Supplementary Information Significance of p53 dynamics in regulating apoptosis in response to ionizing radiation, and polypharmacological strategies Bing Liu 1,, Divesh Bhatt 1,, Zoltan N. Oltvai 2, Joel S. GreenBerger 3, Ivet Bahar 1, Departments of 1 Computational & Systems Biology, 2 Pathology, and 3 Radiation Oncology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA Model Parameters We estimated model parameters based on experimental data [3, 7, 4, 5] or data adopted in previous computational studies [2, 1]. Following Bagei et al [2] the cooperative formation of the apoptosome is modeled by a forward reaction of order 4. The interactions of XIAP are modeled following Albec et al [1]. Among the experimentally deduced parameters used in the model, we note that the dissociation constants, 22/ + 22, for p53 (M) and pro-apoptotic Bcl-2 family members have been given as 160 nm [3] and 535 nm [7], respectively - both measured by surface plasmon resonance. We chose an intermediate value (333 nm). PUMA binds significantly more strongly, and Chipu et al. [3] reported the dissociation constant 23/ + 23 = 10 nm, a value that we use. Edlich et al. [4] have reported the on and off rate of Bax from cytosol and mitochondria as approximately the same, and established a first order reaction (represented by Eq 24, Table S2) with + 24 = 4.9 10 3 s 1 and 24 = 4.7 10 3 s 1. For Eq 26, it is reported that p53 (M) displaces Bax (M) from its complex with Bcl-2 at equimolar concentration, whereas Bax (M) displaces p53 (M) from its complex with Bcl-2 at a 50 times higher concentration. This implies that 26/ + 26 = 50 nm. Since Eqs 22, 25, and 26 are not independent, these values imply that 25/ + 25 17µM. This value is significantly higher than those reported in the literature (0.1 µm [5] and 20 nm [6]). We use a value of 20 nm (and not use Eq 26 explicitly), except where noted - and, as shown below, the results obtained are robust for a wide range of values for this dissociation constant. Half lives of a few proteins have also been reported in vivo. However, the direct use of such an information (for example, to compute the decay rates of the type X Φ) is often not appropriate because the decay rate of protein X in vivo is convoluted with, for example, the decay of the protein Y that upregulates X. These parameters for the p53 module are chosen to reflect the p53 oscillations observed experimentally. 10 and 11 do not affect the dose-dependent oscillatory behavior of p53; however, they are important for coupling the p53 module to the apoptotic machinery that regulates downstream apoptosis. 1
