From 0940e414b3b093fcdbfc531fd0f3d7b750f97315 Mon Sep 17 00:00:00 2001 From: Eshan Mitra Date: Tue, 18 Jun 2019 08:44:45 -0600 Subject: [PATCH] Add Salazar-Cavazos2019 --- .../190127_CHO_EGFR_Epigen.bngl | 336 ++++++++++++ .../190127_CHO_EGFR_best-fit.bngl | 358 +++++++++++++ .../190127_CHO_EGFR_sensitivity.bngl | 478 ++++++++++++++++++ .../190127_CHO_HA_EGFR_L858R.bngl | 334 ++++++++++++ .../Salazar-Cavazos2019/190127_HMEC.bngl | 313 ++++++++++++ .../Salazar-Cavazos2019/190127_HeLa.bngl | 313 ++++++++++++ .../Salazar-Cavazos2019/190127_MCF10A.bngl | 313 ++++++++++++ .../190127_CHO_EGFR_forBNF.bngl | 319 ++++++++++++ .../PyBNF-fitting-setup/EGF_25nM.exp | 4 + .../PyBNF-fitting-setup/dose_resp.exp | 4 + .../PyBNF-fitting-setup/fit_bootstrap.conf | 57 +++ .../PyBNF-fitting-setup/fit_pt.conf | 62 +++ .../PyBNF-fitting-setup/fit_v1_28.conf | 54 ++ Published/Salazar-Cavazos2019/README.md | 25 + README.md | 1 + 15 files changed, 2971 insertions(+) create mode 100644 Published/Salazar-Cavazos2019/190127_CHO_EGFR_Epigen.bngl create mode 100644 Published/Salazar-Cavazos2019/190127_CHO_EGFR_best-fit.bngl create mode 100644 Published/Salazar-Cavazos2019/190127_CHO_EGFR_sensitivity.bngl create mode 100644 Published/Salazar-Cavazos2019/190127_CHO_HA_EGFR_L858R.bngl create mode 100644 Published/Salazar-Cavazos2019/190127_HMEC.bngl create mode 100644 Published/Salazar-Cavazos2019/190127_HeLa.bngl create mode 100644 Published/Salazar-Cavazos2019/190127_MCF10A.bngl create mode 100644 Published/Salazar-Cavazos2019/PyBNF-fitting-setup/190127_CHO_EGFR_forBNF.bngl create mode 100644 Published/Salazar-Cavazos2019/PyBNF-fitting-setup/EGF_25nM.exp create mode 100644 Published/Salazar-Cavazos2019/PyBNF-fitting-setup/dose_resp.exp create mode 100644 Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_bootstrap.conf create mode 100644 Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_pt.conf create mode 100644 Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_v1_28.conf create mode 100644 Published/Salazar-Cavazos2019/README.md diff --git a/Published/Salazar-Cavazos2019/190127_CHO_EGFR_Epigen.bngl b/Published/Salazar-Cavazos2019/190127_CHO_EGFR_Epigen.bngl new file mode 100644 index 00000000..2b2d325f --- /dev/null +++ b/Published/Salazar-Cavazos2019/190127_CHO_EGFR_Epigen.bngl @@ -0,0 +1,336 @@ +begin model + + +# References + +# - Freed, Daniel M., et al. "EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics." Cell 171.3 (2017): 683-695. +# - Fujioka, Aki, et al. "Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes." Journal of biological chemistry 281.13 (2006): 8917-8926. +# - Hause Jr, Ronald J., et al. "Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional +# diversification of ErbB receptors." PloS one 7.9 (2012): e44471. +# - Kim, Youngjoo, et al. "Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of +# gefitinib." Biochemistry 51.25 (2012): 5212-5222. +# - Kleiman, Laura B., et al. "Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding." Molecular cell 43.5 (2011): 723-737. +# - Kovacs, Erika, et al. "Analysis of the role of the C-terminal tail in the regulation of the +# epidermal growth factor receptor." Molecular and cellular biology 35.17 (2015): 3083-3102. +# - Kulak, Nils A., et al. "Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells." Nature methods 11.3 (2014): 319. +# - Low-Nam, Shalini T., et al. "ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding." Nature Structural and Molecular Biology 18.11 (2011): 1244. +# - Morimatsu, Miki, et al. "Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis." Proceedings of +# the National Academy of Sciences 104.46 (2007): 18013-18018. +# - Reddy, Raven J., et al. "Early signaling dynamics of the epidermal growth factor receptor." Proceedings of the National Academy of Sciences 113.11 (2016): 3114-3119. +# - Shi, Tujin, et al. "Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway." Sci. Signal. 9.436 (2016): rs6-rs6. + + +begin parameters + +# Values for free params obtained by fitting to EGFR phosphorylation data obtained by Single-molecule Pull-down (EGF dose response, and kinetics using 25 nM EGF) +GRB2_total__FREE 169853.19422230462 +SHC1_total__FREE 649425.6012099041 +kdephosY1068__FREE 1.6588142136877735 +kdephosYN__FREE 0.01718186979848268 +ratio_kpkd_Y1068__FREE 0.15754585734593093 +ratio_kpkd_YN__FREE 0.44476356871177164 + + +# IMPORTANT NOTE ON EPIGEN: for practical purposes name of ligand was kept as 'EGF', but binding parameters and concentrations +# were changed to represent properties of Epigen ligand binding + +# Epigen-induced dimers were simulated as having 10 times higher off-rate than EGF-induced dimers. This was based on the observation +# that Epigen-induced dimers are much weaker than EGF- induced dimers Freed et al. (2018). Kd for Epigen-EGFR was estimated to be +# about 2 uM by Freed et al. (2018) +offrate_f 4.8 # factor to multiply off-rate for (used during param scan too) +onrate_f 1.0 # factor to multiply on-rate for (used during param scan) + +# Keep constant +kdephosY1068_f 1.0 # factor to multiply kdephos in parameter scan +kdephosY1173_f 1.0 # factor to multiply kdephos in parameter scan +kphos_f 1.0 # factor to multiply kphos in parameter scan +GRB2_f 1.0 # factor to multiply number of Grb2 molecules per cell in parameter scan + +kon__ 5.0e6 # [=] /M/s. A typical association rate constant for a protein-protein interaction. Consistent with Morimatsu et al. (2007). +kon_EGF__ 8.0e6 # [=] /M/s. Association rate for EGF-EGFR interaction. This rate was set so EGFR phosphorylation kinetics occurs similarly as observed by Reddy et. al (2016). +Kd_EGF__ 2.0e-6 # [=] M. This values is for Kd of Epigen binding to EGFR (naming for EGF was kept for simplicity). Value of EC50 for phosphorylation of full receptors induced by Epigen +# which is consistent with previously reported Kd values for EGF binding to EGFR +ratio_kdephosY1173 1.0 # Equal dephosphoryaltion rates for pY1068 and pY1173 were assumed, based on similarity observed by Kleiman et al. (2011). +ratio_kphosY1173 1.0 # Equal phosphoryaltion rates for Y1068 and Y1173 were assumed, based on similarity observed by Kim et al. (2012). + + + + + +# Avogadro constant +NA 6.02214e23 # [=] molecules per mol + +# Fraction of cell to consider in a stochastic simulation +f 1 # [=] dimensionless, 0<=f<=1 + + +# Cytoplasmic volume +# A volume of 1 to 2 pL is typical for a mammalian cell (Fujioka et al., 2006). +Vc f*1.0e-12 # [=] L (1.0 pL) + +# Number of cells per dish +numCells 1.0e7 # [=] cells per 60 mm^2 dish (10 million) + +# Volume of media per dish +volMedia 1.0e-2 # [=] L (10 mL) + +# Volume of extracellular fluid surrounding a cell +Vextra=f*volMedia/numCells + + +GRB2_total_pre GRB2_total__FREE*f # [=] molecules per cell +GRB2_total GRB2_total_pre*GRB2_f # [=] molecules per cell +EGFR_total 7.7e5*f # [=] molecules per cell (as estimated by flow cytometry for CHO EGFR-GFP cell line used in this work) +SHC1_total SHC1_total__FREE*f # [=] molecules per cell + + + + +# Concentration of EGF +#EGFconc 0 # [=] M +EGFconc 20.0e-6 # [=] M + +EGF_total=EGFconc*(NA*Vextra) # [=] molecules per cell + + + + +# A typical association rate constant for a protein-protein interaction +kon kon__ # [=] /M/s + +# Association rate for EGF-EGFR interaction +kon_EGF kon_EGF__ # [=] /M/s + +# Dissocation and association rate constants for EGF-EGFR interaction at the plasma membrane +Kd_EGF Kd_EGF__*(NA*Vextra) # [=] molecules +kp_EGF=kon_EGF/(NA*Vextra) +km_EGF=Kd_EGF*kp_EGF + + +# Equilibrium dissociation constant for EGFR dimerization +# This parameter is set so that EGFR_total/KD_dim >> 1 when number of receptors is high (e.g. 7.7e5, value estimated for CHO EGFR-GFP cells) +KD_dim 7.7e5/20 # [=] molecules per cell + + +# Dissocation and association rate constants for interaction between two liganded (EGF-bound) receptors. +# Rates with suffix '_pre' are multiplied by a factor 'offrate_f' or 'onrate_f' during a parameter scan. +km_dim_L_L_pre 0.273 # [=] /s. Estimated by Low-Nam et al., 2011. +kp_dim_L_L_pre=km_dim_L_L_pre/KD_dim # [=] /(molecule/cell)/s +km_dim_L_L=km_dim_L_L_pre*offrate_f +kp_dim_L_L=kp_dim_L_L_pre*onrate_f + + +# Kd for Grb2-SH2 domain binding to pY1068 EGFR +Kd_GE 0.6e-6*(NA*Vc) # [=] molecules. Kd estimated by Morimatsu et al. (2007). +kp_GE=kon/(NA*Vc) +km_GE=Kd_GE*kp_GE + + +# Kd for SHC1-PTB domain binding to pY1173 EGFR +Kd_SE 0.6e-6*(NA*Vc) # [=] molecules. Kd for SHC1-PTB binding to EGFR assumed to be the same as for Grb2-SH2, based on similarity in dissociation constants observed by Hause et al. (2012). +kp_SE=kon/(NA*Vc) +km_SE=Kd_SE*kp_SE + + +# Generic (pseudo first-order) dephosphorylation rate constant. +kdephosY1068_pre kdephosY1068__FREE +kdephosY1173_pre kdephosY1068_pre*ratio_kdephosY1173 +# YN represents the other Y sites in EGFR +kdephosYN_pre kdephosYN__FREE + +# Generic (pseudo first-order) phosphorylation rate constants. +# ratio_kpkd_Y1068: ratio between kphos and kdephos. +kphosY1068_pre ratio_kpkd_Y1068__FREE*kdephosY1068__FREE + +kphosY1173_pre kphosY1068_pre*ratio_kphosY1173 + +kphosYN_pre ratio_kpkd_YN__FREE*kdephosYN__FREE + +# Multiply by factors '_f' during parameter scan or to simulate changes in parameter values. Doing this at the +# end prevents changes to be carried to other parameters that get calculated using the varied paramater +kdephosY1068 kdephosY1068_pre*kdephosY1068_f +kdephosY1173 kdephosY1173_pre*kdephosY1173_f +kdephosYN kdephosYN_pre + +kphosY1068 kphosY1068_pre*kphos_f +kphosY1173 kphosY1173_pre*kphos_f +kphosYN kphosYN_pre*kphos_f + + +# Relative phosphorylation rate in C-tail for the receiver receptor by itself (relative to phosphorylation for the activator receptor). +ratio_kphos_receiver 0.7 # Estimated from Kovacks et al. (2015). + + +end parameters + +begin molecule types + +# Ligand, growth factor +EGF(EGFL) + +# Receptor tyrosine kinase, Epidermal growth factor receptor +# I_III: domains I and III in the ectodomain for EGF binding +# II: domain II for dimerization through ectodomain. Dimerization reaction will change state of receptor +# from monomer (unbound) to dimer (bound), and dissociation reaction the opposite. +# Kin: Kinase domain in EGFR cytoplasmic domain. When EGFR is a monomer Kin is inactive (Kin~0), when EGFR +# dimerizes kinase domain can adopt a conformation in which it serves as an activator (Kin~act) of the other kinase in +# the dimer pair, which is termed the receiver kinase (Kin~rec). +# +EGFR(I_III,II~u~b,Kin~0~act~rec,Y1068~0~P,Y1173~0~P,YN~0~P) + +# Grb2 adaptor protein +GRB2(SH2) + +# SHC1 adaptor protein +SHC1(PTB) + +end molecule types + + +begin seed species + +EGF(EGFL) EGF_total +EGFR(I_III,II~u,Kin~0,Y1068~0,Y1173~0,YN~0) EGFR_total +GRB2(SH2) GRB2_total +SHC1(PTB) SHC1_total + + +end seed species + +begin observables + +Molecules EGF EGF() +Molecules EGFRtot EGFR() +Molecules Grb2tot GRB2() +Molecules Shc1tot SHC1() +Molecules EGFR_EGF EGFR(I_III!+) +Molecules monR EGFR(II~u,Kin~0) +Molecules dimR EGFR(II~b) +Molecules dimR_act EGFR(II~b,Kin~act) +Molecules dimR_rec EGFR(II~b,Kin~rec) +Molecules pY1068 EGFR(Y1068~P!?) +Molecules pY1173 EGFR(Y1173~P!?) +Molecules pYN EGFR(YN~P!?) +Molecules unphosR EGFR(Y1068~0,Y1173~0,YN~0) +Molecules pY1068_act EGFR(Y1068~P!?,Kin~act) +Molecules pY1068_rec EGFR(Y1068~P!?,Kin~rec) +Molecules Grb2_EGFR GRB2(SH2!+) +Molecules Shc1_EGFR SHC1(PTB!+) + +Molecules pY1068_pY1173 EGFR(Y1068~P!?,Y1173~P!?) +Molecules pY1068_pYN EGFR(Y1068~P!?,YN~P!?) +Molecules pY1173_pYN EGFR(Y1173~P!?,YN~P!?) +Molecules pYpYpYN EGFR(Y1068~P!?,Y1173~P!?,YN~P!?) + +Molecules monR_pYpY EGFR(II~u,Y1068~P!?,Y1173~P!?) +Molecules dimR_pYpY EGFR(II~b,Y1068~P!?,Y1173~P!?) + + + +end observables + + +begin functions + +pY1068_percent() 100*pY1068/EGFRtot +pY1173_percent() 100*pY1173/EGFRtot +pYpY_per() 100*pY1068_pY1173/EGFRtot +random_pYpY_per() 100*(pY1068/EGFRtot)*(pY1173/EGFRtot) +pY1173inP68_per() 100*pY1068_pY1173/(pY1068+1) +#PEnrichment() pY1173inP68_per()/pY1173_percent() + +monR_pYpY_per() 100*monR_pYpY/(monR+1) # +1 to avoid dividing by 0 +dimR_pYpY_per() 100*dimR_pYpY/(dimR+1) # +1 to avoid dividing by 0 + +pYN_percent() 100*pYN/EGFRtot +unphosR_per() 100*unphosR/EGFRtot +phosR_per() 100*(EGFRtot-unphosR)/EGFRtot # Phosphorylated in at least one site + +pY1173inPYN_per() 100*pY1173_pYN/(pYN+1) +pY1173inP68PYN_per() 100*pYpYpYN/(pY1068_pYN+1) +pYpYinPYN_per() 100*pYpYpYN/(pYN+1) + + + +end functions + + +begin reaction rules + +# EGF reversibly binds EGFR monomers +EGF(EGFL)+EGFR(I_III,II~u)<->EGF(EGFL!1).EGFR(I_III!1,II~u) kp_EGF,km_EGF + + +# Dimerization of EGFR for two EGF-bound receptors +# Transition from monomer (II~u, unbound) to dimer (II~b, bound) states. The kinase domain of one of the receptors adopt an activator conformation (Kin~act) +# and the other the receiver conformation (Kin~rec). +# Simplification: dimerization only happens between two EGF-bound receptors +EGFR(I_III!+,II~u,Kin~0)+EGFR(I_III!+,II~u,Kin~0)->EGFR(I_III!+,II~b,Kin~act)+EGFR(I_III!