Cytosolic sensing of aberrant DNA: Arming STING on the endoplasmic reticulum

Expert Opinion on Therapeutic Targets

Volumn 19, Issue 10, 2015, Pages 1397-1409

  Wang, Qiang ^a   Liu, Xing ^a   Zhou, Qin ^b   Wang, Chen ^a  

^a INSTITUTE OF BIOCHEMISTRY AND CELL BIOLOGY   (China)

^b CHONGQING MEDICAL UNIVERSITY   (China)

Author keywords

autoimmune diseases; DNA sensors; DNA vaccines; innate immunity; STING; type I interferon

Indexed keywords

CYCLIC AMP; CYCLIC GMP; DEAD BOX POLYPEPTIDE 41; DEAD BOX PROTEIN; DEOXYRIBONUCLEOPROTEIN; DNA; DNA VACCINE; EXTRACHROMOSOMAL DNA; INTERFERON INDUCIBLE PROTEIN 16; STING PROTEIN; TOLL LIKE RECEPTOR 9; UNCLASSIFIED DRUG; ANTIINFECTIVE AGENT; MEMBRANE PROTEIN; MPYS PROTEIN, HUMAN;

APOPTOSIS; AUTOIMMUNE DISEASE; ENDOPLASMIC RETICULUM; ENDOSOME; GENE; HUMAN; IMMUNOGENICITY; INNATE IMMUNITY; MELANOMA; NONHUMAN; PROTEIN PHOSPHORYLATION; REVIEW; STIMULATOR OF INTERFERON GENE; UBIQUITINATION; ANIMAL; AUTOIMMUNE DISEASES; CYTOSOL; DRUG DESIGN; IMMUNOLOGY; METABOLISM; SIGNAL TRANSDUCTION;

ANIMALS; ANTI-INFECTIVE AGENTS; AUTOIMMUNE DISEASES; CYTOSOL; DNA; DRUG DESIGN; ENDOPLASMIC RETICULUM; HUMANS; IMMUNITY, INNATE; MEMBRANE PROTEINS; SIGNAL TRANSDUCTION; VACCINES, DNA;

EID: 84941805904     PISSN: 14728222     EISSN: 17447631     Source Type: Journal    
DOI: 10.1517/14728222.2015.1067303     Document Type: Review

References (119)

1. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006; 124: 783-801
2. Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol 2009; 21: 317-37
3. Liu X, Wang Q, Chen W, et al. Dynamic regulation of innate immunity by ubiquitin and ubiquitin-like proteins. Cytokine Growth Factor Rev 2013; 24: 559-70
4. Yoneyama M, Fujita T. RNA recognition and signal transduction by RIG-I-like receptors. Immunol Rev 2009; 227: 54-65
5. Loo YM, Gale M Jr. Immune signaling by RIG-I-like receptors. Immunity 2011; 34: 680-92
6. Paludan SR, Bowie AG. Immune sensing of DNA. Immunity 2013; 38: 870-80
7. Sharma S, Fitzgerald KA. Innate immune sensing of DNA. Plos Pathog 2011; 7: e1001310
8. Palm NW, Medzhitov R. Pattern recognition receptors and control of adaptive immunity. Immunol Rev 2009; 227: 221-33
9. Yang YG, Lindahl T, Barnes DE. Trex1 exonuclease degrades ssDNA to prevent chronic checkpoint activation and autoimmune disease. Cell 2007; 131: 873-86
10. Gall A, Treuting P, Elkon KB, et al. Autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease. Immunity 2012; 36: 120-31
11. Rumore PM, Steinman CR. Endogenous circulating DNA in systemic lupus-erythematosus-occurrence as multimeric complexes bound to histone. J Clin Invest 1990; 86: 69-74
12. Smith S, Jefferies C. Role of DNA/RNA sensors and contribution to autoimmunity. Cytokine Growth Factor Rev 2014; 25: 745-57
13. Liu MA. DNA vaccines: an historical perspective and view to the future. Immunol Rev 2011; 239: 62-84
14. Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408: 740-5
15. Gurunathan S, Klinman DM, Seder RA. DNA vaccines: immunology, application, and optimization. Annu Rev Immunol 2000; 18: 927-74
16. Spies B, Hochrein H, Vabulas M, et al. Vaccination with plasmid DNA activates dendritic cells via Toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice. J Immunol 2003; 171: 5908-12
17. Babiuk S, Mookherjee N, Pontarollo R, et al. TLR9-/-and TLR9+/+ mice display similar immune responses to a DNA vaccine. Immunology 2004; 113: 114-20
18. Stetson DB, Medzhitov R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 2006; 24: 93-103
19. Ishii KJ, Coban C, Kato H, et al. A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA. Nat Immunol 2006; 7: 40-8
20. Honda K, Ohba Y, Yanai H, et al. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 2005; 434: 1035-40
21. Kawai T, Sato S, Ishii KJ, et al. Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. Nat Immunol 2004; 5: 1061-8
22. Schroder K, Muruve DA, Tschopp J. Innate Immunity: Cytoplasmic DNA Sensing by the AIM2 Inflammasome. Curr Biol 2009; 19: R262-5
23. Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 2008; 455: 674-8
24. Barber GN. STING-dependent cytosolic DNA sensing pathways. Trends Immunol 2014; 35: 88-93
25. Hornung V, Ablasser A, Charrel-Dennis M, et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 2009; 458: 514-18
26. Fernandes-Alnemri T, Yu JW, Datta P, et al. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 2009; 458: 509-13
27. Burckstummer T, Baumann C, Bluml S, et al. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat Immunol 2009; 10: 266-72
28. Rathinam VA, Jiang ZZ, Waggoner SN, et al. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat Immunol 2010; 11: 395-403
29. Kerur N, Veettil MV, Sharma-Walia N, et al. IFI16 Acts as a Nuclear Pathogen Sensor to Induce the Inflammasome in Response to Kaposi Sarcoma-Associated Herpesvirus Infection. Cell Host Microbe 2011; 9: 363-75
30. Takaoka A, Wang Z, Choi MK, et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 2007; 448: 501-5
31. Zhang Z, Yuan B, Bao M, et al. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat Immunol 2011; 12: 959-65
32. Sun LJ, Wu JX, Du FH, et al. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 2013; 339: 786-91
33. Li XD, Wu JX, Gao DX, et al. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 2013; 341: 1390-4
34. Unterholzner L, Keating SE, Baran M, et al. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol 2010; 11: 997-1004
35. Jakobsen MR, Paludan SR. IFI16: At the interphase between innate DNA sensing and genome regulation. Cytokine Growth Factor Rev 2014; 25: 649-55