symbol p53 (N) p53 (C) p53 A p53 (C) p53 (N) p53 M description p53 in nucleus p53 in cytoplasm stress-induced transcriptionally active p53 for apoptotic activation ubiquitinated p53 in cytoplasm ubiquitinated p53 in nucleus mitochondrial p53 (p53 N ) 4 p53 tetramer for transcription activation of Mdm2 (p53 A ) 4 p53 tetramer for transcription activation of pro-apoptotic PUMA/Bax mrna (N) Mdm2 mrna (C) Mdm2 Mdm2 (N) Mdm2 (C) PUMA Bcl-2 Bax Bax (M) Bax 2 Bax 4 Bid tbid cyt c (M) cyt c (C) Smac c (M) Smac c (C) Apaf XIAP apop mrna of Mdm2 in nucleus mrna of Mdm2 in cytoplasm Mouse double minute 2 homolog/e3 ligase protein in nucleus Mouse double minute 2 homolog/e3 ligase protein in cytoplasm p53-upregulated modulator of apoptosis B-cell lymphoma 2 protein Bcl-2-associated X protein in cytoplasm Bcl-2-associated X protein in mitochondria dimerized Bcl-2-associated X protein tetramerized Bcl-2-associated X protein BH3 interacting-domain death agonist truncated Bid cytochrome c in mitochondria cytochrome c in cytoplasm Smac/Diablo protein in mitochondria Smac/Diablo protein in cytoplasm Apaf-1 pro-apoptotic protein X-lined inhibitor of apoptosis protein apoptosome proc9 procaspase 9 C9 ubiquitinate caspase 9 proc3 procaspase 3 C3 active caspase 3 C3 ubiquitinated caspase 3 C8 active caspase 8 Bcl-x L Bcl-x (M) L ATM B-cell lymphoma-extra large protein in cytoplasm B-cell lymphoma-extra large protein in mitochondira Ataxia telangiectasia mutated protien Table S1: List of proteins in Figure 1 and their descriptions 2
Table S2: Reactions and parameters with default values (no radiation condition). Number Reaction Parameters 1 p53 (C) 1 p53 (N) 1 = 10 4 s 1 2 4p53 (N) + 2 (p53 (N) ) 4 + 2 = 10 µm 3 s 1, 2 = 10 4 s 1 2 3 (p53 (N) ) 3 4 (p53 (N) ) 4 + mrna (N) Mdm2 4 mrna (N) 4 (C) Mdm2 mrna Mdm2 5 mrna (C) 5 Mdm2 Mdm2 (C) 6 Mdm2 (C) 6 Mdm2 (N) 7 p53 (N) + Mdm2 (N) + 7 7 8 p53 (C) + Mdm2 (C) + 8 9 p53 (N) 10 p53 (C) 9 p53 (C) 10 p53 (M) 11 p53 (N) 11 p53 A 8 3 = 10 1 s 1 4 = 2 10 4 s 1 5 = 5 10 4 s 1 6 = 10 4 s 1 p53.mdm2 (N) 7 p53 (N) + Mdm2(N) + 7 = 0.1µM 1 s 1, 7 = 0.0s 1, 7 = 10 3 s 1 p53.mdm2 (C) 8 p53 (C) + Mdm2(C) + 8 = 0.1µM 1 s 1, 8 = 0.0s 1, 8 = 10 3 s 1 9 = 10 4 s 1 10 = 10 5 s 1 11 = 10 5 s 1 + 12 12 4p53 A (p53 A ) 4 + 12 = 10 µm 3 s 1, 12 = 10 4 s 1 12 13 (p53 A ) 13 4 (p53a ) 4 + mrna (N) Mdm2 14 Φ + 14 DNAdamage 14 13 = 10 1 s 1 + 14 = 0.0 µms 1, 14 = 5 10 5 s 1 15 DNAdamage 15 ATM 16 ATM 16 17 ATM 17 p53 (C) 18 p53 (C) 18 19 p53 19 A 20 p53 20 A p53a + PUMA 21 p53 21 A p53a + Bax 22 p53 (M) + Bcl-2 + 22 p53.bcl-2 22 23 PUMA + Bcl-2 + 23 PUMA.Bcl-2 24 Bax + 24 Bax (M) 24 23 25 Bax (M) + Bcl-2 + 25 Bax.Bcl-2 26 p53 (M) + Bax.Bcl-2 + 26 p53.bcl-2 + Bax (M) 27 tbid + 27 tbid (M) 27 25 26 28 tbid (M) + Bax (M) 28 tbid (M) + Bax* 15 = 10 2 s 1 16 = 10 1 s 1 17 = 10 2 s 1 18 = 10 4 s 1 19 = 10 4 s 1 20 = 10 3 s 1 21 = 10 3 s 1 + 22 = 3 10 3 µm 1 s 1, 22 = 10 3 s 1 + 23 = 10 1 µm 1 s 1, 23 = 10 3 s 1 + 24 = 5 10 3 s 1, 24 = 5 10 3 s 1 + 25 = 5 10 2 µm 1 s 1, 25 = 10 3 s 1 + 26 = 1.0µM 1 s 1, 22 = 2 10 2 s 1 + 27 = 5 10 3 µm 1 s 1, 27 = 5 10 3 s 1 28 = 5 10 1 µm 1 s 1 Continued on next page 3