+,II~b,Kin~rec) kp_dim_L_L/2 + + + +# Dissociation of EGFR dimer. Kinase domain becomes inactive (Kin~0) after dissociation +EGFR(II~b,Kin~act)->EGFR(II~u,Kin~0) km_dim_L_L +EGFR(II~b,Kin~rec)->EGFR(II~u,Kin~0) km_dim_L_L + + +# Phosphorylation of C-tail in the activator receptor by the receiver receptor +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~act)->EGFR(II~b,Y1068~P,Kin~act) kphosY1068 +EGFR(II~b,Y1173~0,Kin~act)->EGFR(II~b,Y1173~P,Kin~act) kphosY1173 +EGFR(II~b,YN~0,Kin~act)->EGFR(II~b,YN~P,Kin~act) kphosYN + +# Phosphorylation of C-tail in the receiver receptor by itself. +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~rec)->EGFR(II~b,Y1068~P,Kin~rec) kphosY1068*ratio_kphos_receiver +EGFR(II~b,Y1173~0,Kin~rec)->EGFR(II~b,Y1173~P,Kin~rec) kphosY1173*ratio_kphos_receiver +EGFR(II~b,YN~0,Kin~rec)->EGFR(II~b,YN~P,Kin~rec) kphosYN*ratio_kphos_receiver + + +# Unregulated dephosphorylation of pTyr sites +# (mediated by constitutively active phosphatases) +EGFR(Y1068~P)->EGFR(Y1068~0) kdephosY1068 +EGFR(Y1173~P)->EGFR(Y1173~0) kdephosY1173 +EGFR(YN~P)->EGFR(YN~0) kdephosYN + + +# Binding of Grb2 to pY1068 in EGFR +GRB2(SH2)+EGFR(Y1068~P)<-> GRB2(SH2!1).EGFR(Y1068~P!1) kp_GE,km_GE + + +# Binding of SHC1 to pY1173 in EGFR +SHC1(PTB)+EGFR(Y1173~P)<-> SHC1(PTB!1).EGFR(Y1173~P!1) kp_SE,km_SE + + + +end reaction rules + +end model + +begin actions + +generate_network({overwrite=>1}) + + +# Add 20 uM Epigen, and simulate for 300 seconds +setParameter("EGFconc","20.0e-6") # This refers to the concentration of Epigen (in Molar) +setConcentration("EGF(EGFL)","EGF_total") + +# Save parameters and concentrations before parameter scan +saveParameters("pre_scan_par") +saveConcentrations("pre_scan") + +# Parameter scan varying EGFR dimer off rate (in relation to EGF-induced dimers) to best fit results with Epigen ligand +parameter_scan({suffix=>"km_dim_full",parameter=>"offrate_f",par_scan_vals=>[1,2,3,4,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5,6,7,8,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9,10],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + + +# simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>5,print_functions=>1}) +simulate({suffix=>"Epigen_20uM",method=>"ode",t_start=>0,t_end=>300,n_steps=>300,print_functions=>1}) + +end actions diff --git a/Published/Salazar-Cavazos2019/190127_CHO_EGFR_best-fit.bngl b/Published/Salazar-Cavazos2019/190127_CHO_EGFR_best-fit.bngl new file mode 100644 index 00000000..510cb8d4 --- /dev/null +++ b/Published/Salazar-Cavazos2019/190127_CHO_EGFR_best-fit.bngl @@ -0,0 +1,358 @@ +begin model + + +# References + +# - Freed, Daniel M., et al. "EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics." Cell 171.3 (2017): 683-695. +# - Fujioka, Aki, et al. "Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes." Journal of biological chemistry 281.13 (2006): 8917-8926. +# - Hause Jr, Ronald J., et al. "Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional +# diversification of ErbB receptors." PloS one 7.9 (2012): e44471. +# - Kim, Youngjoo, et al. "Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of +# gefitinib." Biochemistry 51.25 (2012): 5212-5222. +# - Kleiman, Laura B., et al. "Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding." Molecular cell 43.5 (2011): 723-737. +# - Kovacs, Erika, et al. "Analysis of the role of the C-terminal tail in the regulation of the +# epidermal growth factor receptor." Molecular and cellular biology 35.17 (2015): 3083-3102. +# - Kulak, Nils A., et al. "Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells." Nature methods 11.3 (2014): 319. +# - Low-Nam, Shalini T., et al. "ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding." Nature Structural and Molecular Biology 18.11 (2011): 1244. +# - Morimatsu, Miki, et al. "Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis." Proceedings of +# the National Academy of Sciences 104.46 (2007): 18013-18018. +# - Reddy, Raven J., et al. "Early signaling dynamics of the epidermal growth factor receptor." Proceedings of the National Academy of Sciences 113.11 (2016): 3114-3119. +# - Shi, Tujin, et al. "Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway." Sci. Signal. 9.436 (2016): rs6-rs6. + + +begin parameters + +# Values for free params obtained by fitting to EGFR phosphorylation data obtained by Single-molecule Pull-down (EGF dose response, and kinetics using 25 nM EGF) +GRB2_total__FREE 169853.19422230462 +SHC1_total__FREE 649425.6012099041 +kdephosY1068__FREE 1.6588142136877735 +kdephosYN__FREE 0.01718186979848268 +ratio_kpkd_Y1068__FREE 0.15754585734593093 +ratio_kpkd_YN__FREE 0.44476356871177164 + + + + + +# Keep constant +kdephosY1068_f 1.0 # factor to multiply kdephos in parameter scan +kdephosY1173_f 1.0 # factor to multiply kdephos in parameter scan +kphos_f 1.0 # factor to multiply kphos in parameter scan +GRB2_f 1.0 # factor to multiply number of Grb2 molecules per cell in parameter scan + +kon__ 5.0e6 # [=] /M/s. A typical association rate constant for a protein-protein interaction. Consistent with Morimatsu et al. (2007). +kon_EGF__ 8.0e6 # [=] /M/s. Association rate for EGF-EGFR interaction. This rate was set so EGFR phosphorylation kinetics occurs similarly as observed by Reddy et. al (2016). +Kd_EGF__ 2.0e-9 # [=] M. Equilibrium dissocation constant for EGF-EGFR interaction. A value of 2 nM was identified by parameter likelihood profile, +# which is consistent with previously reported Kd values for EGF binding to EGFR +ratio_kdephosY1173 1.0 # Equal dephosphoryaltion rates for pY1068 and pY1173 were assumed, based on similarity observed by Kleiman et al. (2011). +ratio_kphosY1173 1.0 # Equal phosphoryaltion rates for Y1068 and Y1173 were assumed, based on similarity observed by Kim et al. (2012). + + + + + +# Avogadro constant +NA 6.02214e23 # [=] molecules per mol + +# Fraction of cell to consider in a stochastic simulation +f 1 # [=] dimensionless, 0<=f<=1 + + +# Cytoplasmic volume +# A volume of 1 to 2 pL is typical for a mammalian cell (Fujioka et al., 2006). +Vc f*1.0e-12 # [=] L (1.0 pL) + +# Number of cells per dish +numCells 1.0e7 # [=] cells per 60 mm^2 dish (10 million) + +# Volume of media per dish +volMedia 1.0e-2 # [=] L (10 mL) + +# Volume of extracellular fluid surrounding a cell +Vextra=f*volMedia/numCells + + +GRB2_total_pre GRB2_total__FREE*f # [=] molecules per cell +GRB2_total GRB2_total_pre*GRB2_f # [=] molecules per cell +EGFR_total 7.7e5*f # [=] molecules per cell (as estimated by flow cytometry for CHO EGFR-GFP cell line used in this work) +SHC1_total SHC1_total__FREE*f # [=] molecules per cell + + + + +# Concentration of EGF +#EGFconc 0 # [=] M +EGFconc 25.0e-9 # [=] M + +EGF_total=EGFconc*(NA*Vextra) # [=] molecules per cell + + + + +# A typical association rate constant for a protein-protein interaction +kon kon__ # [=] /M/s + +# Association rate for EGF-EGFR interaction +kon_EGF kon_EGF__ # [=] /M/s + +# Dissocation and association rate constants for EGF-EGFR interaction at the plasma membrane +Kd_EGF Kd_EGF__*(NA*Vextra) # [=] molecules +kp_EGF=kon_EGF/(NA*Vextra) +km_EGF=Kd_EGF*kp_EGF + + +# Equilibrium dissociation constant for EGFR dimerization +# This parameter is set so that EGFR_total/KD_dim >> 1 when number of receptors is high (e.g. 7.7e5, value estimated for CHO EGFR-GFP cells) +KD_dim 7.7e5/20 # [=] molecules per cell + + +# Dissocation and association rate constants for interaction between two liganded (EGF-bound) receptors. +# Rates with suffix '_pre' are multiplied by a factor 'offrate_f' or 'onrate_f' during a parameter scan. +km_dim_L_L_pre 0.273 # [=] /s. Estimated by Low-Nam et al., 2011. +kp_dim_L_L_pre=km_dim_L_L_pre/KD_dim # [=] /(molecule/cell)/s +offrate_f 1.0 +onrate_f 1.0 +km_dim_L_L=km_dim_L_L_pre*offrate_f +kp_dim_L_L=kp_dim_L_L_pre*onrate_f + + +# Kd for Grb2-SH2 domain binding to pY1068 EGFR +Kd_GE 0.6e-6*(NA*Vc) # [=] molecules. Kd estimated by Morimatsu et al. (2007). +kp_GE=kon/(NA*Vc) +km_GE=Kd_GE*kp_GE + + +# Kd for SHC1-PTB domain binding to pY1173 EGFR +Kd_SE 0.6e-6*(NA*Vc) # [=] molecules. Kd for SHC1-PTB binding to EGFR assumed to be the same as for Grb2-SH2, based on similarity in dissociation constants observed by Hause et al. (2012). +kp_SE=kon/(NA*Vc) +km_SE=Kd_SE*kp_SE + + +# Generic (pseudo first-order) dephosphorylation rate constant. +kdephosY1068_pre kdephosY1068__FREE +kdephosY1173_pre kdephosY1068_pre*ratio_kdephosY1173 +# YN represents the other Y sites in EGFR +kdephosYN_pre kdephosYN__FREE + +# Generic (pseudo first-order) phosphorylation rate constants. +# ratio_kpkd_Y1068: ratio between kphos and kdephos. +kphosY1068_pre ratio_kpkd_Y1068__FREE*kdephosY1068__FREE + +kphosY1173_pre kphosY1068_pre*ratio_kphosY1173 + +kphosYN_pre ratio_kpkd_YN__FREE*kdephosYN__FREE + +# Multiply by factors '_f' during parameter scan or to simulate changes in parameter values. Doing this at the +# end prevents changes to be carried to other parameters that get calculated using the varied paramater +kdephosY1068 kdephosY1068_pre*kdephosY1068_f +kdephosY1173 kdephosY1173_pre*kdephosY1173_f +kdephosYN kdephosYN_pre + +kphosY1068 kphosY1068_pre*kphos_f +kphosY1173 kphosY1173_pre*kphos_f +kphosYN kphosYN_pre*kphos_f + + +# Relative phosphorylation rate in C-tail for the receiver receptor by itself (relative to phosphorylation for the activator receptor). +ratio_kphos_receiver 0.7 # Estimated from Kovacks et al. (2015). + + +end parameters + +begin molecule types + +# Ligand, growth factor +EGF(EGFL) + +# Receptor tyrosine kinase, Epidermal growth factor receptor +# I_III: domains I and III in the ectodomain for EGF binding +# II: domain II for dimerization through ectodomain. Dimerization reaction will change state of receptor +# from monomer (unbound) to dimer (bound), and dissociation reaction the opposite. +# Kin: Kinase domain in EGFR cytoplasmic domain. When EGFR is a monomer Kin is inactive (Kin~0), when EGFR +# dimerizes kinase domain can adopt a conformation in which it serves as an activator (Kin~act) of the other kinase in +# the dimer pair, which is termed the receiver kinase (Kin~rec). +# +EGFR(I_III,II~u~b,Kin~0~act~rec,Y1068~0~P,Y1173~0~P,YN~0~P) + +# Grb2 adaptor protein +GRB2(SH2) + +# SHC1 adaptor protein +SHC1(PTB) + +end molecule types + + +begin seed species + +EGF(EGFL) EGF_total +EGFR(I_III,II~u,Kin~0,Y1068~0,Y1173~0,YN~0) EGFR_total +GRB2(SH2) GRB2_total +SHC1(PTB) SHC1_total + + +end seed species + +begin observables + +Molecules EGF EGF() +Molecules EGFRtot EGFR() +Molecules Grb2tot GRB2() +Molecules Shc1tot SHC1() +Molecules EGFR_EGF EGFR(I_III!+) +Molecules EGFR_EGF_dim EGFR(I_III!+,II~b) +Molecules monR EGFR(II~u,Kin~0) +Molecules dimR EGFR(II~b) +Molecules dimR_act EGFR(II~b,Kin~act) +Molecules dimR_rec EGFR(II~b,Kin~rec) +Molecules pY1068 EGFR(Y1068~P!?) +Molecules pY1173 EGFR(Y1173~P!?) +Molecules pYN EGFR(YN~P!?) +Molecules unphosR EGFR(Y1068~0,Y1173~0,YN~0) +Molecules pY1068_act EGFR(Y1068~P!?,Kin~act) +Molecules pY1068_rec EGFR(Y1068~P!?,Kin~rec) +Molecules Grb2_EGFR GRB2(SH2!+) +Molecules Shc1_EGFR SHC1(PTB!+) + +Molecules pY1068_pY1173 EGFR(Y1068~P!?,Y1173~P!?) +Molecules pY1068_pYN EGFR(Y1068~P!?,YN~P!?) +Molecules pY1173_pYN EGFR(Y1173~P!?,YN~P!?) +Molecules pYpYpYN EGFR(Y1068~P!?,Y1173~P!?,YN~P!?) + +Molecules pY68_not_73_YN EGFR(Y1068~P!?,Y1173~0,YN~0) +Molecules pY73_not_68_YN EGFR(Y1068~0,Y1173~P!?,YN~0) +Molecules not_73_YN EGFR(Y1173~0,YN~0) +Molecules not_68_YN EGFR(Y1068~0,YN~0) + + + +end observables + + +begin functions + +pY1068_percent() 100*pY1068/EGFRtot +pY1173_percent() 100*pY1173/EGFRtot +pYpY_per() 100*pY1068_pY1173/EGFRtot +pY1173inP68_per() 100*pY1068_pY1173/(pY1068+0.001) +pY1068inP73_per() 100*pY1068_pY1173/(pY1173+0.001) + +pYN_percent() 100*pYN/EGFRtot +unphosR_per() 100*unphosR/EGFRtot +phosR_per() 100*(EGFRtot-unphosR)/EGFRtot # Phosphorylated in at least one site + + + +pY68with73orYN() pY1068-pY68_not_73_YN +pY73with68orYN() pY1173-pY73_not_68_YN +pY73orYN() EGFRtot-not_73_YN +pY68orYN() EGFRtot-not_68_YN +pY73orYN_per() 100*pY73orYN()/EGFRtot +pY68orYN_per() 100*pY68orYN()/EGFRtot +pY73orYNin68_per() 100*pY68with73orYN()/(pY1068+0.001) +pY68orYNin73_per() 100*pY73with68orYN()/(pY1173+0.001) +pY68in73orYN_per() 100*pY68with73orYN()/(pY73orYN()+0.001) +pY73in68orYN_per() 100*pY73with68orYN()/(pY68orYN()+0.001) + + + +end functions + + +begin reaction rules + +# EGF reversibly binds EGFR monomers +EGF(EGFL)+EGFR(I_III,II~u)<->EGF(EGFL!1).EGFR(I_III!1,II~u) kp_EGF,km_EGF + + +# Dimerization of EGFR for two EGF-bound receptors +# Transition from monomer (II~u, unbound) to dimer (II~b, bound) states. The kinase domain of one of the receptors adopt an activator conformation (Kin~act) +# and the other the receiver conformation (Kin~rec). +# Simplification: dimerization only happens between two EGF-bound receptors +EGFR(I_III!+,II~u,Kin~0)+EGFR(I_III!+,II~u,Kin~0)->EGFR(I_III!+,II~b,Kin~act)+EGFR(I_III!+,II~b,Kin~rec) kp_dim_L_L/2 + + + +# Dissociation of EGFR dimer. Kinase domain becomes inactive (Kin~0) after dissociation +EGFR(II~b,Kin~act)->EGFR(II~u,Kin~0) km_dim_L_L +EGFR(II~b,Kin~rec)->EGFR(II~u,Kin~0) km_dim_L_L + + +# Phosphorylation of C-tail in the activator receptor by the receiver receptor +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~act)->EGFR(II~b,Y1068~P,Kin~act) kphosY1068 +EGFR(II~b,Y1173~0,Kin~act)->EGFR(II~b,Y1173~P,Kin~act) kphosY1173 +EGFR(II~b,YN~0,Kin~act)->EGFR(II~b,YN~P,Kin~act) kphosYN + +# Phosphorylation of C-tail in the receiver receptor by itself. +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~rec)->EGFR(II~b,Y1068~P,Kin~rec) kphosY1068*ratio_kphos_receiver +EGFR(II~b,Y1173~0,Kin~rec)->EGFR(II~b,Y1173~P,Kin~rec) kphosY1173*ratio_kphos_receiver +EGFR(II~b,YN~0,Kin~rec)->EGFR(II~b,YN~P,Kin~rec) kphosYN*ratio_kphos_receiver + + +# Unregulated dephosphorylation of pTyr sites +# (mediated by constitutively active phosphatases) +EGFR(Y1068~P)->EGFR(Y1068~0) kdephosY1068 +EGFR(Y1173~P)->EGFR(Y1173~0) kdephosY1173 +EGFR(YN~P)->EGFR(YN~0) kdephosYN + + +# Binding of Grb2 to pY1068 in EGFR +GRB2(SH2)+EGFR(Y1068~P)<-> GRB2(SH2!1).EGFR(Y1068~P!1) kp_GE,km_GE + + +# Binding of SHC1 to pY1173 in EGFR +SHC1(PTB)+EGFR(Y1173~P)<-> SHC1(PTB!1).EGFR(Y1173~P!1) kp_SE,km_SE + + + +end reaction rules + +end model + +begin actions + +generate_network({overwrite=>1}) + + + +# Save parameters and concentrations before parameter scan +saveParameters("pre_scan_par") +saveConcentrations("pre_scan") + +# Perform a parameter scan of EGF ligand concentrations to simulate dose-response experiment with single labeling (pY1068, pY1173 or PY) +parameter_scan({suffix=>"dose_resp",parameter=>"EGFconc",par_scan_vals=>[0.0,0.05e-9,0.5e-9,5.0e-9,50.0e-9],\ +method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + +# Simulate kinetics of EGFR phosphorylation after addition of 25 nM EGF +setParameter("EGFconc","25.0e-9") +setConcentration("EGF(EGFL)","EGF_total") +# simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>5,print_functions=>1}) +simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>300,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + +# Simulate EGFR phosphorylation kinetics if Grb2 is overexpressed at different levels (in relation to WT) +parameter_scan({suffix=>"Grb2",parameter=>"GRB2_f",par_scan_vals=>[1.0,2.0,4.0,8.0],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>300,print_functions=>1}) + + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +# Perform