions (L/IB = 0.23 for NLRP3). Since the L and IB fractions emanate from the same sample, the distribution is an appropriate and direct comparison.
96
Fig 3.7 Affinity purification of His-tagged NLRP3 protein. Panel A. The IB fraction of
ExpiCHO-S-expressed NLRP3 (day 8 harvest) was isolated and affinity-purified on a
Ni SepharoseTM 6 Fast Flow column as described in the Methods. The sample was
loaded on the column and washed in Buffer A (20 mM imidazole) followed by elution
in Buffer B (500 mM imidazole). Protein concentration was monitored in-line by UV
absorbance at 280 nm (A280) (solid line) and also determined by BCA assay (circles)
in the collected 2 mL fractions. Panel B. Integration of the affinity chromatography
elution peaks of the NLRP3 IB (panel A) and L fractions provided a distribution
between the two fractions. The integration value was obtained by multiplication of the
fraction volume and concentration for all of the eluted protein fractions. The L fraction
was obtained using Protocol #1 as described in the Methods. Fig by Dr. Nichols
Similar elution and distribution profiles were observed for the expression, recovery, and affinity purification of ASC (Figure 3.8A) and the caspase-1 p10 domain (Figure 3.9A). The data presented is for an intracellularly expressed form of ASC, in which the gene did not contain an added secretion sequence. The
L/IB protein ratio for non-secreted ASC was 0.18 (Figure 3.8B). Expression of a secreted form of ASC (gene contained secretion sequence) yielded a distribution
(L/IB = 0.30) similar to the non-secreted form (data not shown). No further inflammasome proteins, besides NLRP3 and ASC, were expressed with a secretion sequence in the ExpiCHO-S cell system as the leader sequence did not dramatically increase the yield of protein. A third protein component of the
NLRP3 inflammasome complex, the p10 subunit of caspase-1, was expressed,
97 purified (Figure 3.9A), and also recovered predominantly in the IB fraction with an
L/IB ratio of 0.14 (Figure 3.9B).
Figure 3.8 Affinity purification of His-tagged ASC protein lysate and IB. Panel A. The
L fraction of ExpiCHO-S-expressed ASC (day 11 harvest) was isolated and affinity-
purified on a Ni SepharoseTM 6 Fast Flow column as described in the Methods and in
Figure 3.7 legend. Protein concentration was monitored in-line by A280 (solid line)
during loading, washing, and elution of the sample and also determined by BCA
assay (circles) in the collected 2 mL fractions. Panel B. Integration of the affinity
chromatography elution peaks of the ASC L (Panel A) and IB fractions, as described
in Figure 3.7 legend, provided a distribution between the two fractions. Fig by Dr.
Nichols.
98
Figure 3.9 Affinity purification of His-tagged caspase-1 p10 subunit. Panel A. The IB fraction of ExpiCHO-S-expressed caspase-1 p10 (day 8 harvest) was isolated and affinity-purified on a Ni SepharoseTM 6 Fast Flow column as described in the Methods and in Figure 3 legend. Protein concentration was monitored in-line by A280 (solid line) during loading, washing, and elution of the sample and also determined by BCA assay (circles) in the collected 2 mL fractions. Panel B. Integration of the affinity chromatography elution peaks of the p10 IB (panel A) and L fractions, as described in
Figure 3.7 legend, provided a distribution between the two fractions. Fig by Dr.
Nichols.
99
3.6.2. Fusion proteins enhance solubility of expressed proteins
In order to shift the distribution of expressed inflammasome proteins to the lysate fraction, fusion proteins of ASC were prepared with both small-ubiquitin- related modifier (SUMO) and maltose-binding protein (MBP). These protein tags have been shown to increase expression levels and solubility of difficult proteins in mammalian cells [288, 290]. SUMO from Saccharomyces cerevisiae containing R64T/R71E mutations was added at the 5’ end of ASC with a tobacco etch virus (TEV) protease site engineered between the two proteins. This SUMO mutant has been shown to work effectively as a fusion protein in mammalian cell expression [288]. A similar construct of ASC was prepared containing a mammalianized version of bacterial MBP [290]. A more stringent lysis buffer was also employed that contained additional detergent components including 1%
Triton X-100, 1% deoxycholic acid, and 0.1% SDS. This more stringent lysis buffer shifted the L/IB ratio for ExpiCHO-S-expressed ASC to 0.46 (Figure
3.10A), compared to 0.18 for ASC-expressing ExpiCHO-S cells lysed without detergents (Figure 3.8B). Expression of ASC as a fusion protein with SUMO further enhanced the solubility with an L/IB ratio of 0.79 (Figure 3.10B). Finally, the MBP-ASC demonstrated the best L/IB ratio with a value of 2.85 indicating that more of the protein was found in the lysate fraction compared to the inclusion body fraction (Figure 3.10C). However, it was noted that even with the fusion tags, there was still some degree of protein insolubility. MBP-ASC contained the 10 mutated residues (D82A, K83A, E172A, N173A, K239A,
100
A312V, I317V, E359A, K362A, D363A) [290]. Further literature analysis confirmed that only 2 mutations (A312V, I317V) were necessary for purification and expression. The other mutations facilitate protein crystallization by decreasing protein flexibility hence were not vital for our research purposes.
Follow-up gene constructs for MBP-p20 and MBP-p10 fusion proteins included only the two necessary mutations.
10 10
A B 10 C
ASC, mg ASC, MBP-ASC, mg MBP-ASC, SUMO-ASC, mg 0 0 0 L IB L IB L IB expression fraction expression fraction expression fraction
Figure 3.10 Fusion tags enhance the solubility of the ExpiCHO-S-expressed ASC
protein. L and IB fractions were obtained and affinity-purified from ExpiCHO-S cells
expressing either ASC (Panel A), SUMO-ASC (Panel B), or MBP-ASC (Panel C).
Lysates were obtained using Protocol #2 as described in the Methods. Integration of
the L and IB affinity chromatography elution peaks, as described in the Figure 3.5
legend, for the L and IB fractions provided a distribution between the two fractions.
Figures by Dr. Nichols.
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In conclusion, recombinant expression of the NLRP3 inflammasome proteins in an ExpiCHO-S mammalian system was successful. The original gene design featured a secretion sequence to allow for easy harvesting of the proteins from the expression medium. However, this tag was not included in the later constructs because it did not offer any advantage. The ExpiCHO-S cells maintained good viability during the expression period (optimized expression time from 2-13 days). Dot blot experiments proved that maximum expression was reached as early as day 4 therefore expression times were amended to a maximum of 4 days. I was able to isolate proteins from both the lysates and inclusion bodies. Addition of detergents, reducing agents, and denaturants and implementing solubility fusion tags in the gene constructs greatly shifted the amount of proteins obtained in the lysate fraction compared to the inclusion bodies. High overall yields were obtained for all expressed proteins with slight variations based on the type of the plasmids used as revealed by the affinity purification curves. Thus, ExpiCHO-S cells presented a great expression system for the inflammasome proteins.
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CHAPTER 4: OLIGOMERIZATION STATE DETERMINATION OF
EXPICHO-EXPRESSED INFLAMMASOME PROTEINS
Although quite a few structural studies have been conducted with recombinant NLRP3 inflammasome proteins (as reviewed in Chapter 1), studies are yet to be done to determine the actual stoichiometry of each individual protein and the NLRP3 inflammasome complex. Knowing the stable forms of each protein and the actual components of the inflammasome as a whole can be useful in determining which mechanisms are involved in these processes thereby pinpointing the exact targets during drug discovery. Biochemical and biophysical assays were employed to verify successful expression of proteins and to also characterize the recombinant proteins.
Immunoblotting methods with specific antibodies can verify the presence of the proteins of interest. Size-exclusion chromatography separates species based on their molecular weights and also gives information on whether soluble aggregates are present in the protein sample. Light scattering techniques are used to determine relative sizes and structures of proteins while circular dichroism studies reveal how the proteins are folded. Additional structural information can be obtained by microscopy techniques. In these studies, both denaturing and non-denaturing solution conditions were used in the characterization studies to obtain information on the oligomerization states of the recombinant proteins. Since NLRP3 inflammasome proteins have an inherent propensity to aggregate coupled with the unwanted aggregation brought about by overexpression, various stringent protocols were used to break the preformed
103 aggregates in order to obtain more soluble, small molecular weight oligomeric proteins for analysis. To clarify on the terminology used in this text, oligomerization refers to the formation of small molecular weight species, eventually leading to aggregation (formation of high molecular weight species).
4.1. Analysis of the oligomerization state of the recombinant proteins
under denaturing conditions
4.1.1. Proteins mainly appeared as monomers under denaturing
conditions
Immunoblotting analysis of affinity-purified fractions was employed to identify each of the expressed proteins. Proteins were probed with specific antibodies and selected examples are shown in Figure 4.1 including NLRP3 (118 kD), ASC (22 kD), NLRP3 pyrin domain (12 kD), MBP-ASC monomers (61kD), and caspase-1 proteins [procaspase-1 (45 kD), CARD-p20 (33 kD), MBP-p10 (50 kD), p20 (19.8 kD)]. Under SDS denaturing conditions, the primary species observed was typically in monomeric form.
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Figure 4.1. Immunoblot analysis confirms identity of affinity-purified inflammasome proteins and most of them appeared as monomers under denaturing conditions. Affinity- purified inclusion bodies (IB) were used for these experiments except MBP-p10 where lysate sample was used. Protein samples (15 μL) from affinity elution fractions were separated by gel electrophoresis under denaturing conditions, transferred to PVDF membrane, and probed with antibodies specific for each protein. Selected proteins are shown in the figure at their expected monomeric Mw and include NLRP3 (118 kD),
NLRP3 pyrin domain(12 kD), ASC (22 kD), MBP-ASC (61 kD), procaspase-1 (45 kD), caspase-1 CARDp20 (33 kD), MBP-p10 (50 kD) and caspase-1 p20 (20 kD). Mw values were determined by running Mw markers in the first and last lane of every gel. Specific primary antibodies and full details of the SDS-PAGE/Western blot are described in the
Methods section.
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4.1.2. Expressed proteins showed some degree of oligomerization
A common feature and probably the greatest challenge during the overexpression of proteins is unwanted oligomerization. In this situation, it is most likely that protein concentration and the inherent properties of these proteins contributed to the aggregation process. The expressed proteins exhibited oligomerization even under denaturing conditions as shown in Fig 4.2.
Selected immunoblots show the presence of SDS-resistant oligomers in FL
NLRP3 (Panel A), ASC (Panel B), and NLRP3 pyrin domain (Panel C). It was interesting to note that FL caspase-1 oligomers were responsive to SDS treatment and appeared as single monomeric bands (Panel D).
A. B.
C. D.
106
Fig 4.2. Immunoblots of expressed inflammasome proteins show the presence of
SDS-resistant oligomers except FL caspase-1. Affinity-purified fractions were
analyzed by SDS-PAGE under denaturing conditions and immunoblot experiments
were conducted with antibodies specific for each protein. The peak fractions showed
higher degree of oligomerization (Panels A-C). The red arrows indicate the monomers
Panel A FL NLRP3 (118 kD), Panel B (ASC 22 kD), Panel C NLRP3 Pyrin (12 kD),
Panel D FL procaspase-1 (45 kD).
4.1.3. Inclusion of the secretion sequence resulted in differences in
oligomerization profiles upon analyzing medium samples
Presumably, the absence of a secretion sequence in the gene insert would prohibit the release of expressed proteins into the cell culture medium.