Table S2 continued from previous page Number Reaction Parameters 29 p53 (M) + Bax (M) 29 p53 (M) + Bax* 30 PUMA + Bax (M) 30 PUMA + Bax* 31 Bax* + Bax* + 31 Bax 2 31 + 32 32 Bax 2 + Bax 2 Bax 4 32 33 Bax + 4 + cytc (M) 33 Bax 4 + cytc (C) 34 Bax + 4 + Smac (M) 34 Bax 4 + Smac (C) 35 cytc (C) + Apaf + 35 cytc.apaf 35 36 7 cytcapaf + 36 apop 36 37 apop + proc9 + 37 apop.c9 37 38 apop.c9 + proc3 + 38 39 C3 + Bid + 39 39 40 C8 + Bid + 40 40 38 apop.c3.c9 38 apop.c9 + C3 C3.Bid 39 C3 + tbid C8.Bid 40 C8 + tbid 41 apop.c9 + XIAP + 41 apop.c9.xiap 42 C3 + XIAP + 42 42 41 43 proc9 + XIAP + 43 C3.XIAP 42 C3 + XIAP 43 44 Smac (C) + XIAP + 44 Smac.XIAP 44 C9.XIAP 43 C9 + XIAP 29 = 5 10 2 µm 1 s 1 30 = 5 10 1 µm 1 s 1 + 31 = 6 10 1 µm 1 s 1, 31 = 10 3 s 1 + 32 = 10 1 µm 1 s 1, 32 = 10 3 s 1 33 = 10µM 1 s 1 34 = 10µM 1 s 1 + 35 = 3 10 1 µm 1 s 1, 35 = 10 3 s 1 + 36 = 105 µm 3 s 1, 36 = 10 5 s 1 + 37 = 3 10 2 µm 1 s 1, 37 = 10 4 s 1 + 38 = 3 10 2 µm 1 s 1, 38 = 10 4 s 1, 38 = 1.0 s 1 + 39 = 3 10 1 µm 1 s 1, 39 = 10 3 s 1, 39 = 1.0 s 1 + 40 = 3 10 1 µm 1 s 1, 40 = 10 3 s 1, 40 = 1.0 s 1 + 41 = 3 10 2 µm 1 s 1, 41 = 10 4 s 1 + 42 = 1.0µM 1 s 1, 42 = 10 3 s 1, 42 = 0.1 s 1 + 43 = 1.0µM 1 s 1, 43 = 10 3 s 1, 42 = 0.1 s 1 + 44 = 10 1 µm 1 s 1, 44 = 10 3 s 1 45 p53 A + Mdm2 (N) + 45 45 p53a.mdm2 (N) 45 p53 (N) + Mdm2(N) + 45 = 0.1µM 1 s 1, 45 = 0.0s 1, 45 = 10 3 s 1 + 46 46 PUMA + I PUMA PUMA.I PUMA 46 + 47 47 Bid + I Bid Bid.I Bid 47 + 48 48 C3 + I C3 C3.I C3 48 + 49 49 Bax + I Bax Bax.I Bax 50 Apaf 50 51 Bax 51 52 Bax* 52 53 Bcl-2 53 54 Bid 54 49 + 46 = 1.0µM 1 s 1, 46 = 10 4 s 1 + 47 = 1.0µM 1 s 1, 47 = 10 4 s 1 + 48 = 1.0µM 1 s 1, 48 = 10 4 s 1 + 49 = 1.0µM 1 s 1, 49 = 10 4 s 1 50 = 10 3 s 1 51 = 10 3 s 1 52 = 10 3 s 1 53 = 10 3 s 1 54 = 10 3 s 1 Continued on next page 4
Table S2 continued from previous page Number Reaction Parameters 55 C3 55 56 C3 56 57 proc9 57 58 C9 58 59 cytc (C) 59 60 cytc (M) 60 61 Mdm2 (C) 61 62 Mdm2 (N) 62 63 p53 (C) 63 64 p53 (M) 64 65 p53 (C) 66 p53 (M) 65 66 67 proc3 67 68 PUMA 68 69 Smac (C) 69 70 Smac (M) 70 71 tbid 71 72 XIAP 72 73 Φ 73 Apaf 74 Φ 74 Bax 75 Φ 75 Bcl-2 76 Φ 76 Bid 77 Φ 77 C9 78 Φ 78 cytc (M) 79 Φ 79 Smac (M) 80 Φ 80 XIAP 81 Φ 81 p53 (C) 82 Φ 82 proc3 55 = 10 3 s 1 56 = 10 3 s 1 57 = 10 3 s 1 58 = 10 3 s 1 59 = 10 2 s 1 60 = 10 3 s 1 61 = 5 10 4 s 1 62 = 5 10 4 s 1 63 = 10 4 s 1 64 = 10 3 s 1 65 = 10 3 s 1 66 = 10 3 s 1 67 = 10 3 s 1 68 = 10 3 s 1 69 = 10 3 s 1 70 = 10 3 s 1 71 = 10 3 s 1 72 = 10 3 s 1 73 = 10 4 µm 1 s 1 74 = 10 5 µm 1 s 1 75 = 10 5 µm 1 s 1 76 = 10 5 µm 1 s 1 77 = 10 4 µm 1 s 1 78 = 10 4 µm 1 s 1 79 = 10 4 µm 1 s 1 80 = 10 4 µm 1 s 1 81 = 10 6 µm 1 s 1 82 = 10 4 µm 1 s 1 + 83 83 Bcl-x L 83 Bcl-x (M) L + 83 = 5 10 3 s 1, 83 = 5 10 3 s 1 84 Bcl-x (M) L + Bax (M) 84 Bcl-x (M) L + Bax 84 = 5 10 2 µm 1 s 1 85 Φ 85 Bcl-x L 85 = 10 5 µm 1 s 1 86 Bcl-x 86 L 86 = 10 3 s 1 5
