Trafficking and processing of endosomal TLR (Homo sapiens)

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2, 5, 7, 11, 14...5, 213, 6, 9, 17, 20...1, 5, 9, 18, 22...4, 13, 15, 16, 19...4245, 10, 12, 29Golgi lumenneutral pHcytosolendolysosome lumenendoplasmic reticulum lumenendolysosome lumenintracellularTLR3/7/8/9TLR8 FL-TLR7 dimerCTSL2 UNC93B1H+TLR8 TLR3C-ter TLR7 dimerTLR8 dimerADPApo-GP96 dimerTLR9 HSP90B1 TLR3 intracellularTLR:UNC93B1TLR7 UNC93B1UNC93B1 HSP90B1 CNPY3 TLR9 CTSL(114-288) CTSS-like proteinsTLR3 intracellularTLR:UNC93B1TLR7(?-1049) TLR7 Cathepsins B, K, L, S TLR9 FL-TLR9 dimerTLR3 TLR8 folded FL-TLR7/8/9 TLR8 folded FL-TLR7/8/9 Legumain/CathepsinsUNC93B1 LGMN CTSS TLR9 TLR7/8/9C-ter-TLR9 dimerTLR8 folded FL-TLR7/8/9TLR9(1-?) CNPY3ATP TLR9(?-1032) ATPATP-boundGp96dimer:CNPY3:TLR7/8/9TLR9 TLR7 TLR3TLR7 N-ter TLR9 dimerTLR7 48, 238, 23


Mammalian TLR3, TLR7, TLR8, TLR9 are endosomal receptors that sense nucleic acids that have been released from endocytosed/phagocytosed bacteria, viruses or parasites. These TLRs have a ligand-recognition domain that faces the lumen of the endosome (which is topologically equivalent to the outside of the cell), a transmembrane domain, and a signaling domain that faces the cytosol.

Under normal conditions, self nucleic acids are not recognized by TLRs due to multiple levels of regulation including receptor compartmentalization, trafficking and proteolytic processing (Barton GM et al 2006, Ewald SE et al 2008). At steady state TLR3, TLR7, TLR8, TLR9 reside primarily in the endoplasmic reticulum (ER), however, their activation by specific ligands only occurs within acidified endolysosomal compartments (Hacker H et al 1998, Funami K et al 2004, Gibbard RJ et al 2006). Several chaperon proteins associate with TLRs in the ER to provide efficient translocation to endolysosome. Upon reaching endolysosomal compartments the ectodomains of TLR7 and TLR9 are proteolytically cleaved by cysteine endoproteases. Both full-length and cleaved C-terminus of TLR9 bind CpG-oligodeoxynucleotides, however it has been proposed that only the processed receptor is functional.<p> Although similar cleavage of TLR3 has been reported by Ewald et al 2011, other studies demonstrated that the N-terminal region of TLR3 ectodomain was implicated in ligand binding, thus TLR3 may function as a full-length receptor (Liu L et al 2008, Tokisue T et al 2008).<p> There are no data on TLR8 processing, although the cell biology of TLR8 is probably similar to TLR9 and TLR7 (Gibbard RJ et al 2006, Wei T et al 2009). View original pathway at Reactome.</div>


Pathway is converted from Reactome ID: 1679131
Reactome version: 75
Reactome Author 
Reactome Author: Shamovsky, Veronica