Surprisingly however, some of the non-secretion-designed proteins were recovered in affinity-purified medium samples. A significant amount of the secreted form of ASC was obtained from the medium after affinity purification which was expected. However, the non-secreted form of ASC was also recovered and purified from the medium indicating that ASC can be released from cells. Despite both secreted and non-secreted forms being present in the medium, there were differences in their oligomerization profiles (Figure 4.3).
SDS-PAGE and western blotting indicated that the secreted form of ASC had a substantial portion of monomeric protein under denaturing conditions (Figure
107
4.3A), while the non-secreted form was highly oligomerized and no monomeric
ASC was observed (Figure 4.3B).
A. B.
Fig 4.3 Expressed ASC proteins were present in the ExpiCHO-S medium whether or
not they contained a secretion sequence. Medium was collected after ExpiCHO-S
expression of ASC containing an Ig κ-chain secretion sequence (panel A, secreted
form) or not (panel B, non-secreted form). Immunoblot analysis was affinity-purified
fractions using anti-ASC B-3 primary antibody. Mw markers from the membrane were
aligned with the film images and are shown in lane 1 for both panels while HL60
positive control lysate was used in lane 2. As for lanes 3-5 respectively Panel A.
Fractions 10-12 were used Panel B. Fractions 9-11.
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4.1.4. Oligomerization profiles of NLRP3 lysates and inclusion bodies
are quite distinct
Another interesting observation was the significant difference in oligomerization patterns observed between NLRP3 pyrin lysates and inclusion bodies (Fig 4.4). The lysates showed the presence of a single oligomeric species
(trimers). On the other hand, the inclusion bodies had two distinct species around
12 kD (monomer) and 70 kD (possible hexamer). These differences reveal that the lysate sample contains a stable species whereas the multiple species in the inclusion bodies indicate aggregation which may be due to improper folding.
Fig 4.4. Oligomerization patterns of NLRP3 pyrin lysates and inclusion bodies are quite
distinct. Affinity purified NLRP3 fractions were subjected to SDS-PAGE was followed by
immunoblotting with anti-His antibody. Mw markers are in Lane 1, Lysates fractions 11 &
12 (lane 2-3), and IB fractions 15-17 (lanes 5-7 respectively). Lane 4 is empty.
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4.2. Analysis of inflammasome proteins oligomerization state in solution
Due to the oligomerization propensity of the NLRP3 inflammasome proteins, size-exclusion chromatography (SEC) was employed to separate any soluble protein from pre-formed aggregates. In order to optimize the best conditions to isolate stable monomeric species, various stringent conditions were exploited in an effort to break the pre-formed protein aggregates, and keep the proteins in stable solution states as shown in (Table 4.1) based on previous publications [14, 16, 146, 149, 231, 302, 303].
Table 4.1 Various sample treatment conditions employed in order to obtain stable,
soluble proteins during purification and storage afterwards. Various SEC buffers and
buffer additives were explored that included detergents (SDS, NP40, Tween-20),
reducing agents (2 mM DTT/ TCEP, 5% BME), denaturants (2M GuHCl) and
stabilizers (glycerol, sucrose).
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4.2.1. ASC showed some degree of oligomerization
Since ASC is extremely prone to aggregation, a Superose 6 column which has a high fractionation range (5-5000 kD) was used. The column was pre- blocked with 0.75 mg/mL BSA then 1.5 mg/mL of affinity-purified ASC medium
(peak 1 Fig 3.6) was centrifuged, loaded, and eluted in phosphate buffer. BSA monomer (66 kD) eluted at ~ 17 mL. Overlaying the BSA and ASC elution traces showed that ASC (monomer ~ 22 kD) eluted as various oligomeric species as shown in Fig 4.5.
Because I observed mostly monomeric proteins under denaturing SDS-
PAGE conditions, I predicted that I would possibly see monomers if I performed chromatography under similar conditions. In an effort to reverse the aggregation/ oligomerization process, the same sample was treated with SDS only or SDS/
BME then eluted in Tris/ Glycine buffer (same buffer used when conducting SDS-
PAGE). However, even under these stringent conditions, most of the protein still eluted in the oligomerized form indicating that the oligomers and/or aggregates are SDS-resistant (data not shown).
111
80
BSA 60 ASC
40 mAU
20
0
8 10 12 14 16 18 20 22 24 Volume (mL)
Fig 4.5 Recombinant ASC exists as oligomers of various molecular weights. Size exclusion column purification of ASC was done using a Superose 6 column
(fractionation range 5-5000 kD) pre-blocked with BSA. Sample used was 1.5 mg/mL
ASC (affinity peak #1 Fig 3.6). No denaturing conditions were used in sample treatment. 200 ul protein was centrifuged for 10 mins at 17,000g, loaded, running buffer PBS pH 7.2. The figure above shows an overlay of BSA and ASC elution curves.
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4.2.2. NLRP3 pyrin existed as a mixture of small and high molecular
weight species
For long term storage of proteins with little to no damage to their structural integrity, lyophilization is a commonly used method especially with recombinant proteins. Since the recombinant proteins were aggregating in solution, lyophilization was employed as an alternative storage strategy in order to maintain protein stability. Lyophilized affinity- purified NLRP3 pyrin domain was reconstituted, as described in the Methods, in 6 M GuHCl/2 mM DTT, heated, centrifuged, and the supernatant eluted on a Superdex 200 column pre-blocked with BSA. Multiple peaks were observed in the NLRP3 pyrin domain sample including a high Mw peak (8 mL) likely comprised of oligomerized pyrin protein
(Figure 4.6 Panel A). Based on the elution volume, it was determined that the peak at 17-18 mL (asterisk, Panel A) potentially represented monomeric pyrin.
The collected fractions at the 17-18 mL elution volume were reinjected on SEC and eluted a second time. The second elution resulted in one peak at the expected elution volume for pyrin with a Mw of 12,483 Da (Panel B). BSA (Mw
66,500 Da) was routinely eluted on Superdex 200 at an elution volume of ~14 mL. The separation in elution volumes of pyrin and BSA falls easily into the
Superdex 200 fractionation range of 10- 600 kD. Although I was able to get monomeric NLRP3 pyrin, the amounts were too low to perform functional assays.
Thus, these findings show that NLRP3 pyrin is unstable and prone to aggregation both in solution and lyophilized form.
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150 15 A B BSA NLRP3 pyrin 66.5 kD 12.5 kD
100 10 mAU * mAU 50 5
0 0 5 10 15 20 5 10 15 20 elution volume, mL elution volume, mL
Figure 4.6 Two rounds of SEC purification yielded monomeric NLRP3 pyrin domain.
A lyophilized sample of affinity-purified and dialyzed pyrin protein obtained from the IB
fraction of an ExpiCHO-S expression was reconstituted in PBS containing 6 M GuHCl
and 2 mM DTT. Panel A. The reconstituted pyrin was treated and SEC-purified on a
Superdex 200 column as described in the Methods. Elution in PBS was monitored in-
line by A280 (solid line) and the asterisk corresponds to monomeric pyrin. Panel B. A 1
mL pool of the two peak fractions (asterisk in panel A) was reinjected on Superdex
200 column and eluted a second time. The protein eluted at the expected volume for
pyrin monomer (12.5 kD). For comparison, BSA (66.5 kD) was also purified on
Superdex 200. Fig by Dr. Nichols.
Characterization studies on caspase-1 will be covered in detail in Chapter 5.
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4.2.3. Optimizing various SEC buffers using bovine serum albumin
(BSA)
Multi-angle light scattering (MALS) analysis in line with SEC was used to determine the molecular weights of the proteins. Protein aggregates, however, tend to scatter a lot of light thus distorting data collection. Various sample treatment strategies and buffer conditions listed in Table 4.1 were employed to maximize obtaining stable solution proteins that could be detected by MALS. For the buffer optimization experiments, BSA was eluted on a Superdex 75 column and the elution peaks are showed in Fig 4.7. BSA monomer elution peak was similar in PBS buffer +/- 2 mM DTT. However, there was a noticeable shift in the elution volume in both HEPES and Tris/ Glycine buffers.
1200
1000
800
600 mAU 400
200
0 4 6 8 10 12 14 16 Volume (mL) BSA, 2.5 mg, PBS/DTT BSA, 5 mg, PBS BSA, 5 mg, HEPES BSA, 5 mg, Tris/ Glycine
115
Fig 4.7 BSA has different elution profiles in different buffers. BSA (2.5 mg or 5 mg)
was loaded onto the Sup 75 column and eluted in various buffers in order to optimize
the best buffer conditions to obtain recombinant proteins in their soluble forms. This
information is important when predicting the sizes of eluted proteins based on their
elution volumes in different buffers. It is also important to note that the BSA recoveries
were not consistent.
4.2.4. A large portion of the ASC fusion proteins appeared in the
aggregated form
Since the elution volume of BSA monomer was somehow similar in PBS buffers +/- reducing agent (DTT) without a significant shift in the peak, follow-up experiments were conducted in PBS/ DTT buffer. Fig 4.8 shows the elution profiles of ASC fusion proteins [SUMO-ASC (33 kD), MBP-ASC (61 kD), ASC (22 kD)]. BSA monomer (66 kD) elutes around 10 mL and anything else bigger than
66 kD elutes before BSA. Ideally, the elution peaks of the fusion proteins would have appeared after the BSA elution peak had these proteins been in their monomeric forms. For example, I would have expected monomeric MBP-ASC to elute right around 10 mL based on its Mw. Likewise, SUMO-ASC dimer and intracellular ASC trimer are expected to elute right around the BSA monomer elution volume.
In this case however, a large portion of the proteins appeared as high molecular weights species and eluted in the void volume. The maximum separation threshold for a Sup 75 column is 70 kD and anything bigger than that
116 appears in the void volume as shown by the major elution peaks before 10 mL mark. Small molecular weight species were observed but at very low amounts.
Although the presence of solubility-enhancing fusion tags increased the amounts of proteins obtained in the lysate fraction, that did not improve the yields of monomers and small oligomers.
600 80
500 60 400
300 40 mAU 200 20 100
0 0 4 6 8 10 12 14 Volume (mL) BSA, 2.5 mg, PBS/DTT (primary axis) ASC intra, 0.35 mg SUMO-ASC, 0.35 mg MBP-ASC, 0.08 mg
Fig 4.8 Sup 75 purifications of ASC fusion proteins in PBS/ DTT buffer. All the
proteins eluted in the void BSA monomer elution peak. Amounts of proteins loaded
are as follows: BSA (2.5 mg), MBP-ASC (0.08 mg), SUMO-ASC (0.35 mg) SDS/BME
treatment, intra-ASC (0.35 mg).
117
Further optimization of the best buffer conditions was done with the fusion protein, MBP-ASC. Superdex 75 elution profiles of MBP-ASC in various buffers are shown in Fig 4.9. Just like the previous findings, the proteins mostly eluted as high molecular weight species in the void. Interestingly, all the elution peaks
(except the one in PBS only buffer) showed the signature double-hump suggesting presence of smaller oligomers. Considering the elution in Tris/glycine
(SDS-PAGE buffer) a small monomer peak was observed although a significant amount of the protein was aggregated. Since this finding seemed promising,
Tris/glycine buffer would be later used in subsequent SEC-MALS experiments.
These data show that addition of buffer additives somehow assisted in solubilizing the aggregates into smaller species to some extent.
Fig 4.9 Superdex 75 elutions of
MBP-ASC fusion proteins in 120 various buffers. SDS-BME 100 treatment was done for lysate 80 (0.5 mg) and medium (0.22 mg) 60 mAU samples eluted in PBS and 40 HEPES buffers respectively. 20 0.08 mg medium sample was 0 4 6 8 10 12 14 treated with 6M GuHCl/ 2mM Volume (mL) MBP-ASC, PBS, 0.5 mg DTT prior to loading then eluted MBP-ASC, HEPES, 0.22 mg MBP-ASC, PBS/DTT, 0.08 mg in PBS/DTT, lysate (0.25 mg) MBP-ASC, Tris/Glycine, 0.25 mg was eluted in Tris/glycine.