36. Wang ZC, Choi MK, Ban T, et al. Regulation of innate immune responses by DAI (DLM-1/ZBP1) and other DNA-sensing molecules. Proc Natl Acad Sci USA 2008; 105: 5477-82
37. Ishii KJ, Kawagoe T, Koyama S, et al. TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines. Nature 2008; 451: 725-9
38. Lladser A, Mougiakakos D, Tufvesson H, et al. DAI (DLM-1/ZBP1) as a Genetic Adjuvant for DNA Vaccines That Promotes Effective Antitumor CTL Immunity. Mol Ther 2011; 19: 594-601
39. Parvatiyar K, Zhang ZQ, Teles RM, et al. The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response. Nat Immunol 2012; 13: 1155-61
40. Lee KG, Kim SS, Kui L, et al. Brutons Tyrosine Kinase Phosphorylates DDX41 and Activates Its Binding of dsDNA and STING to Initiate Type 1 Interferon Response. Cell Reports 2015; 10: 1055-65
41. Chen ZH, Schaap P. The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium. Nature 2012; 488: 680-3
42. Karaolis DKR, Rashid MH. Chythanya R, et al. c-di-GMP (3-5-cyclic diguanylic acid) inhibits Staphylococcus aureus cell-cell interactions and biofilm formation. Antimicrob Agents Ch 2005; 49: 1029-38
43. Tamayo R, Pratt JT, Camilli A. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annu Rev Microbiol 2007; 61: 131-48
44. McWhirter SM, Barbalat R, Monroe KM, et al. A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP. J Exp Med 2009; 206: 1899-911
45. Woodward JJ, Iavarone AT, Portnoy DA. c-di-AMP Secreted by Intracellular Listeria monocytogenes Activates a Host Type I Interferon Response. Science 2010; 328: 1703-5
46. Chen WX, KuoLee R, Yan HB. The potential of 3,5-cyclic diguanylic acid (c-di-GMP) as an effective vaccine adjuvant. Vaccine 2010; 28: 3080-5
47. Ebensen T, Libanova R, Schulze K, et al. Bis-(3,5)-cyclic dimeric adenosine monophosphate: Strong Th1/Th2/Th17 promoting mucosal adjuvant. Vaccine 2011; 29: 5210-20
48. Libanova R, Becker PD, Guzman CA. Cyclic di-nucleotides: new era for small molecules as adjuvants. Microb Biotechnol 2012; 5: 168-76
49. Zhang X, Wu J, Du F, et al. The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop. Cell Reports 2014; 6: 421-30
50. Kranzusch PJ, Lee AS, Berger JM, et al. Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity. Cell Reports 2013; 3: 1362-8
51. Wu JX, Sun LJ, Chen X, et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 2013; 339: 826-30
52. Zhang X, Shi HP, Wu JX, et al. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell 2013; 51: 226-35
53. Gao P, Ascano M, Wu Y, et al. Cyclic [G(2,5) pA(3,5)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase. Cell 2013; 153: 1094-107
54. Ablasser A, Goldeck M, Cavlar T, et al. cGAS produces a 2-5-linked cyclic dinucleotide second messenger that activates STING. Nature 2013; 498: 380-4
55. Diner EJ, Burdette DL, Wilson SC, et al. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Reports 2013; 3: 1355-61
56. Skrnjug I, Guzman CA, Ruecker C. Cyclic GMP-AMP displays mucosal adjuvant activity in mice. Plos One 2014; 9 (10): e110150
57. Zhong B, Yang Y, Li S, et al. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity 2008; 29: 538-50
58. Sun WX, Li Y, Chen L, et al. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization. Proc Natl Acad Sci USA 2009; 106: 8653-8
59. Jin L, Waterman PM, Jonscher KR, et al. MPYS, a novel membrane tetraspanner, is associated with major histocompatibility complex class II and mediates transduction of apoptotic signals. Mol Cell Biol 2008; 28: 5014-26
60. Ishikawa H, Ma Z, Barber GN. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 2009; 461: 788-92
61. Sun W, Li Y, Chen L, et al. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization. Proc Natl Acad Sci USA 2009; 106: 8653-8
62. White MJ, McArthur K, Metcalf D, et al. Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production. Cell 2014; 159: 1549-62
63. Rongvaux A, Jackson R, Harman CCD, et al. Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 2014; 159: 1563-77
64. West AP, Khoury-Hanold W, Staron M, et al. Mitochondrial DNA stress primes the antiviral innate immune response. Nature 2015; 520: 553-7