a parameter scan of EGF ligand concentrations to simulate multi-site phosphorylation as a function of EGF, as assessed by SiMPull using pY1068 and PY antibodies. +parameter_scan({suffix=>"dose_resp_2",parameter=>"EGFconc",par_scan_vals=>[0.1e-9,0.2e-9,0.3e-9,0.5e-9,0.7e-9,1.0e-9,2.0e-9,3.0e-9,5.0e-9,7.0e-9,10.0e-9,20.0e-9,30.0e-9,100.0e-9],\ +method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + + + +end actions diff --git a/Published/Salazar-Cavazos2019/190127_CHO_EGFR_sensitivity.bngl b/Published/Salazar-Cavazos2019/190127_CHO_EGFR_sensitivity.bngl new file mode 100644 index 00000000..14c6a4af --- /dev/null +++ b/Published/Salazar-Cavazos2019/190127_CHO_EGFR_sensitivity.bngl @@ -0,0 +1,478 @@ +begin model + + +# References + +# - Freed, Daniel M., et al. "EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics." Cell 171.3 (2017): 683-695. +# - Fujioka, Aki, et al. "Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes." Journal of biological chemistry 281.13 (2006): 8917-8926. +# - Hause Jr, Ronald J., et al. "Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional +# diversification of ErbB receptors." PloS one 7.9 (2012): e44471. +# - Kim, Youngjoo, et al. "Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of +# gefitinib." Biochemistry 51.25 (2012): 5212-5222. +# - Kleiman, Laura B., et al. "Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding." Molecular cell 43.5 (2011): 723-737. +# - Kovacs, Erika, et al. "Analysis of the role of the C-terminal tail in the regulation of the +# epidermal growth factor receptor." Molecular and cellular biology 35.17 (2015): 3083-3102. +# - Kulak, Nils A., et al. "Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells." Nature methods 11.3 (2014): 319. +# - Low-Nam, Shalini T., et al. "ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding." Nature Structural and Molecular Biology 18.11 (2011): 1244. +# - Morimatsu, Miki, et al. "Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis." Proceedings of +# the National Academy of Sciences 104.46 (2007): 18013-18018. +# - Reddy, Raven J., et al. "Early signaling dynamics of the epidermal growth factor receptor." Proceedings of the National Academy of Sciences 113.11 (2016): 3114-3119. +# - Shi, Tujin, et al. "Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway." Sci. Signal. 9.436 (2016): rs6-rs6. + + +begin parameters + +# Values for free params obtained by fitting to EGFR phosphorylation data obtained by Single-molecule Pull-down (EGF dose response, and kinetics using 25 nM EGF) +GRB2_total__FREE 169853.19422230462 +SHC1_total__FREE 649425.6012099041 +kdephosY1068__FREE 1.6588142136877735 +kdephosYN__FREE 0.01718186979848268 +ratio_kpkd_Y1068__FREE 0.15754585734593093 +ratio_kpkd_YN__FREE 0.44476356871177164 + + + + + +# Keep constant +kdephosY1068_f 1.0 # factor to multiply kdephos in parameter scan +kdephosY1173_f 1.0 # factor to multiply kdephos in parameter scan +kphos_f 1.0 # factor to multiply kphos in parameter scan +GRB2_f 1.0 # factor to multiply number of Grb2 molecules per cell in parameter scan + +kon__ 5.0e6 # [=] /M/s. A typical association rate constant for a protein-protein interaction. Consistent with Morimatsu et al. (2007). +kon_EGF__ 8.0e6 # [=] /M/s. Association rate for EGF-EGFR interaction. This rate was set so EGFR phosphorylation kinetics occurs similarly as observed by Reddy et. al (2016). +Kd_EGF__ 2.0e-9 # [=] M. Equilibrium dissocation constant for EGF-EGFR interaction. A value of 2 nM was identified by parameter likelihood profile, +# which is consistent with previously reported Kd values for EGF binding to EGFR +ratio_kdephosY1173 1.0 # Equal dephosphoryaltion rates for pY1068 and pY1173 were assumed, based on similarity observed by Kleiman et al. (2011). +ratio_kphosY1173 1.0 # Equal phosphoryaltion rates for Y1068 and Y1173 were assumed, based on similarity observed by Kim et al. (2012). + + + + + +# Avogadro constant +NA 6.02214e23 # [=] molecules per mol + +# Fraction of cell to consider in a stochastic simulation +f 1 # [=] dimensionless, 0<=f<=1 + + +# Cytoplasmic volume +# A volume of 1 to 2 pL is typical for a mammalian cell (Fujioka et al., 2006). +Vc f*1.0e-12 # [=] L (1.0 pL) + +# Number of cells per dish +numCells 1.0e7 # [=] cells per 60 mm^2 dish (10 million) + +# Volume of media per dish +volMedia 1.0e-2 # [=] L (10 mL) + +# Volume of extracellular fluid surrounding a cell +Vextra=f*volMedia/numCells + + +GRB2_total_pre GRB2_total__FREE*f # [=] molecules per cell +GRB2_total GRB2_total_pre*GRB2_f # [=] molecules per cell +EGFR_total 7.7e5*f # [=] molecules per cell (as estimated by flow cytometry for CHO EGFR-GFP cell line used in this work) +SHC1_total SHC1_total__FREE*f # [=] molecules per cell + + + + +# Concentration of EGF +#EGFconc 0 # [=] M +EGFconc 25.0e-9 # [=] M + +EGF_total=EGFconc*(NA*Vextra) # [=] molecules per cell + + + + +# A typical association rate constant for a protein-protein interaction +kon kon__ # [=] /M/s + +# Association rate for EGF-EGFR interaction +kon_EGF kon_EGF__ # [=] /M/s + +# Dissocation and association rate constants for EGF-EGFR interaction at the plasma membrane +Kd_EGF Kd_EGF__*(NA*Vextra) # [=] molecules +kp_EGF=kon_EGF/(NA*Vextra) +km_EGF=Kd_EGF*kp_EGF + + +# Equilibrium dissociation constant for EGFR dimerization +# This parameter is set so that EGFR_total/KD_dim >> 1 when number of receptors is high (e.g. 7.7e5, value estimated for CHO EGFR-GFP cells) +KD_dim 7.7e5/20 # [=] molecules per cell + + +# Dissocation and association rate constants for interaction between two liganded (EGF-bound) receptors. +# Rates with suffix '_pre' are multiplied by a factor 'offrate_f' or 'onrate_f' during a parameter scan. +km_dim_L_L_pre 0.273 # [=] /s. Estimated by Low-Nam et al., 2011. +kp_dim_L_L_pre=km_dim_L_L_pre/KD_dim # [=] /(molecule/cell)/s +offrate_f 1.0 +onrate_f 1.0 +km_dim=km_dim_L_L_pre*offrate_f +kp_dim=kp_dim_L_L_pre*onrate_f + + +# Kd for Grb2-SH2 domain binding to pY1068 EGFR +Kd_GE 0.6e-6*(NA*Vc) # [=] molecules. Kd estimated by Morimatsu et al. (2007). +kp_GE=kon/(NA*Vc) +km_GE=Kd_GE*kp_GE + + +# Kd for SHC1-PTB domain binding to pY1173 EGFR +Kd_SE 0.6e-6*(NA*Vc) # [=] molecules. Kd for SHC1-PTB binding to EGFR assumed to be the same as for Grb2-SH2, based on similarity in dissociation constants observed by Hause et al. (2012). +kp_SE=kon/(NA*Vc) +km_SE=Kd_SE*kp_SE + + +# Generic (pseudo first-order) dephosphorylation rate constant. +kdephosY1068_pre kdephosY1068__FREE +kdephosY1173_pre kdephosY1068_pre*ratio_kdephosY1173 +# YN represents the other Y sites in EGFR +kdephosYN_pre kdephosYN__FREE + +# Generic (pseudo first-order) phosphorylation rate constants. +# ratio_kpkd_Y1068: ratio between kphos and kdephos. +kphosY1068_pre ratio_kpkd_Y1068__FREE*kdephosY1068__FREE + +kphosY1173_pre kphosY1068_pre*ratio_kphosY1173 + +kphosYN_pre ratio_kpkd_YN__FREE*kdephosYN__FREE + +# Multiply by factors '_f' during parameter scan or to simulate changes in parameter values. Doing this at the +# end prevents changes to be carried to other parameters that get calculated using the varied paramater +kdephosY1068 kdephosY1068_pre*kdephosY1068_f +kdephosY1173 kdephosY1173_pre*kdephosY1173_f +kdephosYN kdephosYN_pre + +kphosY1068 kphosY1068_pre*kphos_f +kphosY1173 kphosY1173_pre*kphos_f +kphosYN kphosYN_pre*kphos_f + + +# Relative phosphorylation rate in C-tail for the receiver receptor by itself (relative to phosphorylation for the activator receptor). +ratio_kphos_receiver 0.7 # Estimated from Kovacks et al. (2015). + + + +# Multiply parameters by a factor '_f' for parameter scan +GRB2_ff 1.0 +EGFR_ff 1.0 +SHC1_ff 1.0 +kp_EGF_ff 1.0 +km_EGF_ff 1.0 +kp_dim_ff 1.0 +km_dim_ff 1.0 +kp_GE_ff 1.0 +km_GE_ff 1.0 +kp_SE_ff 1.0 +km_SE_ff 1.0 +kdephos_ff 1.0 +kphos_ff 1.0 + + + + +GRB2_total_scan GRB2_total*GRB2_ff + +EGFR_total_scan EGFR_total*EGFR_ff + +SHC1_total_scan SHC1_total*SHC1_ff + +kp_EGF_scan kp_EGF*kp_EGF_ff + +km_EGF_scan km_EGF*km_EGF_ff + +kp_dim_scan kp_dim*kp_dim_ff + +km_dim_scan km_dim*km_dim_ff + +kp_GE_scan kp_GE*kp_GE_ff + +km_GE_scan km_GE*km_GE_ff + +kp_SE_scan kp_SE*kp_SE_ff + +km_SE_scan km_SE*km_SE_ff + +kdephosY1068_scan kdephosY1068*kdephos_ff + +kphosY1068_scan kphosY1068*kphos_ff + +kdephosY1173_scan kdephosY1173*kdephos_ff + +kphosY1173_scan kphosY1173*kphos_ff + +kdephosYN_scan kdephosYN*kdephos_ff + +kphosYN_scan kphosYN*kphos_ff + + +end parameters + +begin molecule types + +# Ligand, growth factor +EGF(EGFL) + +# Receptor tyrosine kinase, Epidermal growth factor receptor +# I_III: domains I and III in the ectodomain for EGF binding +# II: domain II for dimerization through ectodomain. Dimerization reaction will change state of receptor +# from monomer (unbound) to dimer (bound), and dissociation reaction the opposite. +# Kin: Kinase domain in EGFR cytoplasmic domain. When EGFR is a monomer Kin is inactive (Kin~0), when EGFR +# dimerizes kinase domain can adopt a conformation in which it serves as an activator (Kin~act) of the other kinase in +# the dimer pair, which is termed the receiver kinase (Kin~rec). +# +EGFR(I_III,II~u~b,Kin~0~act~rec,Y1068~0~P,Y1173~0~P,YN~0~P) + +# Grb2 adaptor protein +GRB2(SH2) + +# SHC1 adaptor protein +SHC1(PTB) + +end molecule types + + +begin seed species + +EGF(EGFL) EGF_total +EGFR(I_III,II~u,Kin~0,Y1068~0,Y1173~0,YN~0) EGFR_total_scan +GRB2(SH2) GRB2_total_scan +SHC1(PTB) SHC1_total_scan + + +end seed species + +begin observables + +Molecules EGF EGF() +Molecules EGFRtot EGFR() +Molecules Grb2tot GRB2() +Molecules Shc1tot SHC1() +Molecules EGFR_EGF EGFR(I_III!+) +Molecules monR EGFR(II~u,Kin~0) +Molecules dimR EGFR(II~b) +Molecules dimR_act EGFR(II~b,Kin~act) +Molecules dimR_rec EGFR(II~b,Kin~rec) +Molecules pY1068 EGFR(Y1068~P!?) +Molecules pY1173 EGFR(Y1173~P!?) +Molecules pYN EGFR(YN~P!?) +Molecules unphosR EGFR(Y1068~0,Y1173~0,YN~0) +Molecules pY1068_act EGFR(Y1068~P!?,Kin~act) +Molecules pY1068_rec EGFR(Y1068~P!?,Kin~rec) +Molecules Grb2_EGFR GRB2(SH2!+) +Molecules Shc1_EGFR SHC1(PTB!+) + +Molecules pY1068_pY1173 EGFR(Y1068~P!?,Y1173~P!?) +Molecules pY1068_pYN EGFR(Y1068~P!?,YN~P!?) +Molecules pY1173_pYN EGFR(Y1173~P!?,YN~P!?) +Molecules pYpYpYN EGFR(Y1068~P!?,Y1173~P!?,YN~P!?) + +Molecules monR_pYpY EGFR(II~u,Y1068~P!?,Y1173~P!?) +Molecules dimR_pYpY EGFR(II~b,Y1068~P!?,Y1173~P!?) + + + +end observables + + +begin functions + +pY1068_percent() 100*pY1068/EGFRtot +pY1173_percent() 100*pY1173/EGFRtot +pYpY_per() 100*pY1068_pY1173/EGFRtot +random_pYpY_per() 100*(pY1068/EGFRtot)*(pY1173/EGFRtot) +pY1173inP68_per() 100*pY1068_pY1173/(pY1068+1) +#PEnrichment() pY1173inP68_per()/pY1173_percent() + +monR_pYpY_per() 100*monR_pYpY/(monR+1) # +1 to avoid dividing by 0 +dimR_pYpY_per() 100*dimR_pYpY/(dimR+1) # +1 to avoid dividing by 0 + +pYN_percent() 100*pYN/EGFRtot +unphosR_per() 100*unphosR/EGFRtot +phosR_per() 100*(EGFRtot-unphosR)/EGFRtot # Phosphorylated in at least one site + +pY1173inPYN_per() 100*pY1173_pYN/(pYN+1) +pY1173inP68PYN_per() 100*pYpYpYN/(pY1068_pYN+1) +pYpYinPYN_per() 100*pYpYpYN/(pYN+1) + + + +end functions + + +begin reaction rules + +# EGF reversibly binds EGFR monomers +EGF(EGFL)+EGFR(I_III,II~u)<->EGF(EGFL!1).EGFR(I_III!1,II~u) kp_EGF_scan,km_EGF_scan + + +# Dimerization of EGFR for two EGF-bound receptors +# Transition from monomer (II~u, unbound) to dimer (II~b, bound) states. The kinase domain of one of the receptors adopt an activator conformation (Kin~act) +# and the other the receiver conformation (Kin~rec). +# Simplification: dimerization only happens between two EGF-bound receptors +EGFR(I_III!+,II~u,Kin~0)+EGFR(I_III!+,II~u,Kin~0)->EGFR(I_III!+,II~b,Kin~act)+EGFR(I_III!+,II~b,Kin~rec) kp_dim_scan/2 + + + +# Dissociation of EGFR dimer. Kinase domain becomes inactive (Kin~0) after dissociation +EGFR(II~b,Kin~act)->EGFR(II~u,Kin~0) km_dim_scan +EGFR(II~b,Kin~rec)->EGFR(II~u,Kin~0) km_dim_scan + + +# Phosphorylation of C-tail in the activator receptor by the receiver receptor +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~act)->EGFR(II~b,Y1068~P,Kin~act) kphosY1068_scan +EGFR(II~b,Y1173~0,Kin~act)->EGFR(II~b,Y1173~P,Kin~act) kphosY1173_scan +EGFR(II~b,YN~0,Kin~act)->EGFR(II~b,YN~P,Kin~act) kphosYN_scan + +# Phosphorylation of C-tail in the receiver receptor by itself. +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~rec)->EGFR(II~b,Y1068~P,Kin~rec) kphosY1068_scan*ratio_kphos_receiver +EGFR(II~b,Y1173~0,Kin~rec)->EGFR(II~b,Y1173~P,Kin~rec) kphosY1173_scan*ratio_kphos_receiver +EGFR(II~b,YN~0,Kin~rec)->EGFR(II~b,YN~P,Kin~rec) kphosYN_scan*ratio_kphos_receiver + + +# Unregulated dephosphorylation of pTyr sites +# (mediated by constitutively active phosphatases) +EGFR(Y1068~P)->EGFR(Y1068~0) kdephosY1068_scan +EGFR(Y1173~P)->EGFR(Y1173~0) kdephosY1173_scan +EGFR(YN~P)->EGFR(YN~0) kdephosYN_scan + + +# Binding of Grb2 to pY1068 in EGFR +GRB2(SH2)+EGFR(Y1068~P)<-> GRB2(SH2!1).EGFR(Y1068~P!1) kp_GE_scan,km_GE_scan + + +# Binding of SHC1 to pY1173 in EGFR +SHC1(PTB)+EGFR(Y1173~P)<-> SHC1(PTB!1).EGFR(Y1173~P!1) kp_SE_scan,km_SE_scan + + + +end reaction rules + +end model + + +begin actions + +generate_network({overwrite=>1}) + + + +# Save parameters and concentrations before parameter scan +saveParameters("pre_scan_par") +saveConcentrations("pre_scan") + + +# Increase values of each of the parameters by 1% (for sensitivity analysis) + +parameter_scan({suffix=>"GRB2",parameter=>"GRB2_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"EGFR",parameter=>"EGFR_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"SHC1",parameter=>"SHC1_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"kp_EGF",parameter=>"kp_EGF_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"km_EGF",parameter=>"km_EGF_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"kp_dim",parameter=>"kp_dim_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"km_dim",parameter=>"km_dim_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"kp_GE",parameter=>"kp_GE_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"km_GE",parameter=>"km_GE_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"kp_SE",parameter=>"kp_SE_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"km_SE",parameter=>"km_SE_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"kdephos",parameter=>"kdephos_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +parameter_scan({suffix=>"kphos",parameter=>"kphos_ff",par_scan_vals=>[1, 