118
4.2.5. SEC-MALS analysis confirmed the existence of protein
aggregates
SEC-MALS experiments with the recombinant proteins would later reveal that light scattering intensities exceeded the threshold thus further confirming the presence of aggregates. Fig 4.10 shows the MALS data obtained with MBP-ASC eluted in Tris/ Glycine and PBS/ DTT buffers. One main challenge encountered when trying to mimic SDS-PAGE conditions during SEC-MALS experiments is that BSA eluted as a dimer in this buffer so that threw off the Mw estimations.
SDS micelles increase the protein size and that explains BSA peak shift and incorrect Mw [304, 305]. Despite our attempts to use different denaturing conditions, the proteins remained as huge aggregates thus distinct species could not be isolated by SEC which made it difficult to properly analyze them by MALS.
Thus, obtaining molecular weights of the aggregated proteins by MALS was not quite successful.
119
Fig 4.10 SEC-MALS experiments with recombinant MBP-ASC protein revealed the presence of aggregates. Superdex 200 elutions were done in PBS/DTT and Tris/ Glycine buffers (same buffer used in SDS-PAGE). BSA (66 kD) eluted as a monomer with
PBS/DTT buffers (blue dotted line) and the Mw read off the Y-axis confirms the protein as monomeric and homogenous (blue straight line). The red dotted line is the BSA elution peak in Tris/Glycine Buffer and it showed a significant shift towards the void (from 14.5 mL to 8.3 mL) and the Mw estimation showed the protein as a dimer in SDS-PAGE buffer. The cyan curves represent MBP-ASC medium (61 kD) eluted in PBS/DTT buffer while purple ones show MBP-ASC lysate sample eluted in Tris/Glycine. Both elution profiles show the major peaks in the void volume and the MALS Mw estimations show the presence of heterogenous, high-order species.
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4.3. Analysis of inflammasome protein oligomer structure and morphology
To further determine the structures of the recombinant proteins, studies were conducted to determine their hydrodynamic radii (dynamic light scattering
DLS), secondary structure (circular dichroism CD), and morphology
(transmission electron microscopy TEM).
4.3.1. DLS analysis of caspase-1 proteins revealed the presence of
monomers and dimers
DLS studies were conducted with caspase-1 proteins purified on a
Superdex 200 column under various conditions as described in the Methods section. The hydrodynamic radii are shown in Table 4. 2.
Table 4.2 DLS experimental data. The experiments indicated that most of
the proteins exist as oligomers. SEC-purified caspase-1 proteins were
analyzed by DLS. In some cases, the DLS-determined sizes did not match
the Mw of the species determined by SEC elution peaks.
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DLS experiments provided some interesting data. Information on the sizes of the proteins was obtained with full-length caspase-1 and the truncated domains as well. In comparison to BSA monomer (66 kD) which has a hydrodynamic radius (RH) of 3.5 nm, the p20 sample had a radius of 2.5 nm and a relative molecular weight of 28 kD. These data indicate that the p20 sample was possibly monomeric. Similarly, the RH values obtained with procaspase-1
(45 kD) and CARDp20 (33 kD) were 5.6 nm and 3.2 nm respectively thus suggested that both proteins were dimers.
However, the data obtained by p10 fractions -26 (predicted to be a dimer) and -32 (predicted to be monomeric) did not match the SEC predictions. These samples showed extremely high hydrodynamic radii which was unexpected. In general, large light scattering intensities are indicative of oligomer formation. DLS studies with other proteins did not yield good readings as the data revealed that most of the proteins existed as aggregates. When conducting DLS measurements, aggregated proteins tend to scatter too much light thus giving unusable readings that are out of range.
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4.3.2. Recombinant ASC exhibits some degree of helicity that is
unaffected by changes in pH
Protein misfolding has been known to induce aggregation. While conducting these studies, one lingering question was whether the proteins were folding properly. CD studies were then conducted to analyze the secondary structures of the expressed proteins. Previous studies by de Alba and colleagues
[17] explored the structure of ASC under neutral (pH 7.4) and acidic (pH 3.8) conditions. Their Nuclear Magnetic Resonance (NMR) and Atomic Force
Microscopy (AFM) studies showed that the structure of ASC was not perturbed by the acidic conditions. Basing on these previous findings, I tested the effect of pH on the structure of recombinant ASC.
CD studies with recombinant ASC revealed some degree of defined structure which was not perturbed by pH changes. CD studies were performed with affinity-purified ASC medium samples (purification traces previously shown in Fig 3.6). Both samples (peak 1 and peak 2) were analyzed at a final concentration of 18.5 µM. Published standard CD spectra shown in Fig 4.11
Panel A were used as references. Under physiological conditions, peak 1 showed some degree of alpha helicity while peak 2 appeared more like a β-sheet
(Panel B). Panel C compares the spectra of peak 1 at both pHs. These data indicate that the structure of ASC is unperturbed by changes in pH. These results were in agreement with the previous studies by de Alba and colleagues [17]. An overlay of peak 1 and standard helix spectra shows that recombinant ASC has
123 some degree of helicity (Panel D). Under normal conditions, no protein exists as a perfect helix or β-sheet. Proteins thus exhibit some degree of helicity and β- sheet structure. It is possible though that improper folding of the proteins during expression may have contributed to protein aggregation.
A. B.
C. D.
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Fig 4.11 CD studies revealed that recombinant ASC had some structure. Studies were
done using a Jasco spectrophotometer as explained in Methods. Affinity-purified
recombinant ASC medium peak fractions (affinity-purification shown in Fig 3.6) were used
for the measurements. BCA assay concentration determined the concentrations of ASC
peaks 1 (1.5 mg/ml) and 2 (0.4 mg/ml). Peak 1 was diluted 4-fold in PBS buffer to match
concentration of peak 2 then the samples were spun for 10mins at 17,000g to get rid of
any aggregates. Panel A. Standard CD spectra obtained from
https://ceberndsen.github.io/CEBerndsen.github.io/CD.html
Panel B. CD spectra of ASC peaks 1 and 2 in PBS buffer pH 7.4. In Panel C, the spectra
of peak 1 were collected under acidic and neutral pH while Panel D compares the
standard helix spectra vs peak 1 at pH 3.
4.3.3. ASC ball-like aggregates were observed by TEM
ASC ball-like aggregates were seen by TEM. To further analyze the structure and morphology of ASC, TEM studies were done. The images showed what looked like ASC ball-like specks/ oligomers (Fig 4.12) thus confirming aggregation. ASC is inherently prone to aggregation at physiological pH as has been previously confirmed by previous studies [3, 4, 17, 128, 147]. Therefore, it was most likely to form aggregates due to overexpression plus under the conditions employed during isolation and purification processes.
125
A. B.
Fig 4.12 Electron microscopy images show recombinant ASC as aggregates. TEM studies were conducted as explained in Methods. Briefly, affinity-purified recombinant
ASC medium peak fraction 1 (affinity-purification shown in Fig 3.6) was diluted to 18
µM in PBS buffer then 10 µl of the sample was placed onto a 200-mesh formvar- coated copper grid (Ted Pella, Inc.), adsorbed for 10 mins, washed and stained with uranyl acetate. After air drying, the grid was imaged. Panel A shows ball-like ASC aggregates at 43k magnification and Panel B is at 35k magnification. The scale bars for both images are 100 nm. Images obtained by Dr. David Osborn.
126
In summary, expressed NLRP3 inflammasome proteins are highly prone to aggregation. Characterization of these proteins to reveal their oligomerization states indicated the presence of HMW aggregates as shown by immunoblotting techniques and biophysical studies. Various harsh conditions were explored in efforts to reverse the aggregation processes with very little success. Because these aggregates were quite resistant to the stringent conditions used, a lot of questions arose. Are the proteins misfolding during the expression? Could it be that the in vitro conditions are not conducive, and that it may not be feasible to obtain stable inflammasome proteins under the conditions employed? What are the stoichiometric components of each individual protein under physiological conditions, and what mechanisms are involved in keeping the proteins in these stable forms? A lot of signaling pathways and a variety of factors are involved in stabilizing these proteins in a cellular environment hence taking away those components may be the cause of aggregation. These and other factors present a very complicated system. Due to their propensity to aggregate, maybe the stable forms of these proteins exist in the aggregated form. Regardless, I was able to obtain some vital structural information on some of the proteins/ domains.
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CHAPTER 5: ISOLATION OF THE ELUSIVE CASPASE-1 ENZYME
Caspase-1 is a cysteine protease that is activated upon assembly of the
NLRP3 inflammasome through a sequence of events leading to IL-1β maturation and subsequently pyroptosis. However, the actual mechanisms surrounding caspase-1 activation are still to be determined. Although a lot of studies have been done on caspase-1, there are still unanswered questions regarding the actual structure of the active enzyme. The general consensus, however, is that procaspase-1 possesses little to no activity thus the goal of these studies is to determine the actual form of the enzyme that is active. Some of the outstanding questions regarding the actual conditions required for enzyme activity include: is the FL procaspase-1 active by itself? If so, do all three proteins need to be present to render the enzyme active? What domains make up the active conformer of caspase-1? To answer these questions, recombinant FL, mutant, and truncated versions of caspase-1 were assessed for enzymatic activity in these studies.
5.1. What is the actual structure of active caspase-1?
Cell-stimulation assays with inflammasome activators revealed the presence of the processed p20 and p10 subunits in the cell-free supernatants and lysates [3, 39, 153, 161, 162, 166, 170, 171, 245, 293, 306]. Processed caspase-1 fragments indicate caspase-1 activation and maturation. Biotin pull- down probes such as biotin valyl-alaninyl-aspartyl chloro/fluoromethylketone (b-
128
VAD-cmk/-fmk) [15, 21, 245] and fluorochrome labeled inhibitors of caspases
(FLICA) have been used to trap caspase-1 in the active form [21, 293, 307-310].
Fig 5.1 shows the possible forms of active caspase-1 gathered from various literature sources.
Fig 5.1 Schematic showing possible active confomers of active caspase-1. Designs
based on the published literature findings. Panel A shows the full-length, unprocessed
proenzyme which is presumed to have little or no activity. Panels B-D show the
various forms of active caspase-1 discussed in the literature. Panel B shows the
p33p10 processed form with an intact CARD domain for anchoring to the
inflammasome. Panel C is the p20p10 heterodimeric form of active caspase-1 while
the heterotetramer is illustrated in Panel D with dimerization occurring via the p10-p10
interface.
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Early studies reported the active enzyme to be p20p10 heterodimer [6] and these findings were confirmed by other groups [9, 15, 16]. In contrast, some groups proposed the active enzyme to be a heterotetramer (p20p10)2 [7, 8, 13].
Two groups reported the existence of an equilibrium between the heterodimer and heterotetramer species [10, 277]. Interestingly, both species were found to be catalytically active. High substrate concentrations and presence of inhibitors promoted heterotetramer assembly [10].
Recent studies by the Schroder group completely dispute previous reports
[21]. Using a bVAD-fmk probe that labels and pulls-down active caspases from stimulated macrophages, they identified the full-length proenzyme p46 and the transient p33p10 heterodimer as the dominant active species. They proposed that the CARD domain anchors the active form of caspase-1 onto the activating inflammasome thus removal of the CARD-domain linker (CDL) releases the enzyme from the inflammasome platform ultimately rendering it inactive. This generates “unstable” p20p10 species [21]. Currently, there are two distinct models for caspase-1 activation as shown in Fig 5.2. Scenario 1 represents the widely-accepted model while Scenario 2 is the most recent model as proposed by Schroder and colleagues [21]. Due to the complexity of the caspase-1 activation process, it is possible that other mechanisms could be involved in these processes. Nonetheless, there are still lingering questions regarding the actual composition and stability of active caspase-1 as discussed in our recent review (Makoni et al, Archives of Biochem. and Biophysics, submitted 2020).