65. Hartlova A, Erttmann SF, Raffi FA, et al. DNA damage primes the type I interferon system via the cytosolic DNA sensor STING to promote anti-microbial innate immunity. Immunity 2015; 42: 332-43
66. Woo SR, Fuertes MB, Corrales L, et al. STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity 2014; 41: 830-42
67. Deng L, Liang H, Xu M, et al. STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors. Immunity 2014; 41: 843-52
68. Ahn J, Gutman D, Saijo S, et al. STING manifests self DNA-dependent inflammatory disease. Proc Natl Acad Sci USA 2012; 109: 19386-91
69. Ahn J, Barber GN. Self-DNA, STING-dependent signaling and the origins of autoinflammatory disease. Curr Opin Immunol 2014; 31: 121-6
70. Evans CJ, Aguilera RJ. DNase II: genes, enzymes and function. Gene 2003; 322: 1-15
71. Nagata S. DNA degradation in development and programmed cell death. Annu Rev Immunol 2005; 23: 853-75
72. Kawane K, Fukuyama H, Kondoh G, et al. Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver. Science 2001; 292: 1546-9
73. Kawane K, Fukuyama H, Yoshida H, et al. Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation. Nat Immunol 2003; 4: 138-44
74. Yoshida H, Okabe Y, Kawane K, et al. Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA. Nat Immunol 2005; 6: 49-56
75. Kawane K, Ohtani M, Miwa K, et al. Chronic polyarthritis caused by mammalian DNA that escapes from degradation in macrophages. Nature 2006; 443: 998-1002
76. Ahn J, Gutman D, Saijo S, et al. STING manifests self DNA-dependent inflammatory disease. Proc Natl Acad Sci USA 2012; 109: 19386-91
77. Lan YY, Londono D, Bouley R, et al. Dnase2a deficiency uncovers lysosomal clearance of damaged nuclear DNA via autophagy. Cell Reports 2014; 9: 180-92
78. Mazur DJ, Perrino FW. Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3->5 exonucleases. J Biol Chem 1999; 274: 19655-60
79. Lee-Kirsch MA, Gong M, Chowdhury D, et al. Mutations in the gene encoding the 3-5 DNA exonuclease TREX1 are associated with systemic lupus erythematosus. Nat Genet 2007; 39: 1065-7
80. Crow YJ, Hayward BE, Parmar R, et al. Mutations in the gene encoding the 3-5 DNA exonuclease TREX1 cause Aicardi-Goutieres syndrome at the AGS1 locus. Nat Genet 2006; 38: 917-20
81. Morita M, Stamp G, Robins P, et al. Gene-targeted mice lacking the Trex1 (DNase III) 3->5 DNA exonuclease develop inflammatory myocarditis. Mol Cell Biol 2004; 24: 6719-27
82. Stetson DB, Ko JS, Heidmann T, et al. Trex1 prevents cell-intrinsic initiation of autoimmunity. Cell 2008; 134: 587-98
83. Yasutomo K, Horiuchi T, Kagami S, et al. Mutation of DNASE1 in people with systemic lupus erythematosus. Nat Genet 2001; 28: 313-14
84. Napirei M, Karsunky H, Zevnik B, et al. Features of systemic lupus erythematosus in Dnase1-deficient mice. Nat Genet 2000; 25: 177-81
85. Sze A, Belgnaoui SM, Olagnier D, et al. Host restriction factor SAMHD1 limits human T cell leukemia virus type 1 infection of monocytes via STING-mediated apoptosis. Cell Host Microbe 2013; 14: 422-34
86. Petrasek J, Iracheta-Vellve A, Csak T, et al. STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease. Proc Natl Acad Sci USA 2013; 110: 16544-9
87. Liu MA. DNA vaccines: an historical perspective and view to the future. Immunol Rev 2011; 239: 62-84
88. Wang Q, Liu X, Cui Y, et al. The E3 ubiquitin ligase AMFR and INSIG1 bridge the activation of TBK1 kinase by modifying the adaptor STING. Immunity 2014; 41: 919-33
89. Shu HB, Wang YY. Adding to the STING. Immunity 2014; 41: 871-3
90. Tsuchida T, Zou JA, Saitoh T, et al. The ubiquitin ligase TRIM56 regulates innate immune responses to intracellular double-stranded DNA. Immunity 2010; 33: 765-76
91. Zhang J, Hu MM, Wang YY, et al. TRIM32 Protein Modulates Type I Interferon Induction and Cellular Antiviral Response by Targeting MITA/STING Protein for K63-linked Ubiquitination. J Biol Chem 2012; 287: 28646-55
92. Zhong B, Zhang Y, Tan B, et al. The E3 Ubiquitin Ligase RNF5 Targets Virus-Induced Signaling Adaptor for Ubiquitination and Degradation. J Immunol 2010; 184: 6249-55
93. Qin Y, Zhou MT, Hu MM, et al. RNF26 temporally regulates virus-triggered type I interferon induction by two distinct mechanisms. Plos Pathog 2014; 10 (9): e1004358