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  1. Kuznik A, Bencina M, Svajger U, Jeras M, Rozman B, Jerala R.; ''Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines.''; PubMed Europe PMC Scholia
  2. Liu L, Botos I, Wang Y, Leonard JN, Shiloach J, Segal DM, Davies DR.; ''Structural basis of toll-like receptor 3 signaling with double-stranded RNA.''; PubMed Europe PMC Scholia
  3. Koehn J, Huesken D, Jaritz M, Rot A, Zurini M, Dwertmann A, Beutler B, Korthäuer U.; ''Assessing the function of human UNC-93B in Toll-like receptor signaling and major histocompatibility complex II response.''; PubMed Europe PMC Scholia
  4. Liu B, Yang Y, Qiu Z, Staron M, Hong F, Li Y, Wu S, Li Y, Hao B, Bona R, Han D, Li Z.; ''Folding of Toll-like receptors by the HSP90 paralogue gp96 requires a substrate-specific cochaperone.''; PubMed Europe PMC Scholia
  5. Ewald SE, Engel A, Lee J, Wang M, Bogyo M, Barton GM.; ''Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase.''; PubMed Europe PMC Scholia
  6. Qi R, Hoose S, Schreiter J, Sawant KV, Lamb R, Ranjith-Kumar CT, Mills J, San Mateo L, Jordan JL, Kao CC.; ''Secretion of the human Toll-like receptor 3 ectodomain is affected by single nucleotide polymorphisms and regulated by Unc93b1.''; PubMed Europe PMC Scholia
  7. Wei T, Gong J, Jamitzky F, Heckl WM, Stark RW, Rössle SC.; ''Homology modeling of human Toll-like receptors TLR7, 8, and 9 ligand-binding domains.''; PubMed Europe PMC Scholia
  8. Chen J, Nag S, Vidi PA, Irudayaraj J.; ''Single molecule in vivo analysis of toll-like receptor 9 and CpG DNA interaction.''; PubMed Europe PMC Scholia
  9. Kim YM, Brinkmann MM, Paquet ME, Ploegh HL.; ''UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes.''; PubMed Europe PMC Scholia
  10. Park B, Brinkmann MM, Spooner E, Lee CC, Kim YM, Ploegh HL.; ''Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9.''; PubMed Europe PMC Scholia
  11. de Bouteiller O, Merck E, Hasan UA, Hubac S, Benguigui B, Trinchieri G, Bates EE, Caux C.; ''Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH.''; PubMed Europe PMC Scholia
  12. Matsumoto F, Saitoh S, Fukui R, Kobayashi T, Tanimura N, Konno K, Kusumoto Y, Akashi-Takamura S, Miyake K.; ''Cathepsins are required for Toll-like receptor 9 responses.''; PubMed Europe PMC Scholia
  13. Staron M, Yang Y, Liu B, Li J, Shen Y, Zúñiga-Pflücker JC, Aguila HL, Goldschneider I, Li Z.; ''gp96, an endoplasmic reticulum master chaperone for integrins and Toll-like receptors, selectively regulates early T and B lymphopoiesis.''; PubMed Europe PMC Scholia
  14. Barton GM, Kagan JC, Medzhitov R.; ''Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA.''; PubMed Europe PMC Scholia
  15. Yang Y, Liu B, Dai J, Srivastava PK, Zammit DJ, Lefrançois L, Li Z.; ''Heat shock protein gp96 is a master chaperone for toll-like receptors and is important in the innate function of macrophages.''; PubMed Europe PMC Scholia
  16. Li Z, Srivastava PK.; ''Tumor rejection antigen gp96/grp94 is an ATPase: implications for protein folding and antigen presentation.''; PubMed Europe PMC Scholia
  17. Casrouge A, Zhang SY, Eidenschenk C, Jouanguy E, Puel A, Yang K, Alcais A, Picard C, Mahfoufi N, Nicolas N, Lorenzo L, Plancoulaine S, Sénéchal B, Geissmann F, Tabeta K, Hoebe K, Du X, Miller RL, Héron B, Mignot C, de Villemeur TB, Lebon P, Dulac O, Rozenberg F, Beutler B, Tardieu M, Abel L, Casanova JL.; ''Herpes simplex virus encephalitis in human UNC-93B deficiency.''; PubMed Europe PMC Scholia
  18. Gibbard RJ, Morley PJ, Gay NJ.; ''Conserved features in the extracellular domain of human toll-like receptor 8 are essential for pH-dependent signaling.''; PubMed Europe PMC Scholia
  19. Liu B, Li Z.; ''Endoplasmic reticulum HSP90b1 (gp96, grp94) optimizes B-cell function via chaperoning integrin and TLR but not immunoglobulin.''; PubMed Europe PMC Scholia
  20. Fukui R, Saitoh S, Matsumoto F, Kozuka-Hata H, Oyama M, Tabeta K, Beutler B, Miyake K.; ''Unc93B1 biases Toll-like receptor responses to nucleic acid in dendritic cells toward DNA- but against RNA-sensing.''; PubMed Europe PMC Scholia
  21. Chockalingam A, Brooks JC, Cameron JL, Blum LK, Leifer CA.; ''TLR9 traffics through the Golgi complex to localize to endolysosomes and respond to CpG DNA.''; PubMed Europe PMC Scholia
  22. Häcker H, Mischak H, Miethke T, Liptay S, Schmid R, Sparwasser T, Heeg K, Lipford GB, Wagner H.; ''CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation.''; PubMed Europe PMC Scholia
  23. Latz E, Verma A, Visintin A, Gong M, Sirois CM, Klein DC, Monks BG, McKnight CJ, Lamphier MS, Duprex WP, Espevik T, Golenbock DT.; ''Ligand-induced conformational changes allosterically activate Toll-like receptor 9.''; PubMed Europe PMC Scholia
  24. Chockalingam A, Cameron JL, Brooks JC, Leifer CA.; ''Negative regulation of signaling by a soluble form of toll-like receptor 9.''; PubMed Europe PMC Scholia
  25. Tokisue T, Watanabe T, Tsujita T, Nishikawa S, Hasegawa T, Seya T, Matsumoto M, Fukuda K.; ''Significance of the N-terminal histidine-rich region for the function of the human toll-like receptor 3 ectodomain.''; PubMed Europe PMC Scholia
  26. Wu S, Hong F, Gewirth D, Guo B, Liu B, Li Z.; ''The molecular chaperone gp96/GRP94 interacts with Toll-like receptors and integrins via its C-terminal hydrophobic domain.''; PubMed Europe PMC Scholia
  27. Brinkmann MM, Spooner E, Hoebe K, Beutler B, Ploegh HL, Kim YM.; ''The interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial for TLR signaling.''; PubMed Europe PMC Scholia
  28. Itoh H, Tatematsu M, Watanabe A, Iwano K, Funami K, Seya T, Matsumoto M.; ''UNC93B1 physically associates with human TLR8 and regulates TLR8-mediated signaling.''; PubMed Europe PMC Scholia
  29. Ewald SE, Lee BL, Lau L, Wickliffe KE, Shi GP, Chapman HA, Barton GM.; ''The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor.''; PubMed Europe PMC Scholia
  30. Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, Lien E, Nilsen NJ, Espevik T, Golenbock DT.; ''TLR9 signals after translocating from the ER to CpG DNA in the lysosome.''; PubMed Europe PMC Scholia
  31. Funami K, Matsumoto M, Oshiumi H, Akazawa T, Yamamoto A, Seya T.; ''The cytoplasmic 'linker region' in Toll-like receptor 3 controls receptor localization and signaling.''; PubMed Europe PMC Scholia
  32. Tabeta K, Hoebe K, Janssen EM, Du X, Georgel P, Crozat K, Mudd S, Mann N, Sovath S, Goode J, Shamel L, Herskovits AA, Portnoy DA, Cooke M, Tarantino LM, Wiltshire T, Steinberg BE, Grinstein S, Beutler B.; ''The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9.''; PubMed Europe PMC Scholia