130
Fig 5.2 Possible scenarios to explain caspase-1 activation mechanisms. The inflammasome facilitates caspase-1 activation via induced proximity mediated autoproteolysis. Scenario 1 proposes that CDL processing is a prerequisite for obtaining an active enzyme. The liberated p20 and p10 subunits recombine to form either the heterodimer or heterotetramer (both which have been found to have enzymatic activity) [6-16]. Presumably, removal of the CDL causes release of the active enzyme from the inflammasome complex to catalyze its substrate elsewhere.
Scenario 2, however, suggests the inflammasome as the site for IL-1β cleavage. The
CARD domain anchors the active caspase onto the inflammasome hence CDL processing to remove the CARD inactivates the enzyme resulting in it dissociating from the activating complex [21].
131
5.2. Recombinant caspase-1 p20p10 reconstitution experiments
5.2.1. Caspase-1 p20p10 reconstitution studies from the literature
Apart from cell-stimulation assays, many groups have utilized recombinant systems to express full-length procaspase-1 and/or the individual domains for functional assays as summarized in Table 5.1. Detailed reports described the various methods of expression, purification, and functionalization of caspases under different conditions [285, 286].
Table 5.1 Recombinant expression of inflammasome proteins from the literature. *In some
papers, the caspase-1 heterotetramer is also referred to as the homodimer/ dimer.
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5.2.2. Caspase-1 stability
Caspase-1 exists as a monomer under normal conditions. Substrates and inhibitors, however, promote dimerization of the enzyme thus stabilizing it and keeping it in its active form [7, 8, 11, 12, 311]. For example, addition of aldehyde- based caspase-1 inhibitor (Ac-YVAD-CHO) or concentrations of malonate exceeding 100 mM prevented autoproteolytic cleavage of the purified refolded enzyme [12]. The main challenge faced by researchers during recombinant expression, purification, and refolding of caspase-1 is to maintain an active enzyme. Various factors to take into consideration include enzyme concentrations, optimum temperatures and pH, ionic strength, inhibitors, half-life, buffer components/ various stabilizing additives (denaturants, detergents, reducing agents) as well as storage conditions [7-9, 11-13, 15, 277, 311].
Despite many reports supporting the fact that inhibitors stabilize caspase-
1 [7, 8, 11, 12, 15], surprising findings revealed that cowpox serpin crmA, a caspase-1 inhibitor, caused dissociation of the active heterotetramer resulting in the loss of the p10 subunit [13]. One group discovered that refolding the enzyme under non-reducing conditions prevents autoproteolysis [9]. In addition, too dilute enzyme solutions below 30 nM result in subunit dissociation while enzyme concentrations exceeding 2 µM promote autoproteolysis [10, 312]. Using cross- linked recombinant caspase-1, Martin and colleagues also reported dissociation of caspase-1 at concentrations below 10 nM and that the enzyme exhibited a very short active life of just 9 mins at 37 oC [15]. Taking all these factors into
133 consideration, finding the right conditions to maintain an active enzyme can be quite a daunting task.
5.2.3. Caspase-1 p20p10 reconstitution assays +/- inhibitors were
unsuccessful
Considering that other groups were able to successfully reconstitute an active enzyme from p20 and p10 fragments, I performed caspase-1 reconstitution experiments with recombinant p20 and p10 fragments. Equimolar amounts of the subunits were solubilized separately, combined then refolded.
Initial caspase-1 reconstitution assays with SEC-purified caspase-1 p10 and p20 fragments were unsuccessful. In follow-up studies, affinity-purified p10 and p20 lysates were solubilized overnight (separately) in 6 M GuHCl/ 20 mM DTT to break any pre-formed aggregates. To test the effect of inhibitors, equimolar amounts of solubilized p10 and p20 were refolded +/- inhibitors (1 µM Ac-YVAD-
CHO, 100 mM malonate). Fig 5.3 shows the SEC traces of the refolded proteins purified on Superdex 75 column. The purification traces show two peaks (with an anomaly peak ~ 5 mL) in the absence of inhibitor while addition of inhibitors yielded a single species that eluted in one peak ~ 8 mL. A possible explanation for the differences in these elution profiles is that presence of inhibitors results in a stable, single species. Since the 8 mL peak slightly overlays with BSA monomer (66 kD), it is most likely a 60 kD species. There were many possibilities regarding the composition of the peak; could it be a p20 trimer, p10 hexamer, or p20p10 heterotetramer?
134
A. B.
Figure 5.3 Analysis of refolded caspase-1 p20p10 +/- inhibitors. Panel A. p20
Equimolar amounts of solubilized p20 & p10 IB samples were combined to a total of 1 mL and added dropwise to 49ml refolding buffer in the presence/ absence of an inhibitor, refolded overnight and affinity purified. 1 mL of affinity peak fraction was centrifuged at 17,000g for 10 mins then loaded onto a Superdex 75 column pre- blocked with BSA. Refolded p20p10 samples were eluted in (10mM Phosphate, 150 mM NaCl, 2mM DTT, 5% glycerol, 0.05% Tween-20) buffer. Panel B. Immunoblots of
SEC-purified refolded p20 and p10 fragments +/- inhibitors. After overnight refolding, proteins were purified by affinity and SEC. Sup 75 peak fraction (13) was used for
SDS-PAGE analysis. Immunoblotting was done with anti-His antibody. Lanes 1 has the MWM, Lane 2 blank, Lane 3 p20p10 (13) no INH, Lane 4 p20p10 (13) Ac-YVAD
CHO, Lane 5 p20p10 (13) malonate.
135
The peak fractions were analyzed by SDS-PAGE followed by immunoblotting with anti-His antibody. Table 5.2 shows the estimated UV250 nm protein concentrations and the amounts of proteins loaded. The immunoblot snippets in Fig 5.3B show similar band patterns with all samples regardless of the presence/ absence of inhibitor which was quite unexpected. The dominant species detected were ~ 20 kD. A minor species (~40 kD) was also detected.
Stripping and re-probing the membrane with an antibody targeting the p20 domain (anti-p20 Ab, Boster Biological, Cat# PA1440) was unsuccessful.
Another problem with the p20 antibody is that it could not distinguish between the p20 and p10 species. Furthermore, crosslinking experiments with a water- insoluble maleimide crosslinker targeting sulfhydryl groups (BMH) did not provide usable data. Therefore, I could not determine whether the bands were entirely p20 or p10 fragments or a combination of the two.
Table 5.2 UV280nm determined concentrations of the refolded caspase-1 p20p10 peak
fractions that were used for the immunoblot assay. Mw used for the calculations is for
the (p20p10)2 heterotetramer (60,176 g/mol)
136
5.3. Caspase-1 activity assays
5.3.1. The enzymatic assay was optimized using commercial active
caspase-1 and inhibitors
Some groups previously reported observing enzyme activity with refolded p20p10 fragments (reviewed above). On the contrary, no single publication as of now has shown activity studies with recombinant pro-caspase-1 since it is assumed to be in an inactive state. To determine which particular domain(s) of the proenzyme are required for activity, caspase-1 activity assays were conducted with recombinant proteins.
To begin, I had to optimize the assay using a purchased human active enzyme and two commercially available coumarin-based caspase-1 tetrapeptide synthetic substrates, Ac-YVAD-AMC and Ac-WEHD-AMC. The 4-residue cleavage site of pro-IL-1β (YVHD116) is quite similar to that of the synthetic tetrapeptide substrates. As Fig 1.8 Panel B shows, amino acid substitutions in the P2 position are tolerable and have no effect on enzyme activity. The fluorescence-based assay showed Ac-WEHD-AMC as a more potent substrate compared to the YVAD version as shown in Fig 5.4 Panel A. Subsequent activity assays were thereby conducted with Ac-WEHD-AMC. To further optimize the assay, caspase-1 inhibitors were introduced. As Panel B shows, enzyme activity
137 was completely diminished in the presence of both reversible and irreversible caspase-1 inhibitors.
600 A. Ac-WEHD-AMC Ac-YVAD-AMC
400
200
450 nm emission 0 0 5 10 15 20 25 30 reaction time, min
1000 B. casp-1 800 casp-1 + fmk casp-1 + cho 600
400
200 450 nm emission 0 0 10 20 30 40 50 60 reaction time, min
Fig 5.4 Optimizing the caspase-1 activity assays with commercial fluorogenic
tetrapeptide substrates and inhibitors. Panel A. Ac-WEHD-AMC proved to be the
more potent synthetic substrate. Panel B. Both the reversible (cho) and irreversible
(fmk) caspase-1 inhibitors erased enzymatic activity. Figs by Dr. Nichols.
138
5.3.2. Recombinant full-length (FL) procaspase-1 exhibited transient
enzyme activity
To verify whether the caspase-1 p20p10 reconstitution experiments
(shown in Fig 5.3) were successful, activity studies were conducted with the synthetic substrate. As Fig 5.5 shows, no activity was observed with the refolded proteins regardless of the conditions in which the refolding occurred i.e presence or absence of inhibitors.
40
30
20
10
450 nm emission nm emission 450 0
-10 0 10 20 30 40 50 60 Time (min) Refolded p20p10 + 100 mM malonate Refolded p20p10 + 1uM Ac-YVAD-CHO Refolded p20p10 no inhibitor Refolded CARDp20 no inhibitor
Fig 5.5: Caspase activity assays of refolded p20p10. Refolded p20p10 (+/- inhibitors)
and CARDp20 have no enzymatic activity. Sup 75 elutions for refolded p20p10 are
shown in Fig 5.3. CARDp20 was purified on a different day but under the same
conditions described in Fig 5.3 legend except that the SEC elution buffer did not
contain glycerol. Peak fractions 13 were assayed for activity.
139
These findings were in contrast to previous published findings that stated that refolded p20p10 possesses enzymatic activity (see Table 5.1). A variety of factors may be responsible for these discrepancies with the main one being the expression system used (previous studies use E. coli). In addition, the experimental conditions employed greatly affect the type of stable species formed and subsequent enzymatic activity. It was not clear if our refolding experiments were successful to begin with. Attempts to obtain activity with the
CARDp20 fragment and refolded CARDp20/p10 (data not shown) proved fruitless too.
After unsuccessful attempts to obtain caspase activity with both the
CARDp20 and the refolded p20p10 fragments, I decided to test enzymatic activity of procaspase-1. To conduct these studies, lysates and refolded inclusion bodies of procaspase-1 were purified by SEC. The Superdex 75 elution profiles in Fig 5.6 Panel A show that all the protein eluted in the void. Surprisingly, FL caspase-1 obtained from both the lysates and inclusion bodies exhibited enzymatic activity when tested with the fluorescence assay. This was the first time any enzymatic activity had been observed with the unprocessed proenzyme.
Panel B shows the activity assay data for the lysate sample. However, there was a drastic loss of activity overnight and over the course of the next few days, activity decreased to none. Addition of a commercial protein stabilizer helped retain some of the activity after 6 days (Panel C). Interestingly though, the active enzyme was a high molecular weight species which eluted in the void whose
140 exact conformation is unknown. These observations were intriguing and only add to the complexity of caspase-1 and its activation mechanisms.