94. Konno H, Konno K, Barber GN. Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STING to prevent sustained innate immune signaling. Cell 2013; 155: 688-98
95. Tanaka Y, Chen ZJ. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci Signal 2012; 5 (214): ra20
96. Liu S, Cai X, Wu J, et al. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science 2015; 347: aaa2630
97. Zhang L, Mo JY, Swanson KV, et al. NLRC3, a Member of the NLR Family of Proteins, Is a Negative Regulator of Innate Immune Signaling Induced by the DNA Sensor STING. Immunity 2014; 40: 329-41
98. Zhou Q, Lin H, Wang SY, et al. The ER-associated protein ZDHHC1 is a positive regulator of DNA virus-triggered, MITA/STING-dependent innate immune signaling. Cell Host Microbe 2014; 16: 450-61
99. Furr SR, Chauhan VS, Moerdyk-Schauwecker MJ, et al. A role for DNA-dependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells. J Neuroinflammation 2011; 8: 99
100. Aguirre S, Maestre AM, Pagni S, et al. DENV inhibits type I IFN production in infected cells by cleaving human STING. Plos Pathog 2012; 8 (10): e1002934
101. Shu C, Li X, Li P. The mechanism of double-stranded DNA sensing through the cGAS-STING pathway. Cytokine Growth Factor Rev 2014; 25: 641-8
102. Liu X, Wang Q, Pan YD, et al. Sensing and responding to cytosolic viruses invasions: An orchestra of kaleidoscopic ubiquitinations. Cytokine Growth Factor Rev 2015; 26 (3): 379-87
103. Nitta S, Sakamoto N, Nakagawa M, et al. Hepatitis C virus NS4B protein targets STING and abrogates RIG-I-mediated type I interferon-dependent innate immunity. Hepatology 2013; 57: 46-58
104. Ding Q, Cao XZ, Lu J, et al. Hepatitis C virus NS4B blocks the interaction of STING and TBK1 to evade host innate immunity. J Hepatol 2013; 59: 52-8
105. Yu CY, Chang TH, Liang JJ, et al. Dengue virus targets the adaptor protein MITA to subvert host innate immunity. Plos Pathog 2012; 8 (6): e1002780
106. Sun L, Xing YL, Chen XJ, et al. Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STING-mediated signaling. Plos One 2012; 7 (2): e30802
107. Qian C, Liu J, Cao X. Innate signaling in the inflammatory immune disorders. Cytokine Growth Factor Rev 2014; 25: 731-8
108. Goubau D, Deddouche S, Reis e Sousa C. Cytosolic sensing of viruses. Immunity 2013; 38: 855-69
109. Chiu YH, MacMillan JB, Chen ZJ. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 2009; 138: 576-91
110. Ablasser A, Bauernfeind F, Hartmann G, et al. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol 2009; 10: 1065-U1040
111. Bowzard JB, Ranjan P, Sambhara S, et al. Antiviral defense: RIG-Ing the immune system to STING. Cytokine Growth Factor Rev 2009; 20: 1-5
112. Chen HH, Sun H, You FP, et al. Activation of STAT6 by STING is critical for antiviral innate immunity. Cell 2011; 147: 436-46
113. Cavlar T, Deimling T, Ablasser A, et al. Species-specific detection of the antiviral small-molecule compound CMA by STING. EMBO J 2013; 32: 1440-50
114. Tozer GM, Kanthou C, Baguley BC. Disrupting tumour blood vessels. Nat Rev Cancer 2005; 5: 423-35
115. Jameson MB, Thompson PI, Baguley BC, et al. Clinical aspects of a phase I trial of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a novel antivascular agent. Br J Cancer 2003; 88: 1844-50
116. Roberts ZJ, Goutagny N, Perera PY, et al. The chemotherapeutic agent DMXAA potently and specifically activates the TBK1-IRF-3 signaling axis. J Exp Med 2007; 204: 1559-69
117. Prantner D, Perkins DJ, Lai W, et al. 5,6-Dimethylxanthenone-4-acetic Acid (DMXAA) Activates Stimulator of Interferon Gene (STING)-dependent Innate Immune Pathways and Is Regulated by Mitochondrial Membrane Potential. J Biol Chem 2012; 287 (47): 39776-88
118. Conlon J, Burdette DL, Sharma S, et al. Mouse, but not Human STING, binds and signals in response to the vascular disrupting agent 5,6-Dimethylxanthenone-4-Acetic Acid. J Immunol 2013; 190: 5216-25
119. Gao P, Zillinger T, Wang W, et al. Binding-pocket and lid-region substitutions render human STING sensitive to the species-specific drug DMXAA. Cell Reports 2014; 8: 1668-76