1.01],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + +end actions \ No newline at end of file diff --git a/Published/Salazar-Cavazos2019/190127_CHO_HA_EGFR_L858R.bngl b/Published/Salazar-Cavazos2019/190127_CHO_HA_EGFR_L858R.bngl new file mode 100644 index 00000000..b20a22a2 --- /dev/null +++ b/Published/Salazar-Cavazos2019/190127_CHO_HA_EGFR_L858R.bngl @@ -0,0 +1,334 @@ +begin model + + +# References + +# - Freed, Daniel M., et al. "EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics." Cell 171.3 (2017): 683-695. +# - Fujioka, Aki, et al. "Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes." Journal of biological chemistry 281.13 (2006): 8917-8926. +# - Hause Jr, Ronald J., et al. "Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional +# diversification of ErbB receptors." PloS one 7.9 (2012): e44471. +# - Kim, Youngjoo, et al. "Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of +# gefitinib." Biochemistry 51.25 (2012): 5212-5222. +# - Kleiman, Laura B., et al. "Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding." Molecular cell 43.5 (2011): 723-737. +# - Kovacs, Erika, et al. "Analysis of the role of the C-terminal tail in the regulation of the +# epidermal growth factor receptor." Molecular and cellular biology 35.17 (2015): 3083-3102. +# - Kulak, Nils A., et al. "Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells." Nature methods 11.3 (2014): 319. +# - Low-Nam, Shalini T., et al. "ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding." Nature Structural and Molecular Biology 18.11 (2011): 1244. +# - Morimatsu, Miki, et al. "Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis." Proceedings of +# the National Academy of Sciences 104.46 (2007): 18013-18018. +# - Reddy, Raven J., et al. "Early signaling dynamics of the epidermal growth factor receptor." Proceedings of the National Academy of Sciences 113.11 (2016): 3114-3119. +# - Shi, Tujin, et al. "Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway." Sci. Signal. 9.436 (2016): rs6-rs6. + + +begin parameters + +# Values for free params obtained by fitting to EGFR phosphorylation data obtained by Single-molecule Pull-down (EGF dose response, and kinetics using 25 nM EGF) +GRB2_total__FREE 169853.19422230462 +SHC1_total__FREE 649425.6012099041 +kdephosY1068__FREE 1.6588142136877735 +kdephosYN__FREE 0.01718186979848268 +ratio_kpkd_Y1068__FREE 0.15754585734593093 +ratio_kpkd_YN__FREE 0.44476356871177164 + + + + + +# Keep constant +kdephosY1068_f 1.0 # factor to multiply kdephos in parameter scan +kdephosY1173_f 1.0 # factor to multiply kdephos in parameter scan +kphos_f 3.6 # factor to multiply kphos in parameter scan +GRB2_f 1.0 # factor to multiply number of Grb2 molecules per cell in parameter scan + +kon__ 5.0e6 # [=] /M/s. A typical association rate constant for a protein-protein interaction. Consistent with Morimatsu et al. (2007). +kon_EGF__ 8.0e6 # [=] /M/s. Association rate for EGF-EGFR interaction. This rate was set so EGFR phosphorylation kinetics occurs similarly as observed by Reddy et. al (2016). +Kd_EGF__ 2.0e-9 # [=] M. Equilibrium dissocation constant for EGF-EGFR interaction. A value of 2 nM was identified by parameter likelihood profile, +# which is consistent with previously reported Kd values for EGF binding to EGFR +ratio_kdephosY1173 1.0 # Equal dephosphoryaltion rates for pY1068 and pY1173 were assumed, based on similarity observed by Kleiman et al. (2011). +ratio_kphosY1173 1.0 # Equal phosphoryaltion rates for Y1068 and Y1173 were assumed, based on similarity observed by Kim et al. (2012). + + + + + +# Avogadro constant +NA 6.02214e23 # [=] molecules per mol + +# Fraction of cell to consider in a stochastic simulation +f 1 # [=] dimensionless, 0<=f<=1 + + +# Cytoplasmic volume +# A volume of 1 to 2 pL is typical for a mammalian cell (Fujioka et al., 2006). +Vc f*1.0e-12 # [=] L (1.0 pL) + +# Number of cells per dish +numCells 1.0e7 # [=] cells per 60 mm^2 dish (10 million) + +# Volume of media per dish +volMedia 1.0e-2 # [=] L (10 mL) + +# Volume of extracellular fluid surrounding a cell +Vextra=f*volMedia/numCells + + +GRB2_total_pre GRB2_total__FREE*f # [=] molecules per cell +GRB2_total GRB2_total_pre*GRB2_f # [=] molecules per cell +EGFR_total 2.7e5*f # [=] molecules per cell (as estimated by flow cytometry for CHO HA-EGFR WT cell line used in this work) +SHC1_total SHC1_total__FREE*f # [=] molecules per cell + + + + +# Concentration of EGF +#EGFconc 0 # [=] M +EGFconc 25.0e-9 # [=] M + +EGF_total=EGFconc*(NA*Vextra) # [=] molecules per cell + + + + +# A typical association rate constant for a protein-protein interaction +kon kon__ # [=] /M/s + +# Association rate for EGF-EGFR interaction +kon_EGF kon_EGF__ # [=] /M/s + +# Dissocation and association rate constants for EGF-EGFR interaction at the plasma membrane +Kd_EGF Kd_EGF__*(NA*Vextra) # [=] molecules +kp_EGF=kon_EGF/(NA*Vextra) +km_EGF=Kd_EGF*kp_EGF + + +# Equilibrium dissociation constant for EGFR dimerization +# This parameter is set so that EGFR_total/KD_dim >> 1 when number of receptors is high (e.g. 7.7e5, value estimated for CHO EGFR-GFP cells) +KD_dim 7.7e5/20 # [=] molecules per cell + + +# Dissocation and association rate constants for interaction between two liganded (EGF-bound) receptors. +# Rates with suffix '_pre' are multiplied by a factor 'offrate_f' or 'onrate_f' during a parameter scan. +km_dim_L_L_pre 0.273 # [=] /s. Estimated by Low-Nam et al., 2011. +kp_dim_L_L_pre=km_dim_L_L_pre/KD_dim # [=] /(molecule/cell)/s +offrate_f 1.0 +onrate_f 1.0 +km_dim_L_L=km_dim_L_L_pre*offrate_f +kp_dim_L_L=kp_dim_L_L_pre*onrate_f + + +# Kd for Grb2-SH2 domain binding to pY1068 EGFR +Kd_GE 0.6e-6*(NA*Vc) # [=] molecules. Kd estimated by Morimatsu et al. (2007). +kp_GE=kon/(NA*Vc) +km_GE=Kd_GE*kp_GE + + +# Kd for SHC1-PTB domain binding to pY1173 EGFR +Kd_SE 0.6e-6*(NA*Vc) # [=] molecules. Kd for SHC1-PTB binding to EGFR assumed to be the same as for Grb2-SH2, based on similarity in dissociation constants observed by Hause et al. (2012). +kp_SE=kon/(NA*Vc) +km_SE=Kd_SE*kp_SE + + +# Generic (pseudo first-order) dephosphorylation rate constant. +kdephosY1068_pre kdephosY1068__FREE +kdephosY1173_pre kdephosY1068_pre*ratio_kdephosY1173 +# YN represents the other Y sites in EGFR +kdephosYN_pre kdephosYN__FREE + +# Generic (pseudo first-order) phosphorylation rate constants. +# ratio_kpkd_Y1068: ratio between kphos and kdephos. +kphosY1068_pre ratio_kpkd_Y1068__FREE*kdephosY1068__FREE + +kphosY1173_pre kphosY1068_pre*ratio_kphosY1173 + +kphosYN_pre ratio_kpkd_YN__FREE*kdephosYN__FREE + +# Multiply by factors '_f' during parameter scan or to simulate changes in parameter values. Doing this at the +# end prevents changes to be carried to other parameters that get calculated using the varied paramater +kdephosY1068 kdephosY1068_pre*kdephosY1068_f +kdephosY1173 kdephosY1173_pre*kdephosY1173_f +kdephosYN kdephosYN_pre + +kphosY1068 kphosY1068_pre*kphos_f +kphosY1173 kphosY1173_pre*kphos_f +kphosYN kphosYN_pre*kphos_f + + +# Relative phosphorylation rate in C-tail for the receiver receptor by itself (relative to phosphorylation for the activator receptor). +ratio_kphos_receiver 0.7 # Estimated from Kovacks et al. (2015). + + +end parameters + +begin molecule types + +# Ligand, growth factor +EGF(EGFL) + +# Receptor tyrosine kinase, Epidermal growth factor receptor +# I_III: domains I and III in the ectodomain for EGF binding +# II: domain II for dimerization through ectodomain. Dimerization reaction will change state of receptor +# from monomer (unbound) to dimer (bound), and dissociation reaction the opposite. +# Kin: Kinase domain in EGFR cytoplasmic domain. When EGFR is a monomer Kin is inactive (Kin~0), when EGFR +# dimerizes kinase domain can adopt a conformation in which it serves as an activator (Kin~act) of the other kinase in +# the dimer pair, which is termed the receiver kinase (Kin~rec). +# +EGFR(I_III,II~u~b,Kin~0~act~rec,Y1068~0~P,Y1173~0~P,YN~0~P) + +# Grb2 adaptor protein +GRB2(SH2) + +# SHC1 adaptor protein +SHC1(PTB) + +end molecule types + + +begin seed species + +EGF(EGFL) EGF_total +EGFR(I_III,II~u,Kin~0,Y1068~0,Y1173~0,YN~0) EGFR_total +GRB2(SH2) GRB2_total +SHC1(PTB) SHC1_total + + +end seed species + +begin observables + +Molecules EGF EGF() +Molecules EGFRtot EGFR() +Molecules Grb2tot GRB2() +Molecules Shc1tot SHC1() +Molecules EGFR_EGF EGFR(I_III!+) +Molecules monR EGFR(II~u,Kin~0) +Molecules dimR EGFR(II~b) +Molecules dimR_act EGFR(II~b,Kin~act) +Molecules dimR_rec EGFR(II~b,Kin~rec) +Molecules pY1068 EGFR(Y1068~P!?) +Molecules pY1173 EGFR(Y1173~P!?) +Molecules pYN EGFR(YN~P!?) +Molecules unphosR EGFR(Y1068~0,Y1173~0,YN~0) +Molecules pY1068_act EGFR(Y1068~P!?,Kin~act) +Molecules pY1068_rec EGFR(Y1068~P!?,Kin~rec) +Molecules Grb2_EGFR GRB2(SH2!+) +Molecules Shc1_EGFR SHC1(PTB!+) + +Molecules pY1068_pY1173 EGFR(Y1068~P!?,Y1173~P!?) +Molecules pY1068_pYN EGFR(Y1068~P!?,YN~P!?) +Molecules pY1173_pYN EGFR(Y1173~P!?,YN~P!?) +Molecules pYpYpYN EGFR(Y1068~P!?,Y1173~P!?,YN~P!?) + +Molecules monR_pYpY EGFR(II~u,Y1068~P!?,Y1173~P!?) +Molecules dimR_pYpY EGFR(II~b,Y1068~P!?,Y1173~P!?) + + + +end observables + + +begin functions + +pY1068_percent() 100*pY1068/EGFRtot +pY1173_percent() 100*pY1173/EGFRtot +pYpY_per() 100*pY1068_pY1173/EGFRtot +random_pYpY_per() 100*(pY1068/EGFRtot)*(pY1173/EGFRtot) +pY1173inP68_per() 100*pY1068_pY1173/(pY1068+1) +#PEnrichment() pY1173inP68_per()/pY1173_percent() + +monR_pYpY_per() 100*monR_pYpY/(monR+1) # +1 to avoid dividing by 0 +dimR_pYpY_per() 100*dimR_pYpY/(dimR+1) # +1 to avoid dividing by 0 + +pYN_percent() 100*pYN/EGFRtot +unphosR_per() 100*unphosR/EGFRtot +phosR_per() 100*(EGFRtot-unphosR)/EGFRtot # Phosphorylated in at least one site + +pY1068inPYN_per() 100*pY1068_pYN/(pYN+1) +pY1173inPYN_per() 100*pY1173_pYN/(pYN+1) +pYpYinPYN_per() 100*pYpYpYN/(pYN+1) + + + +end functions + + +begin reaction rules + +# EGF reversibly binds EGFR monomers +EGF(EGFL)+EGFR(I_III,II~u)<->EGF(EGFL!1).EGFR(I_III!1,II~u) kp_EGF,km_EGF + + +# Dimerization of EGFR for two EGF-bound receptors +# Transition from monomer (II~u, unbound) to dimer (II~b, bound) states. The kinase domain of one of the receptors adopt an activator conformation (Kin~act) +# and the other the receiver conformation (Kin~rec). +# Simplification: dimerization only happens between two EGF-bound receptors +EGFR(I_III!+,II~u,Kin~0)+EGFR(I_III!+,II~u,Kin~0)->EGFR(I_III!+,II~b,Kin~act)+EGFR(I_III!+,II~b,Kin~rec) kp_dim_L_L/2 + + + +# Dissociation of EGFR dimer. Kinase domain becomes inactive (Kin~0) after dissociation +EGFR(II~b,Kin~act)->EGFR(II~u,Kin~0) km_dim_L_L +EGFR(II~b,Kin~rec)->EGFR(II~u,Kin~0) km_dim_L_L + + +# Phosphorylation of C-tail in the activator receptor by the receiver receptor +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~act)->EGFR(II~b,Y1068~P,Kin~act) kphosY1068 +EGFR(II~b,Y1173~0,Kin~act)->EGFR(II~b,Y1173~P,Kin~act) kphosY1173 +EGFR(II~b,YN~0,Kin~act)->EGFR(II~b,YN~P,Kin~act) kphosYN + +# Phosphorylation of C-tail in the receiver receptor by itself. +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~rec)->EGFR(II~b,Y1068~P,Kin~rec) kphosY1068*ratio_kphos_receiver +EGFR(II~b,Y1173~0,Kin~rec)->EGFR(II~b,Y1173~P,Kin~rec) kphosY1173*ratio_kphos_receiver +EGFR(II~b,YN~0,Kin~rec)->EGFR(II~b,YN~P,Kin~rec) kphosYN*ratio_kphos_receiver + + +# Unregulated dephosphorylation of pTyr sites +# (mediated by constitutively active phosphatases) +EGFR(Y1068~P)->EGFR(Y1068~0) kdephosY1068 +EGFR(Y1173~P)->EGFR(Y1173~0) kdephosY1173 +EGFR(YN~P)->EGFR(YN~0) kdephosYN + + +# Binding of Grb2 to pY1068 in EGFR +GRB2(SH2)+EGFR(Y1068~P)<-> GRB2(SH2!1).EGFR(Y1068~P!1) kp_GE,km_GE + + +# Binding of SHC1 to pY1173 in EGFR +SHC1(PTB)+EGFR(Y1173~P)<-> SHC1(PTB!1).EGFR(Y1173~P!1) kp_SE,km_SE + + + +end reaction rules + +end model + +begin actions + +generate_network({overwrite=>1}) + + + +# Save parameters and concentrations before parameter scan +saveParameters("pre_scan_par") +saveConcentrations("pre_scan") + +# Perform parameter scan varying phosphorylation rate (in relation to WT EGFR) to find best fit for experimental results with L858R mutant EGFR +parameter_scan({suffix=>"kphos_full",parameter=>"kphos_f",par_scan_vals=>[1.0,2.0,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,5.0],\ + method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + #parameter_scan({suffix=>"kphos_full",parameter=>"kphos_f",par_scan_vals=>[1.0,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,4.0,5.0],\ + + +resetParameters("pre_scan_par") +saveParameters("pre_scan_par") # IMPORTANT: You have to save parameters in between parameter scans, if not, parameters values will change after scans. +resetConcentrations("pre_scan") + + + +# Add 25 nM EGF, and simulate for 300 seconds +setParameter("EGFconc","25.0e-9") +setConcentration("EGF(EGFL)","EGF_total") +# simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>5,print_functions=>1}) +simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>300,print_functions=>1}) + +end actions diff --git a/Published/Salazar-Cavazos2019/190127_HMEC.bngl b/Published/Salazar-Cavazos2019/190127_HMEC.bngl new file mode 100644 index 00000000..a9328bb6 --- /dev/null +++ b/Published/Salazar-Cavazos2019/190127_HMEC.bngl @@ -0,0 +1,313 @@ +begin model + + +# References + +# - Freed, Daniel M., et al. "EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics." Cell 171.3 (2017): 683-695. +# - Fujioka, Aki, et al. "Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes." Journal of biological chemistry 281.13 (2006): 8917-8926. +# - Hause Jr, Ronald J., et al. "Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional +# diversification of ErbB receptors." PloS one 7.9 (2012): e44471. +# - Kim, Youngjoo, et al. "Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of +# gefitinib." Biochemistry 51.25 (2012): 5212-5222. +# - Kleiman, Laura B., et al. "Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding." Molecular cell 43.5 (2011): 723-737. +# - Kovacs, Erika, et al. "Analysis of the role of the C-terminal tail in the regulation of the +# epidermal growth factor receptor." Molecular and cellular biology 35.17 (2015): 3083-3102. +# - Kulak, Nils A., et al. "Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells." Nature methods 11.3 (2014): 319. +# - Low-Nam, Shalini T., et al. "ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding." Nature Structural and Molecular Biology 18.11 (2011): 1244. +# - Morimatsu, Miki, et al. "Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis." Proceedings of +# the National Academy of Sciences 104.46 (2007): 18013-18018. +# - Reddy, Raven J., et al. "Early signaling dynamics of the epidermal growth factor receptor." Proceedings of the National Academy of Sciences 113.11 (2016): 3114-3119. +# - Shi, Tujin, et al. "Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway." Sci. Signal. 