600 300 A. 500 250 400 200
300 150
mAU mAU 200 100 100 50
0 0 0 5 10 15 20 Volume (mL)
BSA (primary axis) FL lysate FL inclusion bodies
120 B. initial 100 2d, 4C 80 60 40 20
450 nm emission 0
0 1 2 3 4 reaction time, h
150 initial C.
6d w-stabilizer
450 nm emission 0 6d w-o stabilizer 0 50 100 150 reaction time, min
141
Figure 5.6 FL procaspase-1 possesses activity as shown by the fluorogenic peptide
assay. Panel A. Recombinant expressed FL procaspase-1 lysates and inclusion bodies
peak fractions were affinity-purified, centrifuged at 17,000g for 10mins, loaded onto a
Superdex 75 column pre-blocked with BSA. The proteins were eluted in (10 mM
Phosphate, 150 mM NaCl, 2 mM DTT, 5% glycerol, 0.05% Tween-20) buffer and the
collected fractions were assayed for caspase-1 activity. Panel B. SEC purified full-length
procaspase-1 (700 nM) was incubated with 30 μM Ac-WEHD-AMC in caspase-1 buffer at
25 oC and time-dependent caspase activity was monitored. Both lysate and inclusion
bodies samples exhibited activity. Panel C. Protein stabilizer cocktail helps retain
caspase-1 activity. Another ExpiCHO-S expression and purification of FL procaspase-1
was conducted. A slightly modified version of SEC buffer was used (10mM Phosphate
buffer, 150 mM NaCl, 2mM DTT, 0.05% CHAPS). Panels B, C Figs by Dr. Nichols.
5.4. Various conditions were optimized in order to retain a stable, active
enzyme
In an attempt to find out what caused the loss of activity over time, I explored various storage conditions such as concentration, storage temps, buffers, stabilizers, and storing the enzyme in the affinity-purified vs SEC form.
Enzyme concentration plays an important factor when conducting caspase-1 activity studies. Based on my findings, a minimum amount of recombinant enzyme was required to obtain activity (7 µM stock solution amounting to 700 nM final enzyme concentration in the reaction cuvette) and below that threshold,
142 there was no observed activity. That could also explain why the refolded p20p10 and CARDp20 domains did not exhibit any activity as their stock concentrations were 4 µM and 2.8 µM respectively. Higher concentrations of the enzyme may also promote aggregation or auto-degradation so obtaining the right concentrations that yield an active enzyme can be challenging.
To test whether SEC is a requirement to obtain activity, side-by-side activity assays were done with affinity- and -SEC-purified procaspase-1. Affinity- purified samples were found to have no activity but only gained activity after SEC purification. That raised another question whether buffer exchange assisted in yielding an active enzyme. If a simple buffer-exchange process is required, then dialyzed protein (to get rid of the high imidazole in affinity-purified samples) should have activity. Affinity purified FL caspase-1 lysate sample was dialyzed overnight in SEC buffer (10mM Phosphate buffer, 150 mM NaCl, 2mM DTT,
0.05% CHAPS) then assayed for activity for 2 consecutive days. The activity of the dialyzed sample was considerably low by many orders of magnitude compared to the SEC-purified sample (data not shown). Although initial activity of the dialyzed enzyme was low, there was noticeable loss of activity overnight similar to previous observations with SEC-purified enzyme.
Since the enzyme showed significant reduction in activity when stored in
SEC buffers (10 mM Phosphate, 150 mM NaCl, 2mM DTT, 5% glycerol, 0.05%
Tween-20), I decided to test whether storing it in the affinity-purified form had any significance. Two weeks after the initial activity studies, I purified the remaining
143 affinity sample by SEC and tested its activity. The enzyme still retained some activity although the initial activity was not as high as the previously obtained activity in Fig 5.6 Panel B. These findings were quite useful as they indicated that storing the proteins in the affinity-purified form helped in slightly retaining enzyme activity over time compared to storing them as SEC-purified samples.
Another possible explanation for the loss of activity could be aggregation.
To circumvent this, the enzyme was stored in the affinity-purified form at -80 oC
(previously stored at 4 oC) then subjected to SEC after some time. That also proved to be unsuccessful as those samples had no activity. Since aggregation during transfection was another issue, expression times were optimized ranging from 2-13 days. However, there was no difference in total protein yields, and neither was there any improvement in enzymatic activity and/ or stability.
5.5. Recombinant procaspase-1 was unable to cleave the pro form of
interleukin 1-beta (IL-1β)
After observing enzymatic activity with the recombinant procaspase-1 via fluorescence methods, I sought to further test the potency of the enzyme using pro-interleukin-1-beta (pro-IL-1β), well-known caspase-1 substrate. To optimize the cleavage assay, commercial active human recombinant caspase was used.
Time-dependent cleavage of pro-IL-1β was observed as indicated by the disappearance of the 35 kD band (pro-IL-1β) and appearance of a 17 kD band
(mature IL-1β) as Fig 5.7 shows.
144
Fig 5.7 Optimizing IL-1β cleavage assay with active commercial caspase-1. To conduct the assay, 1.8 μg caspase-1 enzyme was mixed with 500 ng/mL IL-1β protein in caspase-assay buffer cocktail (25 mM HEPES, 1 mM EDTA, 25 mM KCl, 10%
Sucrose, 1 mM DTT, pH 7.4) to make a total volume of 100 µL. The reaction was conducted for 4 hours at 25 oC. Aliquots were collected at different time points and the reaction was quenched by adding SDS/BME. Samples were analyzed by SDS-PAGE and immunoblotting (as mentioned in Methods section) then probed with human IL-
1β/ IL-1F2 polyclonal goat antibody. Pro-IL-1β is 35 kD while the cleaved, mature form ~17 kD. Immunoblot done by Shikha Grover.
145
The next goal was to perform the pro-IL-1β cleavage assays using the recombinant procaspase-1. These experiments were performed on the same day as the fluorescence assay. However, no cleavage occurred after a 4hr incubation at room temp (data not shown). Further optimization was done by incubating the reaction vessel overnight to maximize the cleavage reaction. Again, the assay did not give positive results. Although recombinant procaspase-1 showed enzymatic activity with the synthetic substrate in the fluorescence assay, it did not exhibit any activity with the biological substrate. The synthetic substrate has been widely used in caspase-1 activity assays and is considered a good model
[the recognition site in the synthetic fluorogenic substrate (WEHD) and the pro-
IL-1β cleavage site (YVHD)]. Hence, it is still to be determined why the recombinant procaspase-1 did not cleave pro-IL-1β.
5.6. Tobacco etch virus (TEV) protease cleavage reactions to remove
fusion tags
5.6.1. The cleavage experiments showed half maximal cleavage
efficiency
TEV protease cleavage experiments were conducted to remove the MBP and SUMO fusion tags from our proteins of interest. Considering the size of the
MBP protein, its removal would prevent interference with other subsequent assays. For these experiments, affinity-purified MBP-p10 was used. Initial cleavage reactions were conducted with 20 µg of protein and 10 U of enzyme but there was barely any cleavage as revealed by western blotting analysis. Upon
146 realizing that the high amounts of imidazole in the affinity-purified samples (500 mM) were probably inhibiting the enzyme, subsequent cleavage assays were conducted with samples dialyzed overnight in 50 mM Tris-HCl buffer. In addition, amounts of MBP-p10 and enzyme were scaled up 10 times (200 µg and 100 U respectively) to maximize the cleavage reaction.
Under these new conditions, there was time-dependent cleavage of MBP-
P10. Immunoblotting with an antibody recognizing the p10 fragment revealed the appearance of bands ~10 kD (Fig 5.8 Panel A). The cleaved protein appeared as early as 30 minutes into the reaction and maximum cleavage had occurred after
4 hours. Re-probing the same membrane with anti-His antibody showed the time- dependent decrease of His-tagged MBP-p10 (50 kD) coupled with the increase of His-MBP ~40 kD (Panel B). In addition, TEV protease is also His-tagged at the
N-terminus and appeared as bands ~25 kD.
A. B.
147
Fig 5.8 TEV cleavage reactions with MBP-p10 showed time-dependence cleavage
although there were significant amounts of uncleaved protein. Cleavage experiments
were conducted inorder to remove the fusion tags for downstream assays as outlined
in Methods. Briefly, affinity-purified MBP-p10 caspase lysate fraction that was stored
at -80 oC was dialyzed overnight in 50 mM Tris-HCl buffer. 200 ug protein sample
was treated with 100 units TEV (10 µL) protease (1 mL volume total). 15 µL aliquots
were taken at various time points and quenched with 15 µL Laemmli sample buffer
with BME, 95 oC heating for 5 mins then analyzed by SDS-PAGE. Panel A.
Immunoblotting was done with anti-p10 primary and anti-rabbit secondary antibodies.
Cleaved p10 was observed ~10 kD while uncleaved mbp-p10 was detected at ~50
kD. Panel B. Membrane was stripped and re-probed with anti-His and anti-mouse
primary and secondary antibodies respectively. The TEV protease also contains a His
tag and appears ~ 25 kD.
Although there was obvious cleavage, there were still significant amounts of uncleaved protein. Adding the enzyme to the reaction vessel at various time points (instead of adding all the enzyme at once) did not improve the cleavage efficiency either (data not shown). A possible explanation is that due to aggregation, the cleavage site was buried and thereby inaccessible to the TEV protease.
148
5.6.2. Analysis of cleaved MBP-p10
After the cleavage reactions, affinity chromatography was used to separate the cleaved and uncleaved proteins. Fig 5.9 Panel A shows the affinity- purification curves. Peaks 1 and 2 represent the flow through. Cleaved p10 is expected to be in the flow through. On the other hand, the TEV protease, His-
MBP, and uncleaved His-MBP-p10 were expected to appear in the elution peak
3. The peak fractions were collected for dot blot analysis. Due to very low protein amounts, the samples were concentrated first. Dot blotting with both anti-p10 and anti-His antibodies was used to distinguish the fractions in the flow through
(fractions 6, 17, 20) and the elution fraction 62. The caspase-1 p10 antibody detected both the cleaved p10 (fraction 6) and uncleaved His-MBP-p10 with similar intensities as shown in Fig 5.9 Panel B. This could mean that somehow equal amounts of cleaved vs uncleaved protein. Probing with an anti-His antibody showed the presence of the His tag in the eluted fraction 62 as expected (due to residual His-MBP-p10 and the TEV protease). However, the fact that His-tagged was detected in the flow through was quite puzzling suggesting possible affinity column leakage.
149
A. B.
Fig. 5.9 Analysis of cleaved MBP-p10 by affinity purification and immunoblotting.
Panel A. Affinity-purified mbp-p10 caspase lysate peak fractions were dialyzed in
Tris/HCl overnight then concentrated. About 1.26 mg protein was treated with 100 U
(10 µL) TEV protease for 6 hr at 30 oC. The sample was centrifuged at a speed of
17,000g for 10 mins to remove debris then 1 mL supernatant was loaded on affinity column. Collected fractions 5,7 (peak 1), 17-21 (peak 2), and 58-65 (peak 3) for further analysis. Panel B. Dot blot analysis using affinity-purified cleaved mbp-p10 fractions. The dot blot protocol was done as outlined in Methods followed by immunoblotting with anti-p10 antibody. **Assumption is that the flow through fractions
(6, 17, 20) contain cleaved p10 protein and the imidazole-eluted fraction 62 contains the His-MBP, His-MBP-p10, and TEV protease.
