Диссертация (1097599), страница 25
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Результаты анализа представленности функциональных группгенов (functional enrichment analysis) на 20 мин после активации клеток (p<0.05).Приведены гены, для которых обнаружено повышение экспрессии.ФункцияЧислоГеныгеновRegulation of40transcriptionKLF6, ID1, SRF, UBE2V1, FOXC1, IRX3, PUF60,DDIT3, CXXC1, RASD1, HNRPDL, NCOA7,MYC, GTF2IRD2, EGR2, SIX5, FOSB, FOS,CSRNP1,NFKBIA,EGR1,CBX4,JUN,SERTAD3, PRDX2, SIK1, KHSRP, ZNF672,EGR3, NFKBIZ, MSX1, CCNL1, KLF2, NR4A3,EGR4, ATF3, JUNB, SMAD6, SERTAD1, NR4A1Positive18SRF, SERTAD3, FOXC1, DDIT3, KLF2, EGR4,regulation ofNR4A3, MYC, EGR2, CSRNP1, FOS, NFKBIA,transcriptionJUNB, EGR1, NR4A1, SERTAD1, JUN, KLF6Regulation of14cell cycleCKS2,FOXC1, MAD2L1BP, MYC, CDT1, SFRS5,EDN1,JUNB,SMAD6,BAX,SERTAD1,CDKN1A, JUN, SIK1Regulation of19apoptosisFOXC1, DDIT3, DUSP1, MSX1, THBS1, MCL1,IER3, MOAP1, MYC, NFKBIA, SMAD6, CBX4,BAX, NR4A1, CDKN1A, JUN, INTS1, PRDX2Regulation of6MYC, SMAD6, BAX, NR4A1, INTS1, MOAP19SRF, DUSP2, DUSP5, MYC, FOS, DDIT3,caspase activityMAPK signalingDUSP1, NR4A1, JUNTGF-beta5ID1, FOS, GDF15, SMAD6, JUNreceptorsignalingErbBsignalingpathway4RPS6KB2, MYC, CDKN1A, JUN169Таблица П2-7.
Результаты анализа представленности функциональных группгенов (functional enrichment analysis) на 40 мин после активации клеток (p<0.05).Приведены гены, для которых обнаружено повышение экспрессии.ФункцияRegulation ofЧисло генов78transcriptionГеныATF3, BCL10, BHLHE40, CBX4, CCNL1, CEBPB,CGGBP1, CITED2, CSRNP1, DDIT3, DDX5, DNAJB6,EGR1, EGR2, EGR3, EGR4, ELF3, EPC1, ETS2, FHL2,FOS, FOSB, FOSL1, FOXA1, FOXC1, FST, HNRNPAB,HNRPDL, ID3, ING1, ING3, IRX2, IRX3, ISL1, JMJD6,JUN, JUNB, JUND, KDM3A, KLF10, KLF2, KLF6, MAFF,MAFG, MBD1, MED9, MEF2D, MSX1, MYC, NAB2,NCOA7, NFKBIZ, NR4A1, NR4A2, NR4A3, NRBF2,PER2, PPRC1, PUF60, RARA, RASD1, RBM14, RBM15,RBM4, SERTAD1, SERTAD3, SIK1, SRF, TFAP2C,TRIB1, TSC22D2, TSPYL2, UBC, ZNF24, ZNF274,ZNF593, ZNF672, ZNF787Negative21BHLHE40, CBX4, CITED2, DNAJB6, EGR1, EPC1, FOSB,regulation ofFOXA1, FST, HNRNPAB, ID3, JUNB, KLF10, MBD1,transcriptionMSX1, NAB2, RASD1, RBM15, SIK1, ZNF24, ZNF593Positive34BCL10, CEBPB, CITED2, CSRNP1, DDIT3, DDX5, EGR1,regulation