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114912view16:42, 25 January 2021ReactomeTeamReactome version 75
113357view11:43, 2 November 2020ReactomeTeamReactome version 74
112566view15:53, 9 October 2020ReactomeTeamReactome version 73
101479view11:34, 1 November 2018ReactomeTeamreactome version 66
101017view21:14, 31 October 2018ReactomeTeamreactome version 65
100553view19:48, 31 October 2018ReactomeTeamreactome version 64
100101view16:33, 31 October 2018ReactomeTeamreactome version 63
99651view15:04, 31 October 2018ReactomeTeamreactome version 62 (2nd attempt)
99253view12:45, 31 October 2018ReactomeTeamreactome version 62
93870view13:42, 16 August 2017ReactomeTeamreactome version 61
93437view11:23, 9 August 2017ReactomeTeamreactome version 61
88364view16:42, 1 August 2016FehrhartOntology Term : 'pathway pertinent to protein folding, sorting, modification, translocation and degradation' added !
86528view09:20, 11 July 2016ReactomeTeamreactome version 56
83309view10:45, 18 November 2015ReactomeTeamVersion54
81448view12:58, 21 August 2015ReactomeTeamVersion53
76922view08:19, 17 July 2014ReactomeTeamFixed remaining interactions
76627view12:00, 16 July 2014ReactomeTeamFixed remaining interactions
75958view10:01, 11 June 2014ReactomeTeamRe-fixing comment source
75660view10:56, 10 June 2014ReactomeTeamReactome 48 Update
75015view13:52, 8 May 2014AnweshaFixing comment source for displaying WikiPathways description
74659view08:43, 30 April 2014ReactomeTeamNew pathway