150
Summary
These studies revealed, for the first time, enzymatic activity of recombinant procaspase-1. However, the activity was transient, and the enzyme was unstable under the conditions employed for purification and storage. Since the active enzyme eluted in the void volume using a Sup 75 column, this means that the active species had a Mw > 70 kD. These findings suggest the possibility of another active conformation of active caspase-1 apart from the known active forms shown in Fig 5.1. Despite exhibiting enzymatic activity with the fluorescence assay, the recombinant enzyme was unable to cleave pro-IL-β, a well-known caspase-1 substrate. Due to the similarity in the recognition sequences of the synthetic and biological substrates, caspase-1 is expected to cleave both substrates, but this was not the case with our recombinant procaspase-1. Future studies may involve using various caspase-1 substrates to test the potency of our recombinant enzyme. Contrary to previous studies, caspase-1 reconstitution experiments with refolded caspase-1 p20 and p10 fragments were not successful due to variability in experimental conditions.
Subsequent TEV cleavage reactions with caspase-1 domain fusion protein,
MBP-p10, indicated about 50% cleavage efficiency. It is possible that protein aggregation may have concealed the TEV cleavage site thus making it inaccessible to the protease. Nevertheless, caspase-1 studies yielded some useful and interesting results that will help in understanding this enzyme better and the mechanisms surrounding its activation.
151
CHAPTER 6: INTERACTION OF INFLAMMASOME PROTEINS WITH
THE ALZHEIMER’S DISEASE AMYLOID-β PROTEIN
As described in Chapter 1, Aβ is known to activate the NLRP3 inflammasome [19, 23, 37, 53, 56, 100, 115] resulting in caspase-1 activation and IL-1β secretion. Exactly how Aβ stimulates the inflammasome is still unclear and not many studies have been conducted in this area. Either there are direct interactions between Aβ and the inflammasome proteins or there could be indirect interactions involving an alternative pathway. Heneka and colleagues have reported interactions between Aβ and ASC specks [2, 116]. Another group performed cell-free assays to show direct interactions between Aβ and NLRP3
[281]. Since NLRP3 is the sensor protein, it is more likely to interact with Aβ.
Therefore, we decided to investigate these interactions using a two-way approach; in vitro interaction assays with recombinant inflammasome proteins and immune cell stimulation assays to isolate the endogenous inflammasome complex by immunoprecipitation methods.
6.1. There is a direct interaction between Aβ PF and NLRP3 protein
Previous studies from our lab identified Aβ-42 protofibrils (PF) as robust activators of microglia [19]. Additional studies revealed that primary microglia cells preferentially take up Aβ PF compared to monomers [22] hence cell stimulation experiments were conducted with PF. Aβ-42 PF purification was conducted as described in Methods.
152
The purification curves in Fig 6.1 show distinct PF and monomer peaks.
The PF peak was selected for the binding assays. Our studies are quite distinct due to the fact that we use SEC to isolate pure PFs compared to other studies that use a mixture of Aβ species.
600 30
500 25
400 20
300 15 mAU 200 10
100 5
0 0 5 10 15 20 Volume (mL) BSA (primary axis) Amyloid beta-42
Fig. 6.1 Superdex 75 purification of Aβ-42. The column was pre-blocked with 2.5 mg/
mL BSA. The elution trace shows the presence of both protofibrils PF (1st peak) and
monomers (2nd peak). Briefly, 0.9 mg HFIP-treated, lyophilized Aβ1-42 (ERI) was
reconstituted in 0.1 mL 50 mM NaOH/0.9 mL aCSF. The sample was incubated for 30
mins at room temp, centrifuged at 17000g for 10 min, 1 mL supernatant was loaded
and eluted in aCSF running buffer (10 mM Phosphate buffer, 150 mM NaCl, 2 mM pH
7.8).
153
6.1.1. In vitro interaction assays
To conduct in vitro interaction assays, Aβ and recombinant proteins (FL
NLRP3 or pyrin domain only, ASC, caspase-1) were combined in an eppendorf tube as shown in Table 6.1. The final concentration of each protein in the reaction vessel was 2 µM. The experiment proceeded for 2-4 hours at 4 oC
(control tube left at room temperature) followed by co-immunoprecipitation with 1-
2 µg anti-ASC and anti-NLRP3 antibodies. SDS-PAGE and immunoblotting experiments with antibodies targeting Aβ and the inflammasome proteins.
Table 6.1 In vitro inflammasome reconstitution assays were conducted to study
interactions between Aβ binding and the inflammasome. Inflammasome proteins were
purified on a Superdex 200 column in PBS-only buffer. Void peak fractions were used
for in vitro binding assays. All proteins were combined as shown in the table. The final
concentration of each protein in the reaction tube was 2 µM. Samples were incubated
for 2-4 hours or overnight to allow binding (if any) between the proteins, then
proceeded to Immunoprecipitation step using anti-ASC and /or anti-NLRP3 antibodies
(1-2 ug).
154
However, the experiments were not successful as no protein was detected by immunoblotting (data not shown). Increasing the incubation time or quantity of antibodies for co-IP did not help either. This suggested that the protein amounts were below detection limits. Attempts to increase the amounts were quite challenging because that varied with the Aβ isolation preps and yields were not always consistent. Scaling up the protein expression amounts using bigger flasks did not improve the overall recovery. Due to these challenges, I shifted my focus to cell stimulation assays.
6.1.2. Cell stimulation assays
Primary murine microglia, BV-2 microglia, and differentiated THP-1 macrophages were used to conduct the cell stimulation assays as outlined in detail in the Methods. Cells were stimulated with 5-10 µM Aβ PF for 6 hours followed by cell lysate extraction, co-IP assay with anti-NLRP3 antibody, gel electrophoresis under denaturing conditions, and immunoblotting with a conformational-specific antibody generated in the Nichols lab that targets the PF
(AbSL). According to Fig 6.2, the input lanes show that the cell took up the PF and nothing was detected in the control lane. The huge smears indicate high Aβ concentrations, and also the presence of various Aβ (oligomeric) species. The output (IP lanes) revealed a high molecular weight Aβ species that was bound to
NLRP3. These findings thus indicate a direct interaction between the two
155 proteins. Further attempts to detect the inflammasome proteins (ASC, NLRP3, caspase-1) in the crude lysates were unsuccessful (immunoblots not shown).
Fig 6.2 Co-immunoprecipitation assays show direct interactions between Aβ PF and
NLRP3. Cell stimulation assays were conducted with murine primary microglia. 300
μL cells (1x106 cells/ mL) were plated into 24-well plates then allowed to adhere
overnight. After rinsing the cells with sterile PBS, cells were treated with 5 µM-10 µM
Aβ PF (triplicates) and the control wells had assay medium. Cells were incubated for
6 hrs at 37 oC followed by lysate extraction with RIPA buffer. Immunoprecipitation was
done with anti-NLRP3 antibody (2 µg). After running an SDS-PAGE and transfer, PF-
specific apAbSL was used for immunoblotting. Lanes 2-4 represent the input while
lanes 5-7 are IP lysates.
156
The fact that I was not able to detect the proteins in both the crude lysates and immunoprecipitated samples pointed to the possibilities that either the loaded amounts were below detection limits or the cell stimulation assays did not work.
A. B. C.
Table 6.2 Inflammasome proteins were not detected in both the crude and
immunoprecipitated cell lysates. To verify the amounts of total protein present, BCA
assays were conducted. ELISA assays for secreted cytokines verified successful cell
stimulation. Cell stimulation assays were conducted with PMA-differentiated THP-1
monocytes. Cells were plated overnight (500 ul of 1x106 cells/mL) in 24-well plates
then treated with 5 uM and/or 10 uM Aβ PF and control wells had CSF only in assay
medium (triplicates). Cells were incubated for 6 hrs at 37 oC as outlined in Methods.
Immunoprecipitation was done with 2 ug NLRP3 antibody. SDS-PAGE, transfer, and
immunoblotting were performed. Panel A. human TNFα ELISA assay showing
concentration- dependent TNFα release. Panel B. BCA concentration assay Panel C.
To scale up the experiments, 3 mL of 1x106 cells/mL were plated into 6-well plates
overnight then underwent similar treatment as before. BCA assay was conducted with
cell lysates.
157
ELISA assays revealed that the stimulation experiments worked as the untreated wells showed extremely low amounts of secreted TNFα (Table 6.2A).
Various factors affect protein recovery during Co-IP including protein-antibody binding efficiency. In addition, immunoprecipitation does not always provide efficient sample recovery hence large amounts of total proteins must be present in the lysates to rule out any losses during the experimental procedures. In our case, bicinchoninic acid (BCA) protein concentration assays showed that the lysates had low protein amounts to begin with (Table 6.2B). Scaling up the experiments (from 24-well plates to 6-well plates) did not increase the amount total protein present in the lysates (Table 6.2 C).
To conduct further studies of the endogenous inflammasome, another strategy was to stimulate cells with LPS/nigericin. Nigericin is a well-known inflammasome activator. As explained in Methods, five conditions were tested during the cell stimulation assays. As Table 6.3A shows, initial BCA assay revealed low protein amounts in the lysates. Interestingly, total protein amounts in the lysates from stimulated vs unstimulated cells were quite similar except for the noticeably low amounts in the lysates of the wells subjected to 10 µM nigericin treatment. Toxicity assays confirmed that at concentrations of 10 µM, nigericin was toxic to the cells (data not shown).
Trichloroacetic acid (TCA)/ acetone precipitation was explored as an alternative method to improve the amounts of total protein extracted from the lysate samples. As described in the Methods section, samples were mixed in a
1:1 ratio with 20% TCA, incubated on ice for an hour, centrifuged at 4 oC for 10
158 min then went through successive acetone washes and centrifugation 3 more times. Protein pellets were left to air dry, resuspended in SDS buffer and assayed for total protein. Table 6.3B shows significant increase in total protein after TCA precipitation and those samples were used for Co-IP assays. A. B.
Table 6.3 PMA-differentiated THP-1 monocytes were plated overnight (2 mL of 1x106
cells/mL) in 6-well plates then treated with 100 ng/mL LPS for 6 hours followed by 5-
10 uM Nigericin for 0.5 hrs as outlined in Methods. Serum-free medium was used in
this assay. To maximize amount of lysate extracted, both chemical and mechanical
(cell scrapping) methods were used. Total protein in lysates was verified by BCA
assay. TCA precipitation was conducted, as described in Methods, to increase
amount of protein extracted. Protein amounts of crude lysates (Panel A) and TCA
precipitated protein (Panel B) are shown.
Despite the improvement in total protein amounts after TCA precipitation, detecting the inflammasome proteins in the crude lysates and the Co-IP output proved to be difficult. Nonetheless, I was able to demonstrate direct interactions between Aβ and NLRP3 protein.
159
6.2. Glutathione-S- transferase (GST) fusion tag improves protein
solubility
Protein aggregation is the main challenge we have encountered when performing recombinant expression. Addition of MBP- and -SUMO fusion tags slightly improved protein solubility although most of the proteins were still in aggregated form. Due to previous aggregation problems with our recombinant proteins, we introduced a glutathione-s-transferase (GST) solubility tag into our new gene constructs. In addition, two linkers were incorporated in order to further improve protein flexibility and solubility; a GST linker SDGSSMGSS (Temple &
Dong PDB 6N8U) and a His-tag C-terminal linker GSGSLE [4]. Fig 6.3 shows the
GST-ASC constructs: GST-ASC PYD, GST-ASC PYD K21A (mutation prevents oligomerization), and GST-CARD [2-5, 136, 283]. Full-length ASC as well as the individual PYD and CARD domains are known to self-associate forming oligomers and specks [3-5, 17, 135, 136, 142, 283]. To improve ASC solubility, some groups suggested the use of buffers containing 150 mM of potassium
[147], low pH [17], purification at 4 °C and including 2 mM DTT in all buffers [4],
0.9 M NaCl [283] or numerous dialysis steps against H2O at pH 4.0 [135].
GST-ASC proteins were expressed in mammalian ExpiCHO-S cells as explained in the Methods. The proteins were purified by affinity purification on a
Ni2+ column. The overall yields and quantities were quite similar to those seen in previous expressions. Affinity-purified proteins were further subjected to SEC
160 purification in order to isolate soluble monomeric (stable) forms of the proteins.