ofEGR2, EGR4, EPC1, ETS2, FHL2, FOS, FOSL1, FOXA1,transcriptionFOXC1, HNRNPAB, ING1, ISL1, JUN, JUNB, KDM3A,KLF2, KLF6, MYC, NR4A1, NR4A2, NR4A 3, RBM14,RBM15, SERTAD1, SERTAD3, SRF, UBCRegulation ofcell death36AEN, BARD1, BCL10, BIK, CBX4, CDKN1A, CEBPB,CITED2, CYCS, DCUN1D3, DDIT3, DNAJB6, DUSP1,FOSL1, FOXC1, HERPUD1, ID3, IER3, ING3, IP6K2, JUN,KLF10, MCL1, MOAP1, MSX1, MYC, NR4A1, NR4A2,PHLDA1, PIM3, SLC25A4, THBS1, TIMP3, TNFAIP3,TUBB2C, UBC170Positive21AEN,BARD1,BCL10,BIK,CDKN1A,CEBPB,regulation ofDCUN1D3, DDIT3, DUSP1, FOSL1, ID3, ING3, IP6K2,apoptosisJUN, KLF10, MYC, NR4A1, PHLDA1, TIMP3, TUBB2C,UBCNegative16BARD1, BCL10, CBX4, CDKN1A, CEBPB, CITED2,regulation ofFOXC1, IER3, MCL1, MSX1, MYC, NR4A2, PIM3,apoptosisTHBS1, TNFAIP3, UBCRegulation of6CYCS, DNAJB6, HERPUD1, MOAP1, MYC, NR4A123ATP2A2, BARD1, BHLHE40, CBX4, CDKN1A, CEBPB,caspaseactivityUblconjugationFOS, KLF10, MAFG, MBD1, MCL1, MEF2D, MSX1,RNF103, SLC25A4, SLC3A2, TFAP2C, TPM1, UBC,UBE2S, VPS24, WEE1, ZNF24Response to11extracellularADM, CDKN1A, DDIT3, DUSP1, FOS, FOSL1, JUN,NR4A2, RARA, SLC10A3, TIMP3stimuluiMAPK14signalingDDIT3, DUSP1, DUSP2, DUSP4, DUSP5, DUSP8, FOS,GADD45A, JUN, JUND, MAP3K14, MYC, NR4A1, SRFpathwayPhosphataseactivity8DUSP1, DUSP2, DUSP4,DUSP5, DUSP8, CYCS, PFKFB3,PTP4A1Приложение 3.
Модель NRF2 сигнальной системыМодель NRF2 сигнальной системы включает следующие ОДУdNRF2 VNrf2 , syn VNrf2 , KEAP1 VNrf2 , KEAP1ox VNrf2 ,nucl VNrf2 ,degdtdH 2O2 VH 2O2 , prod VPx ,ox VKEAP1,ox VNrf2 , KEAP1ox VH 2O2 ,extdtdH 2O2ext VH 2O2 ,extdt(1)(2)(3)171dKEAP1 VNrf2 , KEAP1 VKEAP1,ox VKEAP1,red + VKEAP1, Nrf2ub ,disdtdNRF2 KEAP1 VNrf2 , KEAP1 VKEAP1, Nrf2ub VNrf2 , KEAP1oxdtdNRF 2 KEAP1ub VKEAP1, Nrf2ub VKEAP1, Nrf2ub ,disdtdKEAP1ox VKEAP1,ox VKEAP1,red VKEAP1ox , Nrf2dtdNRF2 KEAP1ox VKEAP1ox , Nrf2 + VNrf2 , KEAP1oxdtdTRX VTRX ,red VKEAP1,reddtdPx VPx , syn VPx ,ox +VPx ,red VPx ,degdtdNRF2nucl VNrf2 ,nucl VNrf2 , MafdtdNRF2 MAF VNrf2 , Maf VNrf2 , AREdtdMAF2 VMaf2 VMaf2 , AREdtdNRF2 MAF ARE VNrf2 , AREdtdMAF2 ARE VMaf2 , AREdt(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)Таблица П3-1.