9.436 (2016): rs6-rs6. + + +begin parameters + +# Values for free params obtained by fitting to EGFR phosphorylation data obtained by Single-molecule Pull-down (EGF dose response, and kinetics using 25 nM EGF) +kdephosY1068__FREE 1.6588142136877735 +kdephosYN__FREE 0.01718186979848268 +ratio_kpkd_Y1068__FREE 0.15754585734593093 +ratio_kpkd_YN__FREE 0.44476356871177164 + + + + + +# Keep constant +kdephosY1068_f 1.0 # factor to multiply kdephos in parameter scan +kdephosY1173_f 1.0 # factor to multiply kdephos in parameter scan +kphos_f 1.0 # factor to multiply kphos in parameter scan +GRB2_f 1.0 # factor to multiply number of Grb2 molecules per cell in parameter scan + +kon__ 5.0e6 # [=] /M/s. A typical association rate constant for a protein-protein interaction. Consistent with Morimatsu et al. (2007). +kon_EGF__ 8.0e6 # [=] /M/s. Association rate for EGF-EGFR interaction. This rate was set so EGFR phosphorylation kinetics occurs similarly as observed by Reddy et. al (2016). +Kd_EGF__ 2.0e-9 # [=] M. Equilibrium dissocation constant for EGF-EGFR interaction. A value of 2 nM was identified by parameter likelihood profile, +# which is consistent with previously reported Kd values for EGF binding to EGFR +ratio_kdephosY1173 1.0 # Equal dephosphoryaltion rates for pY1068 and pY1173 were assumed, based on similarity observed by Kleiman et al. (2011). +ratio_kphosY1173 1.0 # Equal phosphoryaltion rates for Y1068 and Y1173 were assumed, based on similarity observed by Kim et al. (2012). + + + + + +# Avogadro constant +NA 6.02214e23 # [=] molecules per mol + +# Fraction of cell to consider in a stochastic simulation +f 1 # [=] dimensionless, 0<=f<=1 + + +# Cytoplasmic volume +# A volume of 1 to 2 pL is typical for a mammalian cell (Fujioka et al., 2006). +Vc f*1.0e-12 # [=] L (1.0 pL) + +# Number of cells per dish +numCells 1.0e7 # [=] cells per 60 mm^2 dish (10 million) + +# Volume of media per dish +volMedia 1.0e-2 # [=] L (10 mL) + +# Volume of extracellular fluid surrounding a cell +Vextra=f*volMedia/numCells + + +GRB2_total 0.43e5*f # [=] molecules per cell (estimated by Shi et al., 2016) +EGFR_total 3.54e5*f # [=] molecules per cell (estimated by Shi et al., 2016) +SHC1_total 0.25e5*f # [=] molecules per cell (estimated by Shi et al., 2016) + + + + +# Concentration of EGF +#EGFconc 0 # [=] M +EGFconc 25.0e-9 # [=] M + +EGF_total=EGFconc*(NA*Vextra) # [=] molecules per cell + + + + +# A typical association rate constant for a protein-protein interaction +kon kon__ # [=] /M/s + +# Association rate for EGF-EGFR interaction +kon_EGF kon_EGF__ # [=] /M/s + +# Dissocation and association rate constants for EGF-EGFR interaction at the plasma membrane +Kd_EGF Kd_EGF__*(NA*Vextra) # [=] molecules +kp_EGF=kon_EGF/(NA*Vextra) +km_EGF=Kd_EGF*kp_EGF + + +# Equilibrium dissociation constant for EGFR dimerization +# This parameter is set so that EGFR_total/KD_dim >> 1 when number of receptors is high (e.g. 7.7e5, value estimated for CHO EGFR-GFP cells) +KD_dim 7.7e5/20 # [=] molecules per cell + + +# Dissocation and association rate constants for interaction between two liganded (EGF-bound) receptors. +# Rates with suffix '_pre' are multiplied by a factor 'offrate_f' or 'onrate_f' during a parameter scan. +km_dim_L_L_pre 0.273 # [=] /s. Estimated by Low-Nam et al., 2011. +kp_dim_L_L_pre=km_dim_L_L_pre/KD_dim # [=] /(molecule/cell)/s +offrate_f 1.0 +onrate_f 1.0 +km_dim_L_L=km_dim_L_L_pre*offrate_f +kp_dim_L_L=kp_dim_L_L_pre*onrate_f + + +# Kd for Grb2-SH2 domain binding to pY1068 EGFR +Kd_GE 0.6e-6*(NA*Vc) # [=] molecules. Kd estimated by Morimatsu et al. (2007). +kp_GE=kon/(NA*Vc) +km_GE=Kd_GE*kp_GE + + +# Kd for SHC1-PTB domain binding to pY1173 EGFR +Kd_SE 0.6e-6*(NA*Vc) # [=] molecules. Kd for SHC1-PTB binding to EGFR assumed to be the same as for Grb2-SH2, based on similarity in dissociation constants observed by Hause et al. (2012). +kp_SE=kon/(NA*Vc) +km_SE=Kd_SE*kp_SE + + +# Generic (pseudo first-order) dephosphorylation rate constant. +kdephosY1068_pre kdephosY1068__FREE +kdephosY1173_pre kdephosY1068_pre*ratio_kdephosY1173 +# YN represents the other Y sites in EGFR +kdephosYN_pre kdephosYN__FREE + +# Generic (pseudo first-order) phosphorylation rate constants. +# ratio_kpkd_Y1068: ratio between kphos and kdephos. +kphosY1068_pre ratio_kpkd_Y1068__FREE*kdephosY1068__FREE + +kphosY1173_pre kphosY1068_pre*ratio_kphosY1173 + +kphosYN_pre ratio_kpkd_YN__FREE*kdephosYN__FREE + +# Multiply by factors '_f' during parameter scan or to simulate changes in parameter values. Doing this at the +# end prevents changes to be carried to other parameters that get calculated using the varied paramater +kdephosY1068 kdephosY1068_pre*kdephosY1068_f +kdephosY1173 kdephosY1173_pre*kdephosY1173_f +kdephosYN kdephosYN_pre + +kphosY1068 kphosY1068_pre*kphos_f +kphosY1173 kphosY1173_pre*kphos_f +kphosYN kphosYN_pre*kphos_f + + +# Relative phosphorylation rate in C-tail for the receiver receptor by itself (relative to phosphorylation for the activator receptor). +ratio_kphos_receiver 0.7 # Estimated from Kovacks et al. (2015). + + +end parameters + +begin molecule types + +# Ligand, growth factor +EGF(EGFL) + +# Receptor tyrosine kinase, Epidermal growth factor receptor +# I_III: domains I and III in the ectodomain for EGF binding +# II: domain II for dimerization through ectodomain. Dimerization reaction will change state of receptor +# from monomer (unbound) to dimer (bound), and dissociation reaction the opposite. +# Kin: Kinase domain in EGFR cytoplasmic domain. When EGFR is a monomer Kin is inactive (Kin~0), when EGFR +# dimerizes kinase domain can adopt a conformation in which it serves as an activator (Kin~act) of the other kinase in +# the dimer pair, which is termed the receiver kinase (Kin~rec). +# +EGFR(I_III,II~u~b,Kin~0~act~rec,Y1068~0~P,Y1173~0~P,YN~0~P) + +# Grb2 adaptor protein +GRB2(SH2) + +# SHC1 adaptor protein +SHC1(PTB) + +end molecule types + + +begin seed species + +EGF(EGFL) EGF_total +EGFR(I_III,II~u,Kin~0,Y1068~0,Y1173~0,YN~0) EGFR_total +GRB2(SH2) GRB2_total +SHC1(PTB) SHC1_total + + +end seed species + +begin observables + +Molecules EGF EGF() +Molecules EGFRtot EGFR() +Molecules Grb2tot GRB2() +Molecules Shc1tot SHC1() +Molecules EGFR_EGF EGFR(I_III!+) +Molecules monR EGFR(II~u,Kin~0) +Molecules dimR EGFR(II~b) +Molecules dimR_act EGFR(II~b,Kin~act) +Molecules dimR_rec EGFR(II~b,Kin~rec) +Molecules pY1068 EGFR(Y1068~P!?) +Molecules pY1173 EGFR(Y1173~P!?) +Molecules pYN EGFR(YN~P!?) +Molecules unphosR EGFR(Y1068~0,Y1173~0,YN~0) +Molecules pY1068_act EGFR(Y1068~P!?,Kin~act) +Molecules pY1068_rec EGFR(Y1068~P!?,Kin~rec) +Molecules Grb2_EGFR GRB2(SH2!+) +Molecules Shc1_EGFR SHC1(PTB!+) + +Molecules pY1068_pY1173 EGFR(Y1068~P!?,Y1173~P!?) +Molecules pY1068_pYN EGFR(Y1068~P!?,YN~P!?) +Molecules pY1173_pYN EGFR(Y1173~P!?,YN~P!?) +Molecules pYpYpYN EGFR(Y1068~P!?,Y1173~P!?,YN~P!?) + +Molecules monR_pYpY EGFR(II~u,Y1068~P!?,Y1173~P!?) +Molecules dimR_pYpY EGFR(II~b,Y1068~P!?,Y1173~P!?) + + + +end observables + + +begin functions + +pY1068_percent() 100*pY1068/EGFRtot +pY1173_percent() 100*pY1173/EGFRtot +pYpY_per() 100*pY1068_pY1173/EGFRtot +random_pYpY_per() 100*(pY1068/EGFRtot)*(pY1173/EGFRtot) +pY1173inP68_per() 100*pY1068_pY1173/(pY1068+1) +#PEnrichment() pY1173inP68_per()/pY1173_percent() + +monR_pYpY_per() 100*monR_pYpY/(monR+1) # +1 to avoid dividing by 0 +dimR_pYpY_per() 100*dimR_pYpY/(dimR+1) # +1 to avoid dividing by 0 + +pYN_percent() 100*pYN/EGFRtot +unphosR_per() 100*unphosR/EGFRtot +phosR_per() 100*(EGFRtot-unphosR)/EGFRtot # Phosphorylated in at least one site + +pY1173inPYN_per() 100*pY1173_pYN/(pYN+1) +pY1173inP68PYN_per() 100*pYpYpYN/(pY1068_pYN+1) +pYpYinPYN_per() 100*pYpYpYN/(pYN+1) + + + +end functions + + +begin reaction rules + +# EGF reversibly binds EGFR monomers +EGF(EGFL)+EGFR(I_III,II~u)<->EGF(EGFL!1).EGFR(I_III!1,II~u) kp_EGF,km_EGF + + +# Dimerization of EGFR for two EGF-bound receptors +# Transition from monomer (II~u, unbound) to dimer (II~b, bound) states. The kinase domain of one of the receptors adopt an activator conformation (Kin~act) +# and the other the receiver conformation (Kin~rec). +# Simplification: dimerization only happens between two EGF-bound receptors +EGFR(I_III!+,II~u,Kin~0)+EGFR(I_III!+,II~u,Kin~0)->EGFR(I_III!+,II~b,Kin~act)+EGFR(I_III!+,II~b,Kin~rec) kp_dim_L_L/2 + + + +# Dissociation of EGFR dimer. Kinase domain becomes inactive (Kin~0) after dissociation +EGFR(II~b,Kin~act)->EGFR(II~u,Kin~0) km_dim_L_L +EGFR(II~b,Kin~rec)->EGFR(II~u,Kin~0) km_dim_L_L + + +# Phosphorylation of C-tail in the activator receptor by the receiver receptor +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~act)->EGFR(II~b,Y1068~P,Kin~act) kphosY1068 +EGFR(II~b,Y1173~0,Kin~act)->EGFR(II~b,Y1173~P,Kin~act) kphosY1173 +EGFR(II~b,YN~0,Kin~act)->EGFR(II~b,YN~P,Kin~act) kphosYN + +# Phosphorylation of C-tail in the receiver receptor by itself. +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~rec)->EGFR(II~b,Y1068~P,Kin~rec) kphosY1068*ratio_kphos_receiver +EGFR(II~b,Y1173~0,Kin~rec)->EGFR(II~b,Y1173~P,Kin~rec) kphosY1173*ratio_kphos_receiver +EGFR(II~b,YN~0,Kin~rec)->EGFR(II~b,YN~P,Kin~rec) kphosYN*ratio_kphos_receiver + + +# Unregulated dephosphorylation of pTyr sites +# (mediated by constitutively active phosphatases) +EGFR(Y1068~P)->EGFR(Y1068~0) kdephosY1068 +EGFR(Y1173~P)->EGFR(Y1173~0) kdephosY1173 +EGFR(YN~P)->EGFR(YN~0) kdephosYN + + +# Binding of Grb2 to pY1068 in EGFR +GRB2(SH2)+EGFR(Y1068~P)<-> GRB2(SH2!1).EGFR(Y1068~P!1) kp_GE,km_GE + + +# Binding of SHC1 to pY1173 in EGFR +SHC1(PTB)+EGFR(Y1173~P)<-> SHC1(PTB!1).EGFR(Y1173~P!1) kp_SE,km_SE + + + +end reaction rules + +end model + +begin actions + +generate_network({overwrite=>1}) + +# Add 25 nM EGF, and simulate for 300 seconds +setParameter("EGFconc","25.0e-9") +setConcentration("EGF(EGFL)","EGF_total") +# simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>5,print_functions=>1}) +simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>300,print_functions=>1}) + +end actions diff --git a/Published/Salazar-Cavazos2019/190127_HeLa.bngl b/Published/Salazar-Cavazos2019/190127_HeLa.bngl new file mode 100644 index 00000000..2762138e --- /dev/null +++ b/Published/Salazar-Cavazos2019/190127_HeLa.bngl @@ -0,0 +1,313 @@ +begin model + + +# References + +# - Freed, Daniel M., et al. "EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics." Cell 171.3 (2017): 683-695. +# - Fujioka, Aki, et al. "Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes." Journal of biological chemistry 281.13 (2006): 8917-8926. +# - Hause Jr, Ronald J., et al. "Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional +# diversification of ErbB receptors." PloS one 7.9 (2012): e44471. +# - Kim, Youngjoo, et al. "Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of +# gefitinib." Biochemistry 51.25 (2012): 5212-5222. +# - Kleiman, Laura B., et al. "Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding." Molecular cell 43.5 (2011): 723-737. +# - Kovacs, Erika, et al. "Analysis of the role of the C-terminal tail in the regulation of the +# epidermal growth factor receptor." Molecular and cellular biology 35.17 (2015): 3083-3102. +# - Kulak, Nils A., et al. "Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells." Nature methods 11.3 (2014): 319. +# - Low-Nam, Shalini T., et al. "ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding." Nature Structural and Molecular Biology 18.11 (2011): 1244. +# - Morimatsu, Miki, et al. "Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis." Proceedings of +# the National Academy of Sciences 104.46 (2007): 18013-18018. +# - Reddy, Raven J., et al. "Early signaling dynamics of the epidermal growth factor receptor." Proceedings of the National Academy of Sciences 113.11 (2016): 3114-3119. +# - Shi, Tujin, et al. "Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway." Sci. Signal. 