External references


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NameTypeDatabase referenceComment
ADPMetaboliteCHEBI:456216 (ChEBI)
ATP MetaboliteCHEBI:30616 (ChEBI)


ComplexR-HSA-1679076 (Reactome)
ATPMetaboliteCHEBI:30616 (ChEBI)
Apo-GP96 dimerComplexR-HSA-1678941 (Reactome)
C-ter TLR7 dimerComplexR-HSA-1678924 (Reactome)
C-ter-TLR9 dimerComplexR-HSA-1678956 (Reactome)
CNPY3 ProteinQ9BT09 (Uniprot-TrEMBL)
CNPY3ProteinQ9BT09 (Uniprot-TrEMBL)
CTSL(114-288) ProteinP07711 (Uniprot-TrEMBL)
CTSL2 ProteinO60911 (Uniprot-TrEMBL)
CTSS ProteinP25774 (Uniprot-TrEMBL)
CTSS-like proteinsComplexR-HSA-4127476 (Reactome) This CandidateSet contains sequences identified by William Pearson's analysis of Reactome catalyst entities. Catalyst entity sequences were used to identify analagous sequences that shared overall homology and active site homology. Sequences in this Candidate set were identified in an April 24, 2012 analysis.
Cathepsins B, K, L, S R-HSA-1678971 (Reactome)
FL-TLR7 dimerComplexR-HSA-188168 (Reactome)
FL-TLR9 dimerComplexR-HSA-1679073 (Reactome)
H+MetaboliteCHEBI:15378 (ChEBI)
HSP90B1 ProteinP14625 (Uniprot-TrEMBL)
LGMN ProteinQ99538 (Uniprot-TrEMBL)
Legumain/CathepsinsComplexR-HSA-1678911 (Reactome)
N-ter TLR9 dimerComplexR-HSA-1679007 (Reactome)
TLR3 ProteinO15455 (Uniprot-TrEMBL)
TLR3ProteinO15455 (Uniprot-TrEMBL)
TLR7 ProteinQ9NYK1 (Uniprot-TrEMBL)
TLR7(?-1049) ProteinQ9NYK1 (Uniprot-TrEMBL)
TLR7/8/9ComplexR-HSA-1679009 (Reactome)
TLR8 ProteinQ9NR97 (Uniprot-TrEMBL)
TLR8 dimerComplexR-HSA-188165 (Reactome)
TLR9 ProteinQ9NR96 (Uniprot-TrEMBL)
TLR9(1-?) ProteinQ9NR96 (Uniprot-TrEMBL)
TLR9(?-1032) ProteinQ9NR96 (Uniprot-TrEMBL)
UNC93B1 ProteinQ9H1C4 (Uniprot-TrEMBL)
UNC93B1ProteinQ9H1C4 (Uniprot-TrEMBL)
folded FL-TLR7/8/9 R-HSA-1679066 (Reactome)
folded FL-TLR7/8/9ComplexR-HSA-1679066 (Reactome)
intracellular TLR3/7/8/9ComplexR-HSA-1679054 (Reactome)
intracellular TLR:UNC93B1ComplexR-HSA-1678926 (Reactome)
intracellular TLR:UNC93B1ComplexR-HSA-1679067 (Reactome)