Уравнения скоростей реакцийNРеакцияУравнения скоростей реакций123Null Nrf2KEAP1 + NRF2 -> KEAP1NRF2KEAP1NRF2 -> KEAP1NRF2UbVNrf2,synVNfr,KEAP1 = k1,Nrf2 (KEAP1NRF2 – K1,Nrf2 KEAP1NRF2)VKEAP1,Nfr2ub=k2KEAP1NRF24KEAP1NRF2Ub KEAP1 + NRF2UbVKEAP1,Nfr2ub,dis = k3KEAP1NRF2Ub56789NRF2 NullNull H2O2H2O2 + Px Pxox + H2OKEAP1 + H2O2 KEAP1oxKEAP1ox + TRX KEAP1 + TRXox10TRXox TRXVNrf2,deg =kNrf2,degNRF2VH2O2,prodVPx,ox=kPx,oxPxH2O2VKEAP1,ox=kKEAP,oxKEAP1H2O2VKEAP1,red=kTrxKEAP1oxH2O2kTR TR TRX ox NADPHVTRX ,red KTR ,TRX KTR , NADPH 1 TRX ox / KTR ,TRX NADPH / KTR , NADPH 1112VNrf2,KEAP1ox = k2,Nrf2(2KEAP1oxNRF2 Kd2,Nrf2 NRF2KEAP1ox)VKNrf2,KEAP1ox = kKEAP,oxKEAP1NRF213KEAP1ox + 2 NRF2 NRF2KEAP1oxNRF2KEAP1+ H2O2 NRF2KEAP1oxPxox + PSS Px + PSH14PSH PSSVPSH ,red 15NRF2 NRF2nuclVNrf2,nucl=knucl(NRF2 NRF2nucl)VPx,red=kPx,redPxoxPSSkRe d Red PSH NADPHK Red , PSH K Red , NADPH 1 PSH / K Red , PSH NADPH / K Red , NADPH 17216171819202122NRF2nucl + MAF NRF2MAFNRF2MAF + NAREARE NRF2MAF AREMAF + MAF MAFMAFMAF + NAREARE MAFARENull PxPx NullH2O2 H2O2extVNrf2,Maf = kNrf2,Maf(NRF2nuclMAF – KNrf2,MafNRF2MAF)VNrf2,ARE = kNrf2,ARE( NARENRF2MAFARE –KNrf2,ARENRF2MAFARE)V2Maf = kMaf2(2MAF – Kd,Maf2MAFMAF)VMaf2,ARE = kMaf2,ARE( NAREMAFARE – KMaf2,AREMAFARE)VPx,syn =F NRF2MAFAREVPx,deg = kPx,degPxVH2O2,out=pAcellNcell (H2O2ext –H2O2)Таблица П3-2.
Концентрации ферментов и метаболитов в моделиNОбозначенияНазвание123456789101112131415NRF2, NRF2nuclKEAP1NRF2KEAP1NRF2UbNRF2UbPx, PxoxPSS, PSHH2O2, H2O2extH2OTRX, TRXoxKEAP1oxNRF2KEAP1oxNRF2MAFNRF2MAF AREMAFMAFMAFMAFARECytoplasmic and nuclear NRF2Complex of KEAP1 with NRF2Ubiquitinated complex of KEAP1 with NRF2Ubiquitinated NRF2Reduced and oxidised peroxidaseReduced and oxidised forms of thiol antioxidant (TRX, GSH)Cytoplasmic and external hydrogen peroxideWater moleculeReduced and oxidised thioredoxinOxidised KEAP1Complex of NRF2 with oxidised KEAP1Complex of NRF2 with MAF proteinComplex of NRF2, MAF, and ARE promoter siteMAF homodimerComplex of MAF homodimer and ARE promoter siteТаблица П3-3.