9.436 (2016): rs6-rs6. + + +begin parameters + +# Values for free params obtained by fitting to EGFR phosphorylation data obtained by Single-molecule Pull-down (EGF dose response, and kinetics using 25 nM EGF) +kdephosY1068__FREE 1.6588142136877735 +kdephosYN__FREE 0.01718186979848268 +ratio_kpkd_Y1068__FREE 0.15754585734593093 +ratio_kpkd_YN__FREE 0.44476356871177164 + + + + + +# Keep constant +kdephosY1068_f 1.0 # factor to multiply kdephos in parameter scan +kdephosY1173_f 1.0 # factor to multiply kdephos in parameter scan +kphos_f 1.0 # factor to multiply kphos in parameter scan +GRB2_f 1.0 # factor to multiply number of Grb2 molecules per cell in parameter scan + +kon__ 5.0e6 # [=] /M/s. A typical association rate constant for a protein-protein interaction. Consistent with Morimatsu et al. (2007). +kon_EGF__ 8.0e6 # [=] /M/s. Association rate for EGF-EGFR interaction. This rate was set so EGFR phosphorylation kinetics occurs similarly as observed by Reddy et. al (2016). +Kd_EGF__ 2.0e-9 # [=] M. Equilibrium dissocation constant for EGF-EGFR interaction. A value of 2 nM was identified by parameter likelihood profile, +# which is consistent with previously reported Kd values for EGF binding to EGFR +ratio_kdephosY1173 1.0 # Equal dephosphoryaltion rates for pY1068 and pY1173 were assumed, based on similarity observed by Kleiman et al. (2011). +ratio_kphosY1173 1.0 # Equal phosphoryaltion rates for Y1068 and Y1173 were assumed, based on similarity observed by Kim et al. (2012). + + + + + +# Avogadro constant +NA 6.02214e23 # [=] molecules per mol + +# Fraction of cell to consider in a stochastic simulation +f 1 # [=] dimensionless, 0<=f<=1 + + +# Cytoplasmic volume +# A volume of 1 to 2 pL is typical for a mammalian cell (Fujioka et al., 2006). +Vc f*1.0e-12 # [=] L (1.0 pL) + +# Number of cells per dish +numCells 1.0e7 # [=] cells per 60 mm^2 dish (10 million) + +# Volume of media per dish +volMedia 1.0e-2 # [=] L (10 mL) + +# Volume of extracellular fluid surrounding a cell +Vextra=f*volMedia/numCells + + +GRB2_total 6.28e5*f # [=] molecules per cell (estimated by Kulak et al., 2014) +EGFR_total 0.93e5*f # [=] molecules per cell (estimated by Kulak et al., 2014) +SHC1_total 1.12e5*f # [=] molecules per cell (estimated by Kulak et al., 2014) + + + + +# Concentration of EGF +#EGFconc 0 # [=] M +EGFconc 25.0e-9 # [=] M + +EGF_total=EGFconc*(NA*Vextra) # [=] molecules per cell + + + + +# A typical association rate constant for a protein-protein interaction +kon kon__ # [=] /M/s + +# Association rate for EGF-EGFR interaction +kon_EGF kon_EGF__ # [=] /M/s + +# Dissocation and association rate constants for EGF-EGFR interaction at the plasma membrane +Kd_EGF Kd_EGF__*(NA*Vextra) # [=] molecules +kp_EGF=kon_EGF/(NA*Vextra) +km_EGF=Kd_EGF*kp_EGF + + +# Equilibrium dissociation constant for EGFR dimerization +# This parameter is set so that EGFR_total/KD_dim >> 1 when number of receptors is high (e.g. 7.7e5, value estimated for CHO EGFR-GFP cells) +KD_dim 7.7e5/20 # [=] molecules per cell + + +# Dissocation and association rate constants for interaction between two liganded (EGF-bound) receptors. +# Rates with suffix '_pre' are multiplied by a factor 'offrate_f' or 'onrate_f' during a parameter scan. +km_dim_L_L_pre 0.273 # [=] /s. Estimated by Low-Nam et al., 2011. +kp_dim_L_L_pre=km_dim_L_L_pre/KD_dim # [=] /(molecule/cell)/s +offrate_f 1.0 +onrate_f 1.0 +km_dim_L_L=km_dim_L_L_pre*offrate_f +kp_dim_L_L=kp_dim_L_L_pre*onrate_f + + +# Kd for Grb2-SH2 domain binding to pY1068 EGFR +Kd_GE 0.6e-6*(NA*Vc) # [=] molecules. Kd estimated by Morimatsu et al. (2007). +kp_GE=kon/(NA*Vc) +km_GE=Kd_GE*kp_GE + + +# Kd for SHC1-PTB domain binding to pY1173 EGFR +Kd_SE 0.6e-6*(NA*Vc) # [=] molecules. Kd for SHC1-PTB binding to EGFR assumed to be the same as for Grb2-SH2, based on similarity in dissociation constants observed by Hause et al. (2012). +kp_SE=kon/(NA*Vc) +km_SE=Kd_SE*kp_SE + + +# Generic (pseudo first-order) dephosphorylation rate constant. +kdephosY1068_pre kdephosY1068__FREE +kdephosY1173_pre kdephosY1068_pre*ratio_kdephosY1173 +# YN represents the other Y sites in EGFR +kdephosYN_pre kdephosYN__FREE + +# Generic (pseudo first-order) phosphorylation rate constants. +# ratio_kpkd_Y1068: ratio between kphos and kdephos. +kphosY1068_pre ratio_kpkd_Y1068__FREE*kdephosY1068__FREE + +kphosY1173_pre kphosY1068_pre*ratio_kphosY1173 + +kphosYN_pre ratio_kpkd_YN__FREE*kdephosYN__FREE + +# Multiply by factors '_f' during parameter scan or to simulate changes in parameter values. Doing this at the +# end prevents changes to be carried to other parameters that get calculated using the varied paramater +kdephosY1068 kdephosY1068_pre*kdephosY1068_f +kdephosY1173 kdephosY1173_pre*kdephosY1173_f +kdephosYN kdephosYN_pre + +kphosY1068 kphosY1068_pre*kphos_f +kphosY1173 kphosY1173_pre*kphos_f +kphosYN kphosYN_pre*kphos_f + + +# Relative phosphorylation rate in C-tail for the receiver receptor by itself (relative to phosphorylation for the activator receptor). +ratio_kphos_receiver 0.7 # Estimated from Kovacks et al. (2015). + + +end parameters + +begin molecule types + +# Ligand, growth factor +EGF(EGFL) + +# Receptor tyrosine kinase, Epidermal growth factor receptor +# I_III: domains I and III in the ectodomain for EGF binding +# II: domain II for dimerization through ectodomain. Dimerization reaction will change state of receptor +# from monomer (unbound) to dimer (bound), and dissociation reaction the opposite. +# Kin: Kinase domain in EGFR cytoplasmic domain. When EGFR is a monomer Kin is inactive (Kin~0), when EGFR +# dimerizes kinase domain can adopt a conformation in which it serves as an activator (Kin~act) of the other kinase in +# the dimer pair, which is termed the receiver kinase (Kin~rec). +# +EGFR(I_III,II~u~b,Kin~0~act~rec,Y1068~0~P,Y1173~0~P,YN~0~P) + +# Grb2 adaptor protein +GRB2(SH2) + +# SHC1 adaptor protein +SHC1(PTB) + +end molecule types + + +begin seed species + +EGF(EGFL) EGF_total +EGFR(I_III,II~u,Kin~0,Y1068~0,Y1173~0,YN~0) EGFR_total +GRB2(SH2) GRB2_total +SHC1(PTB) SHC1_total + + +end seed species + +begin observables + +Molecules EGF EGF() +Molecules EGFRtot EGFR() +Molecules Grb2tot GRB2() +Molecules Shc1tot SHC1() +Molecules EGFR_EGF EGFR(I_III!+) +Molecules monR EGFR(II~u,Kin~0) +Molecules dimR EGFR(II~b) +Molecules dimR_act EGFR(II~b,Kin~act) +Molecules dimR_rec EGFR(II~b,Kin~rec) +Molecules pY1068 EGFR(Y1068~P!?) +Molecules pY1173 EGFR(Y1173~P!?) +Molecules pYN EGFR(YN~P!?) +Molecules unphosR EGFR(Y1068~0,Y1173~0,YN~0) +Molecules pY1068_act EGFR(Y1068~P!?,Kin~act) +Molecules pY1068_rec EGFR(Y1068~P!?,Kin~rec) +Molecules Grb2_EGFR GRB2(SH2!+) +Molecules Shc1_EGFR SHC1(PTB!+) + +Molecules pY1068_pY1173 EGFR(Y1068~P!?,Y1173~P!?) +Molecules pY1068_pYN EGFR(Y1068~P!?,YN~P!?) +Molecules pY1173_pYN EGFR(Y1173~P!?,YN~P!?) +Molecules pYpYpYN EGFR(Y1068~P!?,Y1173~P!?,YN~P!?) + +Molecules monR_pYpY EGFR(II~u,Y1068~P!?,Y1173~P!?) +Molecules dimR_pYpY EGFR(II~b,Y1068~P!?,Y1173~P!?) + + + +end observables + + +begin functions + +pY1068_percent() 100*pY1068/EGFRtot +pY1173_percent() 100*pY1173/EGFRtot +pYpY_per() 100*pY1068_pY1173/EGFRtot +random_pYpY_per() 100*(pY1068/EGFRtot)*(pY1173/EGFRtot) +pY1173inP68_per() 100*pY1068_pY1173/(pY1068+1) +#PEnrichment() pY1173inP68_per()/pY1173_percent() + +monR_pYpY_per() 100*monR_pYpY/(monR+1) # +1 to avoid dividing by 0 +dimR_pYpY_per() 100*dimR_pYpY/(dimR+1) # +1 to avoid dividing by 0 + +pYN_percent() 100*pYN/EGFRtot +unphosR_per() 100*unphosR/EGFRtot +phosR_per() 100*(EGFRtot-unphosR)/EGFRtot # Phosphorylated in at least one site + +pY1173inPYN_per() 100*pY1173_pYN/(pYN+1) +pY1173inP68PYN_per() 100*pYpYpYN/(pY1068_pYN+1) +pYpYinPYN_per() 100*pYpYpYN/(pYN+1) + + + +end functions + + +begin reaction rules + +# EGF reversibly binds EGFR monomers +EGF(EGFL)+EGFR(I_III,II~u)<->EGF(EGFL!1).EGFR(I_III!1,II~u) kp_EGF,km_EGF + + +# Dimerization of EGFR for two EGF-bound receptors +# Transition from monomer (II~u, unbound) to dimer (II~b, bound) states. The kinase domain of one of the receptors adopt an activator conformation (Kin~act) +# and the other the receiver conformation (Kin~rec). +# Simplification: dimerization only happens between two EGF-bound receptors +EGFR(I_III!+,II~u,Kin~0)+EGFR(I_III!+,II~u,Kin~0)->EGFR(I_III!+,II~b,Kin~act)+EGFR(I_III!+,II~b,Kin~rec) kp_dim_L_L/2 + + + +# Dissociation of EGFR dimer. Kinase domain becomes inactive (Kin~0) after dissociation +EGFR(II~b,Kin~act)->EGFR(II~u,Kin~0) km_dim_L_L +EGFR(II~b,Kin~rec)->EGFR(II~u,Kin~0) km_dim_L_L + + +# Phosphorylation of C-tail in the activator receptor by the receiver receptor +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~act)->EGFR(II~b,Y1068~P,Kin~act) kphosY1068 +EGFR(II~b,Y1173~0,Kin~act)->EGFR(II~b,Y1173~P,Kin~act) kphosY1173 +EGFR(II~b,YN~0,Kin~act)->EGFR(II~b,YN~P,Kin~act) kphosYN + +# Phosphorylation of C-tail in the receiver receptor by itself. +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~rec)->EGFR(II~b,Y1068~P,Kin~rec) kphosY1068*ratio_kphos_receiver +EGFR(II~b,Y1173~0,Kin~rec)->EGFR(II~b,Y1173~P,Kin~rec) kphosY1173*ratio_kphos_receiver +EGFR(II~b,YN~0,Kin~rec)->EGFR(II~b,YN~P,Kin~rec) kphosYN*ratio_kphos_receiver + + +# Unregulated dephosphorylation of pTyr sites +# (mediated by constitutively active phosphatases) +EGFR(Y1068~P)->EGFR(Y1068~0) kdephosY1068 +EGFR(Y1173~P)->EGFR(Y1173~0) kdephosY1173 +EGFR(YN~P)->EGFR(YN~0) kdephosYN + + +# Binding of Grb2 to pY1068 in EGFR +GRB2(SH2)+EGFR(Y1068~P)<-> GRB2(SH2!1).EGFR(Y1068~P!1) kp_GE,km_GE + + +# Binding of SHC1 to pY1173 in EGFR +SHC1(PTB)+EGFR(Y1173~P)<-> SHC1(PTB!1).EGFR(Y1173~P!1) kp_SE,km_SE + + + +end reaction rules + +end model + +begin actions + +generate_network({overwrite=>1}) + +# Add 25 nM EGF, and simulate for 300 seconds +setParameter("EGFconc","25.0e-9") +setConcentration("EGF(EGFL)","EGF_total") +# simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>5,print_functions=>1}) +simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>300,print_functions=>1}) + +end actions diff --git a/Published/Salazar-Cavazos2019/190127_MCF10A.bngl b/Published/Salazar-Cavazos2019/190127_MCF10A.bngl new file mode 100644 index 00000000..27f85282 --- /dev/null +++ b/Published/Salazar-Cavazos2019/190127_MCF10A.bngl @@ -0,0 +1,313 @@ +begin model + + +# References + +# - Freed, Daniel M., et al. "EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics." Cell 171.3 (2017): 683-695. +# - Fujioka, Aki, et al. "Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes." Journal of biological chemistry 281.13 (2006): 8917-8926. +# - Hause Jr, Ronald J., et al. "Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional +# diversification of ErbB receptors." PloS one 7.9 (2012): e44471. +# - Kim, Youngjoo, et al. "Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of +# gefitinib." Biochemistry 51.25 (2012): 5212-5222. +# - Kleiman, Laura B., et al. "Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding." Molecular cell 43.5 (2011): 723-737. +# - Kovacs, Erika, et al. "Analysis of the role of the C-terminal tail in the regulation of the +# epidermal growth factor receptor." Molecular and cellular biology 35.17 (2015): 3083-3102. +# - Kulak, Nils A., et al. "Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells." Nature methods 11.3 (2014): 319. +# - Low-Nam, Shalini T., et al. "ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding." Nature Structural and Molecular Biology 18.11 (2011): 1244. +# - Morimatsu, Miki, et al. "Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis." Proceedings of +# the National Academy of Sciences 104.46 (2007): 18013-18018. +# - Reddy, Raven J., et al. "Early signaling dynamics of the epidermal growth factor receptor." Proceedings of the National Academy of Sciences 113.11 (2016): 3114-3119. +# - Shi, Tujin, et al. "Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway." Sci. Signal. 9.436 (2016): rs6-rs6. + + +begin parameters + +# Values for free params obtained by fitting to EGFR phosphorylation data obtained by Single-molecule Pull-down (EGF dose response, and kinetics using 25 nM EGF) +kdephosY1068__FREE 1.6588142136877735 +kdephosYN__FREE 0.01718186979848268 +ratio_kpkd_Y1068__FREE 0.15754585734593093 +ratio_kpkd_YN__FREE 0.44476356871177164 + + + + + +# Keep constant +kdephosY1068_f 1.0 # factor to multiply kdephos in parameter scan +kdephosY1173_f 1.0 # factor to multiply kdephos in parameter scan +kphos_f 1.0 # factor to multiply kphos in parameter scan +GRB2_f 1.0 # factor to multiply number of Grb2 molecules per cell in parameter scan + +kon__ 5.0e6 # [=] /M/s. A typical association rate constant for a protein-protein interaction. Consistent with Morimatsu et al. (2007). +kon_EGF__ 8.0e6 # [=] /M/s. Association rate for EGF-EGFR interaction. This rate was set so EGFR phosphorylation kinetics occurs similarly as observed by Reddy et. al (2016). +Kd_EGF__ 2.0e-9 # [=] M. Equilibrium dissocation constant for EGF-EGFR interaction. A value of 2 nM was identified by parameter likelihood profile, +# which is consistent with previously reported Kd values for EGF binding to EGFR +ratio_kdephosY1173 1.0 # Equal dephosphoryaltion rates for pY1068 and pY1173 were assumed, based on similarity observed by Kleiman et al. (2011). +ratio_kphosY1173 1.0 # Equal phosphoryaltion rates for Y1068 and Y1173 were assumed, based on similarity observed by Kim et al. (2012). + + + + + +# Avogadro constant +NA 6.02214e23 # [=] molecules per mol + +# Fraction of cell to consider in a stochastic simulation +f 1 # [=] dimensionless, 0<=f<=1 + + +# Cytoplasmic volume +# A volume of 1 to 2 pL is typical for a mammalian cell (Fujioka et al., 2006). +Vc f*1.0e-12 # [=] L (1.0 pL) + +# Number of cells per dish +numCells 1.0e7 # [=] cells per 60 mm^2 dish (10 million) + +# Volume of media per dish +volMedia 1.0e-2 # [=] L (10 mL) + +# Volume of extracellular fluid surrounding a cell +Vextra=f*volMedia/numCells + + +GRB2_total 0.50e5*f # [=] molecules per cell (estimated by Shi et al., 2016) +EGFR_total 2.29e5*f # [=] molecules per cell (estimated by Shi et al., 2016) +SHC1_total 0.81e5*f # [=] molecules per cell (estimated by Shi et al., 2016) + + + + +# Concentration of EGF +#EGFconc 0 # [=] M +EGFconc 25.0e-9 # [=] M + +EGF_total=EGFconc*(NA*Vextra) # [=] molecules per cell + + + + +# A typical association rate constant for a protein-protein interaction +kon kon__ # [=] /M/s + +# Association rate for EGF-EGFR interaction +kon_EGF kon_EGF__ # [=] /M/s + +# Dissocation and association rate constants for EGF-EGFR interaction at the plasma membrane +Kd_EGF Kd_EGF__*(NA*Vextra) # [=] molecules +kp_EGF=kon_EGF/(NA*Vextra) +km_EGF=Kd_EGF*kp_EGF + + +# Equilibrium dissociation constant for EGFR dimerization +# This parameter is set so that EGFR_total/KD_dim >> 1 when number of receptors is high (e.g. 7.7e5, value estimated for CHO EGFR-GFP cells) +KD_dim 7.7e5/20 # [=] molecules per cell + + +# Dissocation and association rate constants for interaction between two liganded (EGF-bound) receptors. +# Rates with suffix '_pre' are multiplied by a factor 'offrate_f' or 'onrate_f' during a parameter scan. +km_dim_L_L_pre 0.273 # [=] /s. Estimated by Low-Nam et al., 2011. +kp_dim_L_L_pre=km_dim_L_L_pre/KD_dim # [=] /(molecule/cell)/s +offrate_f 1.0 +onrate_f 1.0 +km_dim_L_L=km_dim_L_L_pre*offrate_f +kp_dim_L_L=kp_dim_L_L_pre*onrate_f + + +# Kd for Grb2-SH2 domain binding to pY1068 EGFR +Kd_GE 0.6e-6*(NA*Vc) # [=] molecules. Kd estimated by Morimatsu et al. (2007). +kp_GE=kon/(NA*Vc) +km_GE=Kd_GE*kp_GE + + +# Kd for SHC1-PTB domain binding to pY1173 EGFR +Kd_SE 0.6e-6*(NA*Vc) # [=] molecules. Kd for SHC1-PTB binding to EGFR assumed