Annotated Interactions

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SourceTargetTypeDatabase referenceComment
ADPArrowR-HSA-1678944 (Reactome)


ArrowR-HSA-1678923 (Reactome)


R-HSA-1678944 (Reactome)
ATPR-HSA-1678923 (Reactome)
Apo-GP96 dimerArrowR-HSA-1678944 (Reactome)
Apo-GP96 dimerR-HSA-1678923 (Reactome)
C-ter TLR7 dimerArrowR-HSA-1678920 (Reactome)
C-ter-TLR9 dimerArrowR-HSA-1678920 (Reactome)
CNPY3ArrowR-HSA-1678944 (Reactome)
CNPY3R-HSA-1678923 (Reactome)
CTSS-like proteinsmim-catalysisR-HSA-1678981 (Reactome)
FL-TLR7 dimerArrowR-HSA-1678927 (Reactome)
FL-TLR7 dimerR-HSA-1678920 (Reactome)
FL-TLR9 dimerArrowR-HSA-1678927 (Reactome)
FL-TLR9 dimerR-HSA-1678920 (Reactome)
FL-TLR9 dimerR-HSA-1678981 (Reactome)
H+ArrowR-HSA-1678920 (Reactome)
Legumain/Cathepsinsmim-catalysisR-HSA-1678920 (Reactome)
N-ter TLR9 dimerArrowR-HSA-1678981 (Reactome)
R-HSA-1678920 (Reactome) Endosome maturation (acidification) is required for both the activation of TLR9 and TLR7 through proteolytic cleavage and the disassembly of pathogens, thereby releasing the TLR ligands within them. TLR7 and TLR9 are cleaved within their ectodomains by pH-sensitive cysteine endopeptidases. Cathepsins (CTS) B, K, L, and S, and asparagine endopeptidase (AEP, also known as legumain) have been implicated in endolysosomal TLR processing, however, several groups have reported somewhat controversial results on the role of specific proteases (Matsumoto F et al 2008, Park B et al 2008, Ewald SE et al 2008, Ewald SE et al 2011, Sepulveda FE et al 2009).

One study showed that TLR9 proteolysis is a multistep process with the initial cleavage that can be mediated by AEP or multiple members of the cathepsin family. The second event is mediated exclusively by cathepsins. TLR7 and TLR3 were reported to be cleaved in a similar manner (Ewald SE et al 2011). Cleavage of TLR3 is not shown in this reaction, since other studies demonstrated that the N-terminal region of TLR3 ectodomain was implicated in ligand binding, suggesting that TLR3 may function as a full-length receptor (Liu L et al 2008, Tokisue T et al 2008).

Both full-length receptor and cleaved fragment corresponding to the C-terminal part of TLR9 were capable to bind ligand, however only the processed form (TLR9 C-ter, aa 471-1032) was shown to bind MyD88 and induce signaling in different mouse cells (Ewald SE et al 2008).

R-HSA-1678921 (Reactome) Mammalian UNC93B1, a multi-transmembrane protein, directly associates with transmembrane domains of TLR3, TLR7, TLR8 and TLR9 (and mouse TLR13) in the ER and facilitates their translocation to endolysosome compartments (Brinkmann et al 2007; Kim et al 2008; Itoh H et al 2011). Mutant mouse and human cells that lack functional UNC93B1 showed disrupted signaling via the endosomal TLRs (Taneda K et al 2006; Fukui et al 2009; Kim YM et al 2008; Qi R et al 2010; Koehn J et al 2007). Furthermore, defects in the human gene encoding UNC93B1 are associated with the increased susceptibility to herpes simplex encephalitis (HSE) in children (Casrouge A et al 2006).