Both Sup 75 and 200 columns (with different fractionation ranges) were used.
Fig 6.3 Glutathione s-transferase (GST) fusion tags enhance solubility of ASC
proteins. Apart from introducing the GST tag, the new genes contain a C-terminal His
tag and two linker regions [SDGSSMGSS, GSGSLE ] to improve protein flexibility and
solubility. Three GST-ASC constructs were designed: GST-ASC PYD, GST-ASC PYD
K21A mut (prevents aggregation), and GST-ASC CARD [2-5]. GST-ASC protein
schematics were designed by Dr. Nichols.
6.2.1. Optimizing protein purification conditions using GST-ASC PYD
K21A
Various protein solubilization methods were employed to reverse the aggregation process in a bid to obtain soluble, stable proteins. The GST-ASC
PYD K21A mutant exhibited improved protein solubility and significant changes in elution profiles under various conditions are shown in Fig 6.4.
161
A. B. 600 20 400 10 No prior treatment No prior treatment
500 8 15 300 400 6
300 10 200 mAU mAU 4 200 100 5 2 100
0 0 0 0 6 8 10 12 14 16 18 20 5 10 15 20 Volume (mL) Volume (mL) BSA (primary axis) BSA (primary axis) GST-ASC K21A, 210 ug GST-ASC K21A, 210 ug
C. D. 400 10 600 10 6 M GuHCl/ 2 mM DTT treatment 6 M GuHCl/ 2 mM DTT treatment 500 8 8 300 400 6 6
200 300 mAU mAU 4 4 200 100 2 100 2
0 0 0 0 5 10 15 20 6 8 10 12 14 16 18 20 Volume (mL) Volume (mL) BSA (primary axis) BSA (primary axis) GST-ASC K21A, 84 ug GST-ASC K21A, 84 ug
E. F. 600 600 40 150 mM KCl treatment 14 O/N dialysis 500 500 12 30 400 10 400
300 8 300 20 mAU mAU 6 200 200 4 10 100 100 2 0 0 0 0 6 8 10 12 14 16 18 20 6 8 10 12 14 16 18 20 Volume (mL) Volume (mL) BSA (primary axis) BSA (primary axis) GST-ASC K21A, 100 ug GST-ASC K21A, 230 ug
162
Fig 6.4. Optimizing sample treatment and purification conditions using affinity-purified
GST-ASC PYD K21A mut. All purifications were done in PBS buffer. Panel A. Sup
200, no prior sample treatment. Panel B. Sup 75, no prior sample treatment Panel C.
Sup 75, 6 M GuHCl 2 mM DTT treatment Panel D. Sup 200, 6 M GuHCl 2 mM DTT
Panel E. Sup 200, 150 mM KCl treatment at 4 oC for 2 days Panel F. Sup 200,
overnight dialysis at 4 oC in 20 mM HEPES, 150 mM KCl, 2 mM DTT.
Initially, there was no sample treatment prior to SEC purification resulting in most of the protein eluting in the void volume after purification with Sup 200
(Panel A) and Sup 75 (Panel B) columns. To shift the elution profile, the next strategy involved using a combination of a denaturants and reducing agents (6 M urea/ 6 M GuHCl and 2 mM DTT) followed by overnight incubation at room temp
(Panels C, D) but there was no noticeable improvement. Ionic interactions play a big part in ASC self-assembly and these can be easily disrupted by high amounts of salt. Since previous studies [147] revealed that ASC was soluble at high concentrations of K+ (150 mM), the GST-ASC recombinant proteins were subjected to 150 mM KCl treatment then incubated overnight, 2-or 4-days at 4 oC
(Panel E). Just like the prior sample treatments, there was not a significant shift.
Ultimately, overnight dialysis at 4 oC in 20 mM HEPES buffer containing 2 mM
DTT & 150 mM KCl, pH 8.0 yielded the best results. As seen in Panel F, there was a huge shift towards the included volume.
163
Although the solubilization methods yielded stable protein with the PYD mutant, there was not much improvement with GST-ASC PYD and GST-ASC
CARD domains as most of the protein still eluted in the void volume thus indicating high degrees of aggregation.
6.2.2. The fusion proteins exhibited concentration-dependent
aggregation
Protein concentration is another important factor when looking at protein aggregation. SEC traces of affinity-purified peak fractions vs side fractions showed concentration-dependent aggregation (Fig 6.5). Considering GST-ASC
PYD, the elution profiles for the peak and side fractions look very similar (Panel
A). The WT protein showed high levels of aggregation even at low concentrations. On the other hand, there are distinct differences between the peak and side fractions of the K21A MUT (Panel B). The peak fraction showed significant amounts of aggregated protein (void) while most of the protein in the less-concentrated side fraction eluted in the included volume. The included peak was proposed to be a dimer. As expected, the K21A mutation in the pyrin domain greatly enhanced solubility of the domain in comparison with the WT.
164
A. B.
600 50 600 50
500 40 500 40 400 400 30 30
300 300 mAU 20 mAU 20 200 200 10 100 100 10
0 0 0 0 6 8 10 12 14 16 18 20 6 8 10 12 14 16 18 20 Volume (mL) Volume (mL)
BSA (primary axis) BSA (primary axis) GST-ASC PYD, 280 ug GST-ASC PYD K21A, 480 ug GST-ASC PYD, 110 ug GST-ASC PYD K21A, 210 ug
Fig 6.5 Superdex 200 elutions of GST-ASC proteins show concentration-dependent
aggregation. Briefly, proteins were harvested 2 days post-transfection. Lysates were
affinity-purified the same day. Peak fractions were immediately pooled then dialyzed
overnight at 4 oC in 20 mM HEPES buffer containing 2 mM DTT & 150 mM KCl,
samples concentrated 10x, centrifuged for 10 mins at 17,000g, 0.5 mL sup loaded onto
column.
6.2.3. The soluble and insoluble fractions of GST-ASC CARD showed
distinct elution profiles
Additional interesting observations were made with GST-ASC CARD.
Overnight dialysis of the affinity-purified peak fractions in HEPES buffer resulted in some of the protein precipitating out of solution. Centrifugation for 10 mins at
17,000g yielded a significant pellet which was solubilized in 6 M GuHCl. Both the supernatant and solubilized pellet were eluted in PBS and the elution traces are
165 shown in Fig 6.6. The pellet represents the insoluble fraction of the protein as evidenced by most of the sample eluting in the void. In contrast, a larger percentage of the supernatant eluted in the included volume thus indicating soluble protein.
600 30
500 25
400 20
300 15
mAU mAU 200 10
100 5
0 0 6 8 10 12 14 16 18 20 Volume (mL) BSA (primary axis) GST-ASC CARD pellet, 105 ug GST-ASC CARD sup, 105 ug
Fig 6.6 Elution profiles of GST-ASC CARD pellet and supernatant samples are
distinct. Dialysis was done right after affinity purification in 20 mM HEPES buffer
containing 2 mM DTT & 150 mM KCl. The sample precipitated out of the solution
during dialysis. After 17,000g centrifugation for 10 mins, a pellet was observed and
was later solubilized in 6 M GuHCl. Both solubilized pellet and supernatant were
eluted in PBS on a Sup 200 column.
166
6.2.4. SEC-purified GST-ASC proteins were fairly stable over time
Since full-length ASC and/or domains are prone to aggregation over time, stability studies were conducted after 1 month of storing the proteins at 4 oC. For these studies, I compared the stabilities of affinity-purified samples (dialyzed overnight in HEPES KCl buffer) and the included fractions after Sup 200- purification. As Fig 6.7 Panel A shows, dialyzed, affinity-purified GST-ASC PYD
K21A was unstable over time as evidenced by the drastic decrease in the dimer peak when compared with the original sample. On the other hand, the re-purified
SEC fraction 25 was quite stable during the same period of time. Its elution profile matched that of the original sample although the yields were quite low.
This was also confirmed by DLS experiments as shown in Table 6.4. These findings highlight the importance of SEC to remove pre-formed aggregates that may promote seeding and subsequent oligomerization leading to aggregate formation.
In comparison, dialyzed GST-ASC CARD (Panel B) was stable over time.
The elution traces of the dialyzed sample after a month were quite similar to that of the original dialyzed sample. Re-purification of the included fraction 25 dimer resulted in extremely low yields. Interestingly, data obtained from DLS experiments revealed that the GST-ASC CARD was not stable over time as evidenced by the increase in the hydrodynamic radius (Table 6.4). These findings, however, were in disagreement with the SEC data. The DLS data also indicated that the WT PYD is highly aggregated compared to the K21A MUT thus
167 confirming the importance of the single mutation in preventing self-association of the domain.
600 50
500 40 A. 400 30
300 mAU 20 mAU 200
100 10
0 0 6 8 10 12 14 16 18 20 Volume (mL) BSA (primary axis) GST-ASC PYD K21A, 210 ug GST-ASC PYD K21A frac 25 GST-ASC PYD K21A, 1 month
600 30
500 25
400 20 B. 300 15
mAU
mAU 200 10
100 5
0 0 6 8 10 12 14 16 18 20 Volume (mL)
BSA (primary axis) GST-ASC CARD sup, 105 ug GST-ASC CARD sup, 105 ug, 1 month Sup frac 25, 1 month later
Fig 6.7 Stability of GST-ASC fusion proteins. To check protein stability over a 1-month period, both dialyzedB affinity - purified and Sup 200 included fraction 25 samples were assessed. BSA was used as a control protein (blue trace). Panel A. Sup 200 elution
traces for GST-ASC K21A MUT; dialyzed sample on day 1 (dark green trace), dialyzed
sample after 1 month (light purple trace), re-purification of SEC-purified fraction 25 after
1 month (red trace). Panel B. Sup 200 elution traces for GST-ASC CARD; original
dialyzed sample ( grey trace) and 1-month later (light purple trace), re-purification of frac
25 dimer had extremely low recovery (light green trace).
168
Table 6.4 DLS data of GST-ASC fusion proteins. DLS experiments
were conducted with Sup 200-purified proteins to test the changes in
the sizes of the proteins over a 1-month period. Included (GST-ASC
CARD & PYD K21A MUT) or void fractions ( GST-ASC PYD) were
assessed in these studies.
6.2.5. The oligomerization state of SEC-purified GST-ASC fusion
proteins affects detection by immunoblotting methods
SEC-purified GST-ASC fusion proteins were also assessed by western blotting as shown in Fig 6.8. For these studies, void and included peak fractions were analyzed. The void and included peak fractions of GST-ASC PYD K21A were detected as monomers using the anti-His antibody. However, the same antibody did not recognize any of the GST-ASC PYD WT samples. While the included sample of the CARD domain was detected, the void sample was not recognized by the antibody just like the PYD WT samples. These findings show
169 that the antibody only recognizes certain oligomeric forms of the proteins and that the binding epitope may be inaccessible in some conformations. The WT
PYD and the CARD domains are more prone to aggregation compared to the
PYD K21A MUT thus these findings make sense. Similar trends were observed using anti-GST antibody hence these findings confirm the importance of the oligomerization state of the proteins when it comes to detection by immunoblotting.
Fig 6.8 Western blot of SEC-purified GST-ASC fusion proteins. Sup 200-purified void
and included peak fractions were analyzed by SDS-PAGE followed by immunoblotting
with anti-His primary antibody and anti-mouse secondary antibody. Loaded amounts
were above minimum detection limit (usually 1 µg). All GST-ASC monomeric proteins
~39 kD.