to be the same as for Grb2-SH2, based on similarity in dissociation constants observed by Hause et al. (2012). +kp_SE=kon/(NA*Vc) +km_SE=Kd_SE*kp_SE + + +# Generic (pseudo first-order) dephosphorylation rate constant. +kdephosY1068_pre kdephosY1068__FREE +kdephosY1173_pre kdephosY1068_pre*ratio_kdephosY1173 +# YN represents the other Y sites in EGFR +kdephosYN_pre kdephosYN__FREE + +# Generic (pseudo first-order) phosphorylation rate constants. +# ratio_kpkd_Y1068: ratio between kphos and kdephos. +kphosY1068_pre ratio_kpkd_Y1068__FREE*kdephosY1068__FREE + +kphosY1173_pre kphosY1068_pre*ratio_kphosY1173 + +kphosYN_pre ratio_kpkd_YN__FREE*kdephosYN__FREE + +# Multiply by factors '_f' during parameter scan or to simulate changes in parameter values. Doing this at the +# end prevents changes to be carried to other parameters that get calculated using the varied paramater +kdephosY1068 kdephosY1068_pre*kdephosY1068_f +kdephosY1173 kdephosY1173_pre*kdephosY1173_f +kdephosYN kdephosYN_pre + +kphosY1068 kphosY1068_pre*kphos_f +kphosY1173 kphosY1173_pre*kphos_f +kphosYN kphosYN_pre*kphos_f + + +# Relative phosphorylation rate in C-tail for the receiver receptor by itself (relative to phosphorylation for the activator receptor). +ratio_kphos_receiver 0.7 # Estimated from Kovacks et al. (2015). + + +end parameters + +begin molecule types + +# Ligand, growth factor +EGF(EGFL) + +# Receptor tyrosine kinase, Epidermal growth factor receptor +# I_III: domains I and III in the ectodomain for EGF binding +# II: domain II for dimerization through ectodomain. Dimerization reaction will change state of receptor +# from monomer (unbound) to dimer (bound), and dissociation reaction the opposite. +# Kin: Kinase domain in EGFR cytoplasmic domain. When EGFR is a monomer Kin is inactive (Kin~0), when EGFR +# dimerizes kinase domain can adopt a conformation in which it serves as an activator (Kin~act) of the other kinase in +# the dimer pair, which is termed the receiver kinase (Kin~rec). +# +EGFR(I_III,II~u~b,Kin~0~act~rec,Y1068~0~P,Y1173~0~P,YN~0~P) + +# Grb2 adaptor protein +GRB2(SH2) + +# SHC1 adaptor protein +SHC1(PTB) + +end molecule types + + +begin seed species + +EGF(EGFL) EGF_total +EGFR(I_III,II~u,Kin~0,Y1068~0,Y1173~0,YN~0) EGFR_total +GRB2(SH2) GRB2_total +SHC1(PTB) SHC1_total + + +end seed species + +begin observables + +Molecules EGF EGF() +Molecules EGFRtot EGFR() +Molecules Grb2tot GRB2() +Molecules Shc1tot SHC1() +Molecules EGFR_EGF EGFR(I_III!+) +Molecules monR EGFR(II~u,Kin~0) +Molecules dimR EGFR(II~b) +Molecules dimR_act EGFR(II~b,Kin~act) +Molecules dimR_rec EGFR(II~b,Kin~rec) +Molecules pY1068 EGFR(Y1068~P!?) +Molecules pY1173 EGFR(Y1173~P!?) +Molecules pYN EGFR(YN~P!?) +Molecules unphosR EGFR(Y1068~0,Y1173~0,YN~0) +Molecules pY1068_act EGFR(Y1068~P!?,Kin~act) +Molecules pY1068_rec EGFR(Y1068~P!?,Kin~rec) +Molecules Grb2_EGFR GRB2(SH2!+) +Molecules Shc1_EGFR SHC1(PTB!+) + +Molecules pY1068_pY1173 EGFR(Y1068~P!?,Y1173~P!?) +Molecules pY1068_pYN EGFR(Y1068~P!?,YN~P!?) +Molecules pY1173_pYN EGFR(Y1173~P!?,YN~P!?) +Molecules pYpYpYN EGFR(Y1068~P!?,Y1173~P!?,YN~P!?) + +Molecules monR_pYpY EGFR(II~u,Y1068~P!?,Y1173~P!?) +Molecules dimR_pYpY EGFR(II~b,Y1068~P!?,Y1173~P!?) + + + +end observables + + +begin functions + +pY1068_percent() 100*pY1068/EGFRtot +pY1173_percent() 100*pY1173/EGFRtot +pYpY_per() 100*pY1068_pY1173/EGFRtot +random_pYpY_per() 100*(pY1068/EGFRtot)*(pY1173/EGFRtot) +pY1173inP68_per() 100*pY1068_pY1173/(pY1068+1) +#PEnrichment() pY1173inP68_per()/pY1173_percent() + +monR_pYpY_per() 100*monR_pYpY/(monR+1) # +1 to avoid dividing by 0 +dimR_pYpY_per() 100*dimR_pYpY/(dimR+1) # +1 to avoid dividing by 0 + +pYN_percent() 100*pYN/EGFRtot +unphosR_per() 100*unphosR/EGFRtot +phosR_per() 100*(EGFRtot-unphosR)/EGFRtot # Phosphorylated in at least one site + +pY1173inPYN_per() 100*pY1173_pYN/(pYN+1) +pY1173inP68PYN_per() 100*pYpYpYN/(pY1068_pYN+1) +pYpYinPYN_per() 100*pYpYpYN/(pYN+1) + + + +end functions + + +begin reaction rules + +# EGF reversibly binds EGFR monomers +EGF(EGFL)+EGFR(I_III,II~u)<->EGF(EGFL!1).EGFR(I_III!1,II~u) kp_EGF,km_EGF + + +# Dimerization of EGFR for two EGF-bound receptors +# Transition from monomer (II~u, unbound) to dimer (II~b, bound) states. The kinase domain of one of the receptors adopt an activator conformation (Kin~act) +# and the other the receiver conformation (Kin~rec). +# Simplification: dimerization only happens between two EGF-bound receptors +EGFR(I_III!+,II~u,Kin~0)+EGFR(I_III!+,II~u,Kin~0)->EGFR(I_III!+,II~b,Kin~act)+EGFR(I_III!+,II~b,Kin~rec) kp_dim_L_L/2 + + + +# Dissociation of EGFR dimer. Kinase domain becomes inactive (Kin~0) after dissociation +EGFR(II~b,Kin~act)->EGFR(II~u,Kin~0) km_dim_L_L +EGFR(II~b,Kin~rec)->EGFR(II~u,Kin~0) km_dim_L_L + + +# Phosphorylation of C-tail in the activator receptor by the receiver receptor +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~act)->EGFR(II~b,Y1068~P,Kin~act) kphosY1068 +EGFR(II~b,Y1173~0,Kin~act)->EGFR(II~b,Y1173~P,Kin~act) kphosY1173 +EGFR(II~b,YN~0,Kin~act)->EGFR(II~b,YN~P,Kin~act) kphosYN + +# Phosphorylation of C-tail in the receiver receptor by itself. +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~rec)->EGFR(II~b,Y1068~P,Kin~rec) kphosY1068*ratio_kphos_receiver +EGFR(II~b,Y1173~0,Kin~rec)->EGFR(II~b,Y1173~P,Kin~rec) kphosY1173*ratio_kphos_receiver +EGFR(II~b,YN~0,Kin~rec)->EGFR(II~b,YN~P,Kin~rec) kphosYN*ratio_kphos_receiver + + +# Unregulated dephosphorylation of pTyr sites +# (mediated by constitutively active phosphatases) +EGFR(Y1068~P)->EGFR(Y1068~0) kdephosY1068 +EGFR(Y1173~P)->EGFR(Y1173~0) kdephosY1173 +EGFR(YN~P)->EGFR(YN~0) kdephosYN + + +# Binding of Grb2 to pY1068 in EGFR +GRB2(SH2)+EGFR(Y1068~P)<-> GRB2(SH2!1).EGFR(Y1068~P!1) kp_GE,km_GE + + +# Binding of SHC1 to pY1173 in EGFR +SHC1(PTB)+EGFR(Y1173~P)<-> SHC1(PTB!1).EGFR(Y1173~P!1) kp_SE,km_SE + + + +end reaction rules + +end model + +begin actions + +generate_network({overwrite=>1}) + +# Add 25 nM EGF, and simulate for 300 seconds +setParameter("EGFconc","25.0e-9") +setConcentration("EGF(EGFL)","EGF_total") +# simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>5,print_functions=>1}) +simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>300,print_functions=>1}) + +end actions diff --git a/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/190127_CHO_EGFR_forBNF.bngl b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/190127_CHO_EGFR_forBNF.bngl new file mode 100644 index 00000000..fc56e477 --- /dev/null +++ b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/190127_CHO_EGFR_forBNF.bngl @@ -0,0 +1,319 @@ +begin model + + +# References + +# - Freed, Daniel M., et al. "EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics." Cell 171.3 (2017): 683-695. +# - Fujioka, Aki, et al. "Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes." Journal of biological chemistry 281.13 (2006): 8917-8926. +# - Hause Jr, Ronald J., et al. "Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional +# diversification of ErbB receptors." PloS one 7.9 (2012): e44471. +# - Kim, Youngjoo, et al. "Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of +# gefitinib." Biochemistry 51.25 (2012): 5212-5222. +# - Kleiman, Laura B., et al. "Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding." Molecular cell 43.5 (2011): 723-737. +# - Kovacs, Erika, et al. "Analysis of the role of the C-terminal tail in the regulation of the +# epidermal growth factor receptor." Molecular and cellular biology 35.17 (2015): 3083-3102. +# - Kulak, Nils A., et al. "Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells." Nature methods 11.3 (2014): 319. +# - Low-Nam, Shalini T., et al. "ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding." Nature Structural and Molecular Biology 18.11 (2011): 1244. +# - Morimatsu, Miki, et al. "Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis." Proceedings of +# the National Academy of Sciences 104.46 (2007): 18013-18018. +# - Reddy, Raven J., et al. "Early signaling dynamics of the epidermal growth factor receptor." Proceedings of the National Academy of Sciences 113.11 (2016): 3114-3119. +# - Shi, Tujin, et al. "Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway." Sci. Signal. 9.436 (2016): rs6-rs6. + + +begin parameters + + +# Values for free parameters obtained by fitting to EGFR phosphorylation data obtained by Single-molecule Pull-down (EGF dose response, and kinetics using 25 nM EGF) + + + + + + +# Keep constant +kdephosY1068_f 1.0 # factor to multiply kdephos in parameter scan +kdephosY1173_f 1.0 # factor to multiply kdephos in parameter scan +kphos_f 1.0 # factor to multiply kphos in parameter scan +GRB2_f 1.0 # factor to multiply number of Grb2 molecules per cell in parameter scan + +kon__ 5.0e6 # [=] /M/s. A typical association rate constant for a protein-protein interaction. Consistent with Morimatsu et al. (2007). +kon_EGF__ 8.0e6 # [=] /M/s. Association rate for EGF-EGFR interaction. This rate was set so EGFR phosphorylation kinetics occurs similarly as observed by Reddy et. al (2016). +Kd_EGF__ 2.0e-9 # [=] M. Equilibrium dissocation constant for EGF-EGFR interaction. A value of 2 nM was identified by parameter likelihood profile, +# which is consistent with previously reported Kd values for EGF binding to EGFR +ratio_kdephosY1173 1.0 # Equal dephosphoryaltion rates for pY1068 and pY1173 were assumed, based on similarity observed by Kleiman et al. (2011). +ratio_kphosY1173 1.0 # Equal phosphoryaltion rates for Y1068 and Y1173 were assumed, based on similarity observed by Kim et al. (2012). + + + + + +# Avogadro constant +NA 6.02214e23 # [=] molecules per mol + +# Fraction of cell to consider in a stochastic simulation +f 1 # [=] dimensionless, 0<=f<=1 + + +# Cytoplasmic volume +# A volume of 1 to 2 pL is typical for a mammalian cell (Fujioka et al., 2006). +Vc f*1.0e-12 # [=] L (1.0 pL) + +# Number of cells per dish +numCells 1.0e7 # [=] cells per 60 mm^2 dish (10 million) + +# Volume of media per dish +volMedia 1.0e-2 # [=] L (10 mL) + +# Volume of extracellular fluid surrounding a cell +Vextra=f*volMedia/numCells + + +GRB2_total_pre GRB2_total__FREE*f # [=] molecules per cell +GRB2_total GRB2_total_pre*GRB2_f # [=] molecules per cell +EGFR_total 7.7e5*f # [=] molecules per cell (as estimated by flow cytometry for CHO EGFR-GFP cell line used in this work) +SHC1_total SHC1_total__FREE*f # [=] molecules per cell + + + + +# Concentration of EGF +#EGFconc 0 # [=] M +EGFconc 25.0e-9 # [=] M + +EGF_total=EGFconc*(NA*Vextra) # [=] molecules per cell + + + + +# A typical association rate constant for a protein-protein interaction +kon kon__ # [=] /M/s + +# Association rate for EGF-EGFR interaction +kon_EGF kon_EGF__ # [=] /M/s + +# Dissocation and association rate constants for EGF-EGFR interaction at the plasma membrane +Kd_EGF Kd_EGF__*(NA*Vextra) # [=] molecules +kp_EGF=kon_EGF/(NA*Vextra) +km_EGF=Kd_EGF*kp_EGF + + +# Equilibrium dissociation constant for EGFR dimerization +# This parameter is set so that EGFR_total/KD_dim >> 1 when number of receptors is high (e.g. 7.7e5, value estimated for CHO EGFR-GFP cells) +KD_dim 7.7e5/20 # [=] molecules per cell + + +# Dissocation and association rate constants for interaction between two liganded (EGF-bound) receptors. +# Rates with suffix '_pre' are multiplied by a factor 'offrate_f' or 'onrate_f' during a parameter scan. +km_dim_L_L_pre 0.273 # [=] /s. Estimated by Low-Nam et al., 2011. +kp_dim_L_L_pre=km_dim_L_L_pre/KD_dim # [=] /(molecule/cell)/s +offrate_f 1.0 +onrate_f 1.0 +km_dim_L_L=km_dim_L_L_pre*offrate_f +kp_dim_L_L=kp_dim_L_L_pre*onrate_f + + +# Kd for Grb2-SH2 domain binding to pY1068 EGFR +Kd_GE 0.6e-6*(NA*Vc) # [=] molecules. Kd estimated by Morimatsu et al. (2007). +kp_GE=kon/(NA*Vc) +km_GE=Kd_GE*kp_GE + + +# Kd for SHC1-PTB domain binding to pY1173 EGFR +Kd_SE 0.6e-6*(NA*Vc) # [=] molecules. Kd for SHC1-PTB binding to EGFR assumed to be the same as for Grb2-SH2, based on similarity in dissociation constants observed by Hause et al. (2012). +kp_SE=kon/(NA*Vc) +km_SE=Kd_SE*kp_SE + + +# Generic (pseudo first-order) dephosphorylation rate constant. +kdephosY1068_pre kdephosY1068__FREE +kdephosY1173_pre kdephosY1068_pre*ratio_kdephosY1173 +# YN represents the other Y sites in EGFR +kdephosYN_pre kdephosYN__FREE + +# Generic (pseudo first-order) phosphorylation rate constants. +# ratio_kpkd_Y1068: ratio between kphos and kdephos. +kphosY1068_pre ratio_kpkd_Y1068__FREE*kdephosY1068__FREE + +kphosY1173_pre kphosY1068_pre*ratio_kphosY1173 + +kphosYN_pre ratio_kpkd_YN__FREE*kdephosYN__FREE + +# Multiply by factors '_f' during parameter scan or to simulate changes in parameter values. Doing this at the +# end prevents changes to be carried to other parameters that get calculated using the varied paramater +kdephosY1068 kdephosY1068_pre*kdephosY1068_f +kdephosY1173 kdephosY1173_pre*kdephosY1173_f +kdephosYN kdephosYN_pre + +kphosY1068 kphosY1068_pre*kphos_f +kphosY1173 kphosY1173_pre*kphos_f +kphosYN kphosYN_pre*kphos_f + + +# Relative phosphorylation rate in C-tail for the receiver receptor by itself (relative to phosphorylation for the activator receptor). +ratio_kphos_receiver 0.7 # Estimated from Kovacks et al. (2015). + + +end parameters + +begin molecule types + +# Ligand, growth factor +EGF(EGFL) + +# Receptor tyrosine kinase, Epidermal growth factor receptor +# I_III: domains I and III in the ectodomain for EGF binding +# II: domain II for dimerization through ectodomain. Dimerization reaction will change state of receptor +# from monomer (unbound) to dimer (bound), and dissociation reaction the opposite. +# Kin: Kinase domain in EGFR cytoplasmic domain. When EGFR is a monomer Kin is inactive (Kin~0), when EGFR +# dimerizes kinase domain can adopt a conformation in which it serves as an activator (Kin~act) of the other kinase in +# the dimer pair, which is termed the receiver kinase (Kin~rec). +# +EGFR(I_III,II~u~b,Kin~0~act~rec,Y1068~0~P,Y1173~0~P,YN~0~P) + +# Grb2 adaptor protein +GRB2(SH2) + +# SHC1 adaptor protein +SHC1(PTB) + +end molecule types + + +begin seed species + +EGF(EGFL) EGF_total +EGFR(I_III,II~u,Kin~0,Y1068~0,Y1173~0,YN~0) EGFR_total +GRB2(SH2) GRB2_total +SHC1(PTB) SHC1_total + + +end seed species + +begin observables + +Molecules EGF EGF() +Molecules EGFRtot EGFR() +Molecules Grb2tot GRB2() +Molecules Shc1tot SHC1() +Molecules EGFR_EGF EGFR(I_III!