TLR7 and TLR9 compete for UNC931-dependent trafficking and under normal circumstances TLR9 predominates over TLR7. This preference for TLR9 is mediated by an N-terminal domain in UNC93B1 and is reversed to TLR7 if UNC93B1 loses the preferential N-terminal binding site via mutation of aspartate at position 34. Loss of binding to TLR9 and preferential association with TLR7 resulted in hyperresponsiveness to RNA ligands (Fukui et al 2009).

TLR3 appears to translocate to the endosomal compartment with equal efficiency regardless of the presence or absence of the N-terminal domain that mediates preference for TLR9. Thus, endosomal TLR trafficking is orchestrated by UNC93B1 which determines how efficiently each TLR is able to move from the ER to the endolysosomes to initiate host responses.

R-HSA-1678923 (Reactome) GP96 (also known as GRP94, HSP90b1), a paralogue of HSP90 in the endoplasmic reticulum, acts as a chaperone for some integrines and Toll-like receptors. Macrophages or B-cells from gp96 knockout mice have abrogated function of TLR2, 4, 5, 7 and 9, but not TLR3 (Yang Y et al 2007, Liu B and Li Z 2008, Staron M et al 2010). GP96 interacts with TLRs and integrines via its C-terminal hydrophobic domain, formed by residues 652-678 (Wu S et al 2012). GP96 functions as a V-shaped dimer in ATP-dependent manner, however it remains unclear how ATP hydrolysis-dependent conformational changes of GP96 are regulated (Li Z and Srivastava PK 1993).

GP96 forms a complex with co-chaperone CNPY3, also known as PRAT4A. GP96-CNPY3 promotes the proper post-translational ectodomain folding of TLRs, but not TLR3 (Liu B et al 2010).

R-HSA-1678927 (Reactome) TLR3, 7, 8 and 9 activation occurs within acidified endolysosomal compartments. Inhibition of endosome acidification with bafilomicin A or chloroquine abrogated TLR's-mediated responses to pathogen-derived nucleic acids (Hacker H et al 1998, Funami K et al 2004, Gibbard RJ et al 2006, Kuznik A et al 2011). Upon stimulation, TLR3, 7, and 9 (and possibly TLR8) are transported to the signaling endosomes by UNC93B1, whereby they become functional receptors and bind to their specific ligands (Kim et al 2008, Ewald et al 2011). Although UNC93B1 is critically involved in TLRs trafficking it was dispensable for ligand binding by these TLRs (Kim YM et al 2008).
R-HSA-1678944 (Reactome) Folded TLR9 dissociates from GP96:CNPY3 complex (Liu B et al 2010) and translocates to the endolysosome with the aid of the membrane protein UNC93b. Here we assume that TLR7 and TLR8 behave in a similar manner.
R-HSA-1678981 (Reactome) TLR9 traffics to an endosomal vesicle where it is processed by cathepsin S at neural pH to generate an N-terminal product (TLR9 N-ter, aa 1-723). The N-terminal fragment of TLR9 also binds ligand, but in contrast to the C-terminal fragment it inhibits TLR9 signaling. Thus, a proper balance between the two proteolytic events probably regulates TLR9-mediated host responses. (Chockalingam A et al 2011).
R-HSA-1678998 (Reactome) TLRs traffic through the Golgi complex by the conventional secretory pathway and are routed to endolysosomes where they bind their ligands (Chockalingam A et al 2008, Ewald SE et al 2011). .
TLR3ArrowR-HSA-1678927 (Reactome)
TLR7/8/9R-HSA-1678923 (Reactome)
TLR8 dimerArrowR-HSA-1678927 (Reactome)
UNC93B1ArrowR-HSA-1678927 (Reactome)
UNC93B1R-HSA-1678921 (Reactome)
folded FL-TLR7/8/9ArrowR-HSA-1678944 (Reactome)
intracellular TLR3/7/8/9R-HSA-1678921 (Reactome)
intracellular TLR:UNC93B1ArrowR-HSA-1678921 (Reactome)
intracellular TLR:UNC93B1ArrowR-HSA-1678998 (Reactome)
intracellular TLR:UNC93B1R-HSA-1678927 (Reactome)
intracellular TLR:UNC93B1R-HSA-1678998 (Reactome)

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