Parameter No radiation (normal) Radiation (WT) Low frequency p53 oscillation (LF) 1 (s 1 ) 10 4 10 3 10 4 4 (s 1 ) 2 10 4 5 10 3 2 10 4 6 (s 1 ) 10 4 5 10 4 10 4 + 14 (µm s 1 ) 0 5 10 7 2 10 7 18 (s 1 ) 10 4 10 5 10 4 19 (s 1 ) 10 4 10 5 10 4 Table S3: Parameter values for different conditions 1 2 L F c o n d itio n IR 1 0 p 5 3 (N ) c o n c. [n M ] 8 6 4 2 0-4 0-2 0 0 2 0 4 0 6 0 8 0 1 0 0 tim e [h ] Figure S1: Simulated p53 (N) profile under LF condition. 6
1.0 0.8 0.6 5 1 S e n s itiv itie s 0.4 1 1 2 0 - + 2 4 2 4 3 0-3 1 + 3 1 7 4 4 5 0.2 0.0 + 2 3 + - 2 7 2 7 2 8-3 2 + 3 2 3 3 + 3 8 P a ra m e te rs + 4 1-4 1 + 4 2 8 1 6 2 8 0 5 9 Figure S2: Parameter sensitivities. The system behavior is highly sensitive to the reaction rates indicated by the peas. See Table S2 for the corresponding reactions. 7
C3 conc. [nm] 0.6 LF WT p53 (C) IR 0.4 0.2 0.0-40 -20 0 20 40 60 80 100 time [h] Figure S3: Simulated profile of p53 (C) under LF and WT conditions. 1.0 0.8 2 3 = 0.1 2 3 = 0 IR C 3 c o n c. [n M ] 0.6 0.4 0.2 0.0-4 0-2 0 0 2 0 4 0 6 0 8 0 1 0 0 tim e [h ] Figure S4: Simulated profile of caspase-3 under the condition of nocing down PUMA and Bcl-2 interactions. 8
The BTLT expressions of dynamical properties Property 1 Mdm2 reaches its pea after p53. P r 0.95 (p53 (N) 0.01nM Mdm2 (N) 0.01nM F 300h (p53 (N) 1nM Mdm2 (N) 0.01nM F 300h (p53 (N) 3nM Mdm2 (N) 0.4nM F 300h (p53 (N) 4nM Mdm2 (N) 0.4nM)))) The above property specifies that the level of nuclear p53 reaches a pea value between 3 and 4 nm before the level of nuclear Mdm2 reaching a pea value around 0.4 nm. This properties was verified to be true. Property 2 Increased DNA damage induces more p53 pulses. P r 0.95 (p53 (N) 5nMU 300h (F 12h (p53 (N) 6nM (F 12h (p53 (N) 5nM (F 12h (p53 (N) 6nM (F 12h (p53 (N) 5nM (F 88h (G 88h (p53 (N) 5nM))))))) The above property specifies that upon 12 h IR exposure there are at least two p53 pluses induced. For insufficient amount of DNA damage, this property was verified to be false, while it was verified to be true for sufficient amount of DNA damage. This suggests that increased DNA damage induces more p53 pulses. Property 3 Sustained caspase-3 once its level reaches certain threshold. P r 0.95 (C3 0.01nMU 300h (F 56h (C3 0.3nM G 44h (C3 0.3nM)))) The above property specifies that after caspase-3 concentration reaches 0.3nM, it will sustain for at least 44 h and triggers downstream apoptotic cascade. This property was verified to be true. Property 4 Mutated Bax prevents the p53-mediated apoptosis. P r 0.95 (C3 0.01nMU 300h (F 56h (G 100h (C3 0.3nM)))) The above property specifies the case that p53-mediated apoptosis is triggered after IR. It was verified to be true when 11 = 2 10 5 s 1. However, if we mutate Bax by reducing the activation rates of Bax, the property was verified to be false, suggesting that mutant Bax prevents the p53-mediated apoptosis. Property 5 Inhibition of XIAP enhances p53-mediated apoptosis. P r 0.95 (C3 0.01nMU 300h (F 56h (G 100h (C3 1nM)))) The above property specifies a high steady state of caspase-3 after IR. It was verified to be false when the degradation rate of XIAP equals to 10 4 s 1. However, it was verified to be true when the degradation rate of XIAP equals to 10 2 s 1, showing that the inhibition of XIAP enhances p53-mediated apoptosis. Property 6 p53 pulses induce differential expression of target genes. P r 0.95 (Bax 0.001nM P UMA 0.0011nMU 300h (F 56h (Bax 0.06nM P UMA 0.06nM (F 56h (Bax 0.04nM P UMA 0.04nM (F 56h (Bax 0.06nM P UMA 0.06nM (F 56h (Bax 0.04nM P UMA 0.04nM))))) 9
The above property specifies the oscillatory behavior of Bax and PUMA. It was verified to be true for IR condition, indicating that the expression profiles of p53 target genes mirrors the oscillation of p53 level. In contrast, for the condition of sustained p53 activation (implemented by inhibiting Mdm2), this property was verified to be false. This suggests that p53 pulses induce differential expression of target genes. Property 7 Intermittent Nutlin-3 addition induce sustained p53 activation. P r 0.95 (p53 (N) 5nMU 300h (F 56h (p53 (N) 6nM))) Nutlin-3 is a small molecule that binds to Mdm2 and inhibits degradation of p53. Without Nutlin-3 treatment, the above property was verified to be false. However, by applying a sequential treatments of Nutlin-3 at 2.5 h, 3.5 h, 4.5h,... after IR, the property was verified to be true. References [1] J. G. Albec, J. M. Bure, S. L. Spencer, D. A. Lauffenburger, and P. K. Sorger. Modeling a snap-action, variable-delay switch controlling extrinsic cell death. PLoS Biol, 6:e299, 2008. [2] E. Z. Bagci, Y. Vodovotz, T. R. Billiar, G. B. Ermentrout, and I. Bahar. Bistability in apoptosis: Roles of bax, bcl-2, and mitochondrial permeability transition pores. Biophys J, 90:1546 1559, 2006. [3] J. E. Chipu, L. Bouchier-Hayes, T. Kuwana, D. D. Newmeyer, and D. R. Green. PUMA couples the nuclear and cytoplasmic proapoptotic function of p53. Science, 309:1732 1735, 2005. [4] F. Edlich, S. Banerjee, M. Suzui, M. M. Cleland, D. Amoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle. Bcl-xl retrotranslocates Bax from the mitochondria into the cytosol. Cell, 145:104 116, 2100. [5] J. I. Fletcher, S. Meusburger, C. J. Hawins, D. T. Riglar, E. F. Lee, W. D. Fairlie, D. C. S. Huang, and J. M. Adams. Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax. Proc Natl Acad Sci, 105:1808118087, 2008. [6] B. Ku, C. Liang, J. U. Jung, and B.-H. Oh. Evidence that inhibition of Bax activation by Bcl-2 involves its tight and preferential interaction with the BH3 domain of Bax. Cell Res, 21:627641, 2011. [7] Y. Tomita, N. Marchen, S. Erster, A. Nemajerova, A. Dehner, C. Klein, H. Pan, H. Kessler, P. Pancosa, and U. M. Moll. Wt p53, but not tumor-derived mutants, bind to Bcl2 via the dna binding domain and induce mitochondrial permeabilization. J Biol Chem, 281:8600 8606, 2006. 10