+) +Molecules monR EGFR(II~u,Kin~0) +Molecules dimR EGFR(II~b) +Molecules dimR_act EGFR(II~b,Kin~act) +Molecules dimR_rec EGFR(II~b,Kin~rec) +Molecules pY1068 EGFR(Y1068~P!?) +Molecules pY1173 EGFR(Y1173~P!?) +Molecules pYN EGFR(YN~P!?) +Molecules unphosR EGFR(Y1068~0,Y1173~0,YN~0) +Molecules pY1068_act EGFR(Y1068~P!?,Kin~act) +Molecules pY1068_rec EGFR(Y1068~P!?,Kin~rec) +Molecules Grb2_EGFR GRB2(SH2!+) +Molecules Shc1_EGFR SHC1(PTB!+) + +Molecules pY1068_pY1173 EGFR(Y1068~P!?,Y1173~P!?) +Molecules pY1068_pYN EGFR(Y1068~P!?,YN~P!?) +Molecules pY1173_pYN EGFR(Y1173~P!?,YN~P!?) +Molecules pYpYpYN EGFR(Y1068~P!?,Y1173~P!?,YN~P!?) + +Molecules monR_pYpY EGFR(II~u,Y1068~P!?,Y1173~P!?) +Molecules dimR_pYpY EGFR(II~b,Y1068~P!?,Y1173~P!?) + + + +end observables + + +begin functions + +pY1068_percent() 100*pY1068/EGFRtot +pY1173_percent() 100*pY1173/EGFRtot +pYpY_per() 100*pY1068_pY1173/EGFRtot +random_pYpY_per() 100*(pY1068/EGFRtot)*(pY1173/EGFRtot) +pY1173inP68_per() 100*pY1068_pY1173/(pY1068+1) +#PEnrichment() pY1173inP68_per()/pY1173_percent() + +monR_pYpY_per() 100*monR_pYpY/(monR+1) # +1 to avoid dividing by 0 +dimR_pYpY_per() 100*dimR_pYpY/(dimR+1) # +1 to avoid dividing by 0 + +pYN_percent() 100*pYN/EGFRtot +unphosR_per() 100*unphosR/EGFRtot +phosR_per() 100*(EGFRtot-unphosR)/EGFRtot # Phosphorylated in at least one site + +pY1173inPYN_per() 100*pY1173_pYN/(pYN+1) +pY1173inP68PYN_per() 100*pYpYpYN/(pY1068_pYN+1) +pYpYinPYN_per() 100*pYpYpYN/(pYN+1) + + + +end functions + + +begin reaction rules + +# EGF reversibly binds EGFR monomers +EGF(EGFL)+EGFR(I_III,II~u)<->EGF(EGFL!1).EGFR(I_III!1,II~u) kp_EGF,km_EGF + + +# Dimerization of EGFR for two EGF-bound receptors +# Transition from monomer (II~u, unbound) to dimer (II~b, bound) states. The kinase domain of one of the receptors adopt an activator conformation (Kin~act) +# and the other the receiver conformation (Kin~rec). +# Simplification: dimerization only happens between two EGF-bound receptors +EGFR(I_III!+,II~u,Kin~0)+EGFR(I_III!+,II~u,Kin~0)->EGFR(I_III!+,II~b,Kin~act)+EGFR(I_III!+,II~b,Kin~rec) kp_dim_L_L/2 + + + +# Dissociation of EGFR dimer. Kinase domain becomes inactive (Kin~0) after dissociation +EGFR(II~b,Kin~act)->EGFR(II~u,Kin~0) km_dim_L_L +EGFR(II~b,Kin~rec)->EGFR(II~u,Kin~0) km_dim_L_L + + +# Phosphorylation of C-tail in the activator receptor by the receiver receptor +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~act)->EGFR(II~b,Y1068~P,Kin~act) kphosY1068 +EGFR(II~b,Y1173~0,Kin~act)->EGFR(II~b,Y1173~P,Kin~act) kphosY1173 +EGFR(II~b,YN~0,Kin~act)->EGFR(II~b,YN~P,Kin~act) kphosYN + +# Phosphorylation of C-tail in the receiver receptor by itself. +# Occurs only within a dimer +EGFR(II~b,Y1068~0,Kin~rec)->EGFR(II~b,Y1068~P,Kin~rec) kphosY1068*ratio_kphos_receiver +EGFR(II~b,Y1173~0,Kin~rec)->EGFR(II~b,Y1173~P,Kin~rec) kphosY1173*ratio_kphos_receiver +EGFR(II~b,YN~0,Kin~rec)->EGFR(II~b,YN~P,Kin~rec) kphosYN*ratio_kphos_receiver + + +# Unregulated dephosphorylation of pTyr sites +# (mediated by constitutively active phosphatases) +EGFR(Y1068~P)->EGFR(Y1068~0) kdephosY1068 +EGFR(Y1173~P)->EGFR(Y1173~0) kdephosY1173 +EGFR(YN~P)->EGFR(YN~0) kdephosYN + + +# Binding of Grb2 to pY1068 in EGFR +GRB2(SH2)+EGFR(Y1068~P)<-> GRB2(SH2!1).EGFR(Y1068~P!1) kp_GE,km_GE + + +# Binding of SHC1 to pY1173 in EGFR +SHC1(PTB)+EGFR(Y1173~P)<-> SHC1(PTB!1).EGFR(Y1173~P!1) kp_SE,km_SE + + + +end reaction rules + +end model + +begin actions + +generate_network({overwrite=>1}) + + +# Perform a parameter scan for EGF ligand concentrations +parameter_scan({suffix=>"dose_resp",parameter=>"EGFconc",par_scan_vals=>[0.5e-9,5.0e-9,50.0e-9],\ +method=>"ode",t_start=>0,t_end=>300,n_steps=>3,print_functions=>1}) + + +# Add 25 nM EGF, and simulate for 300 seconds +setParameter("EGFconc","25.0e-9") +setConcentration("EGF(EGFL)","EGF_total") +# simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,n_steps=>5,print_functions=>1}) +simulate({suffix=>"EGF_25nM",method=>"ode",t_start=>0,t_end=>300,sample_times=>[60,120,300],print_functions=>1}) + + +end actions diff --git a/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/EGF_25nM.exp b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/EGF_25nM.exp new file mode 100644 index 00000000..a3d2dbd6 --- /dev/null +++ b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/EGF_25nM.exp @@ -0,0 +1,4 @@ +# time pY1068_percent pY1173_percent phosR_per pY1068_percent_SD pY1173_percent_SD phosR_per_SD + 60 9.35 17.96 41.92 0.67 0.79 0.20 + 120 9.47 19.15 42.64 0.69 0.88 0.26 + 300 12.07 21.38 50.59 0.85 1.02 0.34 diff --git a/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/dose_resp.exp b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/dose_resp.exp new file mode 100644 index 00000000..0e9f2358 --- /dev/null +++ b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/dose_resp.exp @@ -0,0 +1,4 @@ +# EGFconc pY1068_percent pY1173_percent phosR_per pY1068_percent_SD pY1173_percent_SD phosR_per_SD + 5.0e-10 3.30 4.19 6.29 0.29 0.31 0.61 + 5.0e-09 12.42 17.68 26.09 0.66 0.66 0.97 + 5.0e-08 19.62 23.50 40.83 0.98 0.80 1.30 diff --git a/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_bootstrap.conf b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_bootstrap.conf new file mode 100644 index 00000000..ce04b301 --- /dev/null +++ b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_bootstrap.conf @@ -0,0 +1,57 @@ + + +############# +### PATHS ### +############# + +# The directory to which job output will be written +output_dir=fit_bootstrap + +# The BioNetGen executable - taken from system $BNGPATH + +# The model file to be used in fitting simulations, and the experimental data to be fit +model=190127_CHO_EGFR_forBNF.bngl : dose_resp.exp, EGF_25nM.exp + + +####################### +### General Options ### +####################### + + +# Default setting for General Options + + +####################### +### Fitting Options ### +####################### + +# Which algorithm to use +fit_type=de # Synchronous differential evolution + +# Which objective function to minimize in fitting. A complete list of objective functions is described in GenFit documentation. +objfunc=chi_sq # Sum of squares + +# Number of simulations to run in parallel. +# Change parellel_count to the number of CPU cores on your machine for increased performance. +parallel_count=36 + +# The maximum number of generations to run. +max_iterations=1000 + +# The number of unique parameter sets generated in a given generation. +population_size=200 + +# Bootstrap settings +bootstrap = 1000 +bootstrap_max_obj = 500 + +delete_old_files=2 + + +# The free parameters. These are generated on a random log scale between numbers indicated. +loguniform_var=GRB2_total__FREE 1E4 1E6 +loguniform_var=SHC1_total__FREE 1E4 1E6 +loguniform_var=kdephosY1068__FREE 0.1 100 +loguniform_var=ratio_kpkd_Y1068__FREE 0.01 100 +loguniform_var=kdephosYN__FREE 0.001 100 +loguniform_var=ratio_kpkd_YN__FREE 0.01 100 diff --git a/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_pt.conf b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_pt.conf new file mode 100644 index 00000000..8f3f2cc3 --- /dev/null +++ b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_pt.conf @@ -0,0 +1,62 @@ + + +############# +### PATHS ### +############# + +# The directory to which job output will be written +output_dir=fit_pt + +# The BioNetGen executable - taken from system $BNGPATH + +# The model file to be used in fitting simulations, and the experimental data to be fit +model=190127_CHO_EGFR_forBNF.bngl : dose_resp.exp, EGF_25nM.exp + + +####################### +### General Options ### +####################### + + +# Default setting for General Options + + +####################### +### Fitting Options ### +####################### + +# Which algorithm to use +fit_type=pt # Parallel tempering + +# Which objective function to minimize in fitting. A complete list of objective functions is described in GenFit documentation. +objfunc=chi_sq # Chi Square + +# Number of simulations to run in parallel. +# Change parellel_count to the number of CPU cores on your machine for increased performance. +parallel_count=36 + +# The maximum number of generations to run. +max_iterations=200000 + +# The number of unique parameter sets generated in a given generation. +population_size=36 +reps_per_beta=4 +beta_range=0.05 1 + +burn_in=50000 # (default is 10000) + +sample_every=25 +output_hist_every=400 +hist_bins=50 + +delete_old_files=2 + +step_size=0.02 + +# The free parameters. These are generated on a random log scale between numbers indicated. +loguniform_var=GRB2_total__FREE 1E4 1E6 +loguniform_var=SHC1_total__FREE 1E4 1E6 +loguniform_var=kdephosY1068__FREE 0.1 100 +loguniform_var=ratio_kpkd_Y1068__FREE 0.01 100 +loguniform_var=kdephosYN__FREE 0.001 100 +loguniform_var=ratio_kpkd_YN__FREE 0.01 100 diff --git a/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_v1_28.conf b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_v1_28.conf new file mode 100644 index 00000000..b380cbdc --- /dev/null +++ b/Published/Salazar-Cavazos2019/PyBNF-fitting-setup/fit_v1_28.conf @@ -0,0 +1,54 @@ + + +############# +### PATHS ### +############# + +# The directory to which job output will be written +output_dir=fit_v1_28_regular + +# The BioNetGen executable - taken from system $BNGPATH + +# The model file to be used in fitting simulations, and the experimental data to be fit +model=190127_CHO_EGFR_forBNF.bngl : dose_resp.exp, EGF_25nM.exp + + +####################### +### General Options ### +####################### + + +# Default setting for General Options + + +####################### +### Fitting Options ### +####################### + +# Which algorithm to use +fit_type=de # Synchronous differential evolution + +# Which objective function to minimize in fitting. A complete list of objective functions is described in GenFit documentation. +objfunc=chi_sq # Sum of squares + +# Number of simulations to run in parallel. +# Change parellel_count to the number of CPU cores on your machine for increased performance. +parallel_count=4 + +# The maximum number of generations to run. +max_iterations=1000 + +# The number of unique parameter sets generated in a given generation. +population_size=200 + + +delete_old_files=1 + + +# The free parameters. These are generated on a random log scale between numbers indicated. +loguniform_var=GRB2_total__FREE 1E4 1E6 +loguniform_var=SHC1_total__FREE 1E4 1E6 +loguniform_var=kdephosY1068__FREE 0.1 100 +loguniform_var=ratio_kpkd_Y1068__FREE 0.01 100 +loguniform_var=kdephosYN__FREE 0.001 100 +loguniform_var=ratio_kpkd_YN__FREE 0.01 100 diff --git a/Published/Salazar-Cavazos2019/README.md b/Published/Salazar-Cavazos2019/README.md new file mode 100644 index 00000000..bfc5a140 --- /dev/null +++ b/Published/Salazar-Cavazos2019/README.md @@ -0,0 +1,25 @@ +## Name +Salazar-Cavazos2019 + +## Reference +Salazar-Cavazos E, Nitta CF, Mitra ED, Wilson BS, Lidke KA, Hlavacek WS, Lidke DS. Integrating Multiplex SiMPull and Computational Modeling to Evaluate Combinatorial Aspects of EGFR Signaling. Under review. + +## Description +A model of EGFR signaling that was parameterized using SiMPull measurements of multisite phosphorylation dynamics. + +## Files +* [190127_CHO_EGFR_best-fit.bngl](190127_CHO_EGFR_best-fit.bngl): Model of EGFR signaling in CHO cells expressing EGFR-GFP, using the parameter values for best fit obtained through PyBNF. +* [190127_HeLa.bngl](190127_HeLa.bngl), [190127_HMEC.bngl](190127_HMEC.bngl) and [190127_MCF10A.bngl](190127_MCF10A.bngl): Models of EGFR signaling for the differents cell lines. These models use as a base the model "190127\_CHO\_EGFR\_best-fit.bngl", with protein copy numbers for EGFR, Grb2 and Shc1 specific to each cell line. +* [190127_CHO_EGFR_sensitivity.bngl](190127_CHO_EGFR_sensitivity.bngl): Modification of the model "190127\_CHO\_EGFR\_best-fit.bngl" to perform sensitity analysis by increasing each of the parameter values in the model by 1%. +* [190127_CHO_EGFR_Epigen.bngl](190127_CHO_EGFR_Epigen.bngl): Model of EGFR signaling in CHO cells expressing EGFR-GFP, but in contrast to model "190127_CHO_EGFR_best-fit.bngl", it simulates EGFR activation with 20uM Epigen instead of 25nM EGF. The parameter value for EGFR dimer off-rate was varied to find the best fit for SiMPull results with Epigen ligand. +* [190127_CHO_HA_EGFR_L858R.bngl](190127_CHO_HA_EGFR_L858R.bngl): Model of EGFR signaling in CHO cells expressing HA-EGFR. The number of EGFR per cell was set to the experimentally estimated value for this cell line. The parameter value for kinase activity (phosphorylation rate) was varied to find best fit for experimental results with L858R mutant EGFR. +* [PyBNF-fitting-setup](PyBNF-fitting-setup): Files required to run parameterization of the base model in PyBioNetFit + * [190127_CHO_EGFR_forBNF.bngl](PyBNF-fitting-setup/190127_CHO_EGFR_forBNF.bngl): Model file with unknown parameters + * [dose_resp.exp](PyBNF-fitting-setup/dose_resp.exp), [EGF_25nM.exp](PyBNF-fitting-setup/EGF_25nM.exp): Experimental data files + * [fit_v1_28.conf](PyBNF-fitting-setup/fit_v1_28.conf): PyBNF configuration file to perform fitting + * [fit_bootstrap.conf](PyBNF-fitting-setup/fit_bootstrap.conf): PyBNF configuration file to perform bootstrapping + * [fit_pt.conf](PyBNF-fitting-setup/fit_pt.conf): PyBNF configuration file to perform Bayesian uncertainty quantification by parallel tempering + + + +## Notes diff --git a/README.md b/README.md index 788b1d68..bdb3b3a5 100644 --- a/README.md +++ b/README.md @@ -5,6 +5,7 @@ Model name| Description [Faeder2003](Published/Faeder2003) | Model of early events in FcεRI signaling, including Lyn recruitment and Syk activation. [Mitra2019](Published/Mitra2019) | Suite of benchmark parameter fitting problems for PyBioNetFit [Thomas2016](Published/Thomas2016) | Parameter fitting problems published with BioNetFit v1.0 +[Salazar-Cavazos2019](Published/Salazar-Cavazos2019) | Model of EGFR signaling fit to Single Molecule Pull-down (SiMPull) data ## Contributed models Model name| Description