Параметры в модели (fp – свободные параметры модели, выбранныена основе наилучшего согласие с экспериментальными данными)Параметр ОписаниеРедокс системаVH2O2,prod Production rate of H2O2kPx,oxRate constant of peroxidase oxidationkPx,redkRedRate constant of peroxidase reductionTurnover number of reductase, RedKRed,PSHMechaelis-Menten constant ofreductase, Red (substrate PSH)Mechaelis-Menten constant ofreductase, Red (substrate NADPH)Turnover number of thioredoxinreductaseMechaelis-Menten constant of TR(substrate TRX)KRed,NADPHkTRKTR,TRXЗначение в моделиЛитературные данные0.1 µM/min in normal cells;7 µM/min in cancer cells0.19-0.45 µM/min in normalcells;4.5 – 8.3 µM/min in cancercells (Qutub & Popel, 2008)(Adimora, Jones, & Kemp,2010)k12 (Adimora et al., 2010)25.78 s-1(www.brenda-enzymes.org)1.4-34 µM(www.brenda-enzymes.org)88 µM(www.brenda-enzymes.org)25.78 s-1(www.brenda-enzymes.org)1.4-34 µM(www.brenda-enzymes.org)2.4 103 µM-1 min-1(peroxiredixin rate constant)120 µM-1 min-11500 min-11.8 µM88 µM1500 min-11.8 µM173KTR,NADPHMechaelis-Menten constant of TR(substrate NADPH)kPx,degDegradation rate of PxpPermeability coefficient for H2O2through the cellular membraneAcellThe surface area of the cellNcellNumber of cells in the assayNRF2-KEAP1 системаVsyn,Nrf2Synthesis rate of NRF288 µMk1,Nrf20.1 µM-1 min-1Reaction rate of the binding of NRF2with KEAP1K1,Nrf2Dissociation constant of NRF2 withETGE motif of KEAP1k2,Nrf2Reaction rate of the binding of NRF2with oxidised KEAP1K2,Nrf2Dissociation constant of the NRF2binding with DLG motif of KEAP1kKEAP1,oxReaction rate of KEAP1-SHoxidationkKEAP1,redReaction rate of KEAP-(SS)2reductionkNrf2,degDegradation rate of NRF2NRF2-MAF-ARE системаkMaf2Reaction rate of the formation ofMAF-MAF homodimerKd,Maf2Dissociation constant of theformation of MAF-MAF homodimerkMaf2,AREReaction rate of the binding of MAFMAF homodimer with ARE siteKMaf2,AREDissociation constant of the bindingof MAF-MAF homodimer with AREsitekNrf2,MafReaction rate of the binding of NRF2with MAFKNrf2,MafDissociation constant of the NRF2binding with MAFkNrf2,AREReaction rate of the binding ofNRF2-MAF transcription complexwith ARE siteKNrf2,AREDissociation constant of NRF2-MAFtranscription complex with ARE siteNARENumber of ARE sitesFTranscription strength of ARE siteknuclRate of cytoplasm/nucleus exchangeof NRF20.1 min-1210-4 cm s-188 µM(www.brenda-enzymes.org)fp(Bienert, Schjoerring, & Jahn,2006)1.510-5 cm2102 µL-12 10-1 µM/min5 nMk28= 4.2 10-2 µM/min (Adimoraet al., 2010)fp0.1 µM-1 min-1(Y.
Chen, Inoyama,Beamer, & Hu, 2011)fp1 µM(Y. Chen et al., 2011)2.4 µM-1 min-1k14 (Adimora et al., 2010)6 µM-1 min-1k19 (Adimora et al., 2010)0.05 min-1fp1 µM-1 min-1(Yamamoto et al., 2006)20 µM(Yamamoto et al., 2006)4.2 µM-1 min-1(Yamamoto et al., 2006)50 µM(Yamamoto et al., 2006)1 µM-1 min-1(Yamamoto et al., 2006)10 µM(Yamamoto et al., 2006)1.2 µM-1 min-1(Yamamoto et al., 2006)20 µM(Yamamoto et al., 2006)1031040.1 min-1(Chorley et al., 2012)fpfpKong,174Таблица П3-4. Концентрации (µМ) ферментов и метаболитов в модели (fp –свободные параметры модели, выбранные на основе наилучшего согласие сэкспериментальными данными)Обозначение ОписаниеЗначение вмоделиPxPSSPeroxidase concentrationantioxidants containingthiol groups (GSH andTRX)Reductase concentration20 µM (fp)200 µM (fp)0.3 µM0.4 µM (fp)(Adimora et al., 2010)(Adimora et al., 2010)TRNADPH concentrationThioredoxinconcentrationThioredoxin reductase4 µM (fp)H2O2oBasal level of H2O20.5 µM4.75 µM (Pillay, Hofmeyr, & Rohwer,2011)<1 (10-3-0.7) µM in normal cells;0.2 µM in tumour cells (Qutub &Popel, 2008)NRF2KEAP1MAFNRF2 concentrationKEAP1 concentrationMAF concentration1.8 µM (fp)2 µM (fp)4 µM (fp)RedNADPHTRXЛитературные данные12 µM (fp)175Список цитированной литературыAdimora, N.
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