170
Summary
A lot of valuable information was obtained from the GST-ASC fusion domains. After testing a variety of buffers and sample treatment conditions, overnight dialysis of affinity-purified proteins in HEPES KCl buffer prior to SEC purification emerged as the best strategy for obtaining stable, soluble proteins.
Inclusion of the K21A mutation immensely helped with the solubility of the PYD as shown by the SEC, DLS, and immunoblotting experiments. The WT PYD, however, was highly aggregated and resistant to all the solubilizing methods that were employed. Interesting findings were also obtained from the CARD domain sample which precipitated during dialysis. The soluble form of the protein proved to be very stable while the insoluble fraction (in the pellet) was highly aggregated.
From these findings, the aggregation trend of these GST-ASC fusion proteins is as follows: PYD> CARD> PYD K21A MUT. This means that the PYD of ASC is mainly responsible for its aggregation.
Besides aggregation, another huge challenge with the GST-ASC proteins were the extremely low percent recoveries < 40% during SEC indicating that most of the protein was lost during the purification process. Since this trend was not observed in the previous preps with other constructs, it is possible that the
GST tag was interfering with the SEC purifications. Nonetheless, including the
GST solubility tags proved to be extremely helpful as that shifted the elution profiles from the void towards the included volume when compared to other fusion tags (MBP, SUMO). Of course, it is quite evident that overnight dialysis in
HEPES KCl buffers also played a major part in solubilizing the aggregates. Due
171 to the promising data with the ASC domains, follow-up studies will involve incorporating the GST solubility tag and dialysis strategies in future expressions of full-length ASC, NLRP3, and procaspase-1.
6.3. Aβ-ASC aggregation assays
A complex relationship exists between the NLRP3 inflammasome
(proteins) and Aβ. Studies have revealed that ASC cross-seeding promotes deposition of Aβ fibrils. Co-immunoprecipitation experiments showed that ASC specks co-sedimented with Aβ forming plaques [2, 35, 56, 116, 158]. Hence, I studied the effect of the GST-ASC domains on Aβ monomer aggregation.
Aggregation assays were conducted as outlined in detail in the Methods section and the increase in thioflavin T (ThT) fluorescence was measured over time.
6.3.1. GST-ASC domains did not promote aggregation of Aβ
monomer
GST-ASC proteins were purified by SEC and eluted in PBS. The void and included peak fractions were used for the aggregation assays. Preliminary studies show that the ASC domains have no effect on Aβ aggregation (Fig 6.9).
Aggregation studies were conducted with Sup 75 oligomer (Panel A) and void peak fractions (Panel B). Studies in Panel C were conducted with ASC fusion proteins from a different purification, and Sup 200 void peak fractions were
172 selected for both GST-ASC PYD and GST-CARD domains. The included peak fraction of GST-ASC PYD K21A was used.
A.
B.
C.
173
Fig 6.9 ASC domains had no effect on Aβ aggregation. GST-ASC proteins were
purified by SEC under various conditions and eluted in PBS Panel A. Oligomer peak
fractions (16) from sup 75 purifications (no prior sample treatment) were used.
Aggregation assays were conducted at 37 oC as described in Methods. In this assay,
the ratio of Aβ:ASC in the reaction vessel was 100:1. Panel B. Void peak fractions
(13) from the same SEC purification as Panel A were used and assays conducted in a
similar manner. Panel C. Another purification was done using Sup 200 column. Prior
GST-ASC sample treatment was done in 6 M GuHCl/ 2 mM DTT and elutions were
done in PBS. Void peak fractions (12 or 13) were selected for PYD and CARD
domains. Included fraction 23 was used for PYD K21A mut. In these assays, the ratio
of Aβ:ASC in the reaction vessel was 100:1 for the PYD WT and MUT domains.
Aβ:ASC ratio of 10:1 was used for the CARD domain. Aggregation assays were
conducted by Shikha Grover.
Summary
Although these preliminary studies have shown that the GST-ASC fusion proteins have no effect on Aβ monomer aggregation, we are yet to determine whether the GST tag interferes with these assays. Important data can be obtained from the aggregation studies with ASC domains i.e the contribution of each domain in promoting aggregation of Aβ or vice versa. However, experiments with the individual ASC domains do not model the actual physiological processes. Follow up studies will need to be conducted with both soluble and insoluble fractions of full-length ASC to get a better understanding of the interplay between Aβ aggregation and ASC speck formation.
174
CHAPTER 7: OVERALL DISCUSSIONS, CONCLUSIONS, & FUTURE
DIRECTIONS
The NLRP3 inflammasome still remains a mystery in terms of its actual structure and the exact processes leading to its activation. So far, no group has done comprehensive structural studies of the NLRP3 inflammasome as a whole to determine its actual stoichiometry. In addition, the exact mechanisms employed by NLRP3 activators are still not clear. Understanding the mechanistic pathways leading to the activation of the NLRP3 inflammasome and procaspase-
1 is crucial in establishing targets for therapeutic intervention in an effort to treat inflammatory disorders. To study the NLRP3 inflammasome in detail, this project involved overexpressing the three inflammasome proteins in a mammalian cell system, conducting structural studies to study the stoichiometry of the recombinant proteins, and ultimately using the recombinant proteins for functional studies.
7.1. Conclusions
Recombinant expression of NLRP3 inflammasome proteins is quite a challenging process due to their inherent propensity to aggregate. Some studies have shown attempts to reconstitute the inflammasome in vitro by co-expressing the proteins with pro-IL-1β and monitoring cleavage of caspase-1 and IL-1β as a measure of inflammasome activity. However, no group so far has attempted to reconstitute the inflammasome using the individual recombinant proteins.
175
Conducting these studies in vitro is quite a challenging task considering the fact that a variety of cell signaling pathways and regulatory proteins normally found in vivo are not available under laboratory settings.
Endogenous inflammasome protein amounts in immune cells are very low and not enough for large scale functional studies thus we resorted to recombinant overexpression of the proteins. We specifically selected the
ExpiCHO-S mammalian cell system for our studies because the cell line has been successfully used to express secreted antibodies. Because the cells grow in suspension and survive at high cellular densities, this allows for more efficient overexpression of proteins without drastic changes in cell viability. Hence, I was able to successfully express full-length, truncated, and mutant versions of the inflammasome proteins. Proteins isolated from both the lysates and inclusion bodies were selectively purified by affinity chromatography on a Ni2+ column. I obtained relatively large yields of all the proteins and most of them were found in the lysates.
Subsequent analysis of the purified samples by SDS-PAGE and immunoblotting revealed the presence of monomeric proteins and SDS-resistant aggregates. I was able to obtain some structural information on some of the proteins pertaining to their sizes and conformations using biophysical techniques
(SEC, MALS, DLS, CD, TEM). The main challenge, however, was obtaining stable, solution proteins for subsequent functional assays since majority of the proteins existed as aggregates in solution. Efforts to break these aggregates using stringent conditions and inclusion of solubility tags were successful to a
176 certain extend as small amounts of the proteins were obtained in monomeric form.
Expressing the proteins for various periods of times did not produce any differences in the amounts expressed or degree of aggregation. Thus, protein aggregation most likely occurred during expression. Inherent properties of the inflammasome proteins tend to promote aggregation under physiological conditions. In addition, various groups that have recombinantly expressed the inflammasome proteins also reported aggregation. Therefore, in vitro studies with inflammasome proteins can be quite challenging. Our attempts to reconstitute the
NLRP3 inflammasome in vitro were not successful. This also brings about the question whether this is actually feasible, and also if the ExpiCHO-S cells present a good expression system for these proteins.
In other studies, caspase-1 activity assays produced some interesting findings. For the first time ever, I observed enzyme activity with the recombinant procaspase-1 using a fluorescence assay. This was quite unexpected and raised more questions regarding the actual form of the active caspase and what domains are required for activity. Whether procaspase-1 activity is relevant under physiological conditions is yet to be determined. Since recent literature findings contradicted the precious findings on the exact form of active caspase-1, this raises the possibility that there could be many forms of the active enzyme, some of which may not have been identified yet. In that case, this makes caspase-1 a difficult target for therapeutic intervention when treating inflammatory disorders.
177
Another important aspect of my studies was to study the interactions between Aβ and the inflammasome proteins. What makes this study distinct from others is the fact that I used SEC-purified Aβ-42 protofibrils which have been previously identified as the most robust form of Aβ. Other studies, however, used a mixture of different Aβ species in their experiments. I was able to show direct interactions between Aβ protofibrils and NLRP3. These interactions are relevant in the immune response pathway and blocking these interactions seems a plausible approach towards preventing AD-related neuroinflammation.
Although direct interactions between Aβ and NLRP3 have been studied, more studies are leaning towards Aβ-ASC interactions. ASC studies are important since formation of ASC specks is a crucial step in the assembly of the
NLRP3 inflammasome. For these studies, I used ASC domains tagged with a
GST tag for added solubility. Compared with the previous expressions, the yields obtained with the GST-tagged proteins were comparatively low. The PYD K21A
MUT (does not oligomerize) and CARD domains showed enhanced solubility and appeared as dimers in solution. In addition, the soluble forms of these domains were quite stable when stored at 4 oC. Meanwhile, the PYD WT was highly aggregated even in the presence of the GST fusion tag. Although the single mutation in the PYD binding surfaces prevents self-oligomerization and may not represent actual physiological states, I was able to obtain important structural information using the PYD K21A mutant with enhanced solubility. With regards to the GST-ASC domains, the overall propensity for oligomer formations is as follows: PYD> CARD> PYD K21A MUT. These data also reveal that PYD of ASC
178 has the greatest contribution to the overall aggregation of full-length ASC compared to the CARD domain.
Preliminary studies showed that the ASC domains had no effect on Aβ monomer aggregation contrary to previous published studies. However, it is yet to be established whether the GST tag may be interfering with the assays hence future aggregation experiments will be conducted after removal of the fusion tag.
It will be interesting to explore the stability of these domains in solution over time and how they affect Aβ aggregation. Since the current studies have utilized ASC domains, follow-up aggregation studies with the full-length GST-ASC will be conducted to get a better model system for the actual physiological events. In sum, the studies presented above revealed some crucial findings pertaining to the interplay between Aβ and the NLRP3 inflammasome. In addition, they also brought about interesting possible mechanisms governing caspase-1 structure and function.
179
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VITA
Nyasha was born and raised in Rusape, a small town located in the eastern part of Zimbabwe. She received her elementary education from Chiwetu and Vengere Primary Schools then proceeded to Vengere High School for her
Ordinary Level studies. Later, she enrolled at St. David’s Girls High (Bonda) in
Mutasa District for her Advanced Level studies and obtained her high school diploma in 2008. After taking a gap year, she came to the US in July 2010 and settled in Memphis, TN. She began her undergraduate studies that fall at the
LeMoyne-Owen College (LOC), a liberal arts college. During her time at LOC, she conducted undergraduate research with Dr. Yahia Hamada studying copper/glycine complexes and their roles in prion diseases. She was also an undergraduate summer research scholar (2012 and 2013) at the University of
Tennessee Health Science Center (UTHSC) in the lab of Dr. Michio Kurosu. In the Kurosu lab she utilized M. smegmatis as an assay tool to develop serine/threonine inhibitors for multi-drug resistant M. tuberculosis infections. She graduated Summa Cum Laude with a Bachelor of Science degree in Chemistry
(and a minor in Biology) in May 2014. She joined the UMSL Chemistry doctoral program in the fall semester of 2015 and later joined the Nichols lab the same semester. She obtained a Master of Science in Chemistry in August 2017.
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