Fas-induced apoptosis pathway

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CSML1.9 version: ZIP | CSML [2007-06-05]
CSML3.0 version: ZIP | CSML [2008-01-29]
Launch on CIOPlayer [2009-08-05]
Launch on CIO [2009-08-05]

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  • Created by Atsushi Doi.

Description

We shall show that the notion of HFPN and Cell Illustrator are also useful to represent and simulate signal transduction pathways.

Apoptosis, programmed cell death, is known to participate in various biological processes such as development, maintenance of tissue homeostasis and elimination of cancer cells.Malfunctions of apoptosis have been implicated in many forms of human diseases such as neurodegenerative diseases, AIDS and ischemic stroke. Reportedly, apoptosis is caused by various inducers such as chemical compounds, proteins or removal of NGF. The biochemical pathways of apoptosis are complex and depend on both the cells and the inducers.

Fas-induced apoptosis has been studied in detail and its mechanism has been proposed as shown in Figure 1 [4]. We will represent this mechanism as a HFPN with Cell Illustrator.

Figure 1: Proposed steps of apoptosis induced by Fas ligand: Fas ligands, which usually exist as trimmers, bind and activate their receptors by inducing receptor trimerization. Activated receptors recruit adaptor molecules such as Fas-associating protein with death domain (FADD), which recruit procaspase 8 to the receptor complex, where it undergoes autocatalytic activation. Activated caspase 8 activates caspase 3 through two pathways; The complex one is that caspase 8 cleaves Bcl-2 interacting protein (Bid) and its COOH-terminal part translocates to mitochondria where it triggers cytochrome c release. The released cytochrome c bind to apoplectic protease activating factor-1 (Apaf-1) together with dATP and procaspase 9 and activates caspase 9. The caspase 9 cleaves procaspase 3 and activates caspase 3. The another pathway is that caspase 8 cleaves procaspase 3 directly and activates it. The caspase 3 cleaves DNA fragmentation factor (DFF) 45 in a heterodimeric factor of DFF40 and DFF45. Cleaved DFF45 dissociates from DFF40, inducing oligomerization of DFF40 that has DNase activity. The active DFF40 oligomer causes the internucleosomal DNA fragmentation, which is an apoptotic hallmark indicative of chromatin condensation.


The pathways consist of several steps where two different pathways from caspase 8 are assumed and many molecules including Fas receptors, caspase family which includes aspartic acid-dependent cysteine proteases and produced from their zymogens, Bcl-2 family which includes pro- and anti-apoptotic proteins, cytochrome c and DNA fragmentation factor. The apoptosis starts from the Fas ligand binding to Fas receptors and ends in the fragmentation of genomic DNA, which is used as a hallmark of apoptosis. Thus the amount of DNA fragmentation can be assumed to be proportional to the cell death.

We have designed a HFPN model by using the facts about the Fas-induced apoptosis pathways shown in Figure 1 and biochemical knowledge about reactions. Figure 2 shows the whole HFPN representation that we have described with Cell Illustrator.

Figure 2: A HFPN representing the Fas-induced apoptosis by HFPN obtained from Figure 1: For Bid (m11), Procaspase-9 (m21), Procaspase-3 (m25), DFF (m30), DNA (m37), the initial concentration of each compound is assumed to be 100. On the other hand, for FADD (m4), Procaspase-8 (m5), Apaf-1 (m17), dATP/ADP (m18), when two compounds react together without the stimulation of apoptosis, the initial concentrations and the rate are assumed to be 39.039 and m1*m2/5000, respectively to keep the stable state condition. Each compound is assumed to be produced by the rate of 0.5 (represented by a transition without any incoming arc) and to degrade by the rate of its concentration divided by 200 (represented by a transition without any outgoing arc), which will keep its concentration at 100 under the stable state condition. This degradation rate also applies to other compounds in the network. The rate of other processes are determined roughly by following Table 1. Synthesis and catabolism processes are added in the model for all proteins. Autocatalytic processes are also added in the model to all caspases since they exist as proenzymes. The pathway from caspase 8 to caspase 3 is assumed when the caspase 8 concentration is over 30. Protease is often synthesized as a proenzyme (zymogen) and changed to active form by other enzymes or by itself. So autocatalytic process is added to every caspase reaction.


Table1: Functions assigned to continuous transitions in the simulation of apoptosis induced by Fas ligand, where mA and mB represent the contents of the corresponding continuous places.

Rate Unimolecular reaction Bimolecular reaction
Self-effacement mA/200
Oligomer mA/20 mA*mB/10000
Monomer mA/10 mA*mB/5000
Enzyme binding mA/5 mA*mB/2500
Enzyme reaction mA*10

Results

By using the apoptosis scheme modeled as a HFPN, we simulated the DNA fragmentation amount by varying the Fas ligand concentration and Figure 3 shows the simulated relationship. It shows that under very weak stimulation (very low amount of Fas ligand), DNA fragmentation does not occur since the stimulation stops at the intermediate point because of the assumption of degradation processes. With the increase of the stimulation, the reaction proceeds to the backward intermediates and DNA fragmentation (cell death) occurs finally, which increases with the increase of the Fas ligand concentration.

Figure 3: Simulated relationship between the DNA fragmentation amount and the Fas ligand concentration: At higher concentration of Fas ligand, the direct pathway from caspase 8 to caspase 3 contributes to the fragmentation. To examine the effect of autocatalytic process of caspases, DNA fragmentation is simulated for both cases of the presence and absence in this process.


There are two pathways from activated caspase 8 to caspase 3, one through several steps including the cytochrome c release from mitochondria when the concentration of activated caspase 8 is low, and the direct one to caspase 3 when the concentration of activated caspase 8 is high [3]. We assume arbitrarily that the direct pathway starts when the concentration of activated caspase 8 is larger than 30. Reportedly the removal of the Bid by gene knockout method increases the resistance of liver cell apoptosis by Fas ligand, while it does not affect the apoptosis of thymus and embryonic cells. If the second pathway is included to the scheme, DNA fragmentation increases slightly, especially, when the Fas ligand concentration is high (Figure 3). However the detailed mechanism of the selection of these two pathways from caspase 8 are still unclear and necessary to be studied in future.

Since the presence of autocatalytic process is proposed in caspases [2], it is included in our model (Figure 4), which increases the DNA fragmentation as shown in Figure 3.

Figure 4: A HFPN representation of autocatalytic process in Figure 2.


However, if the large rate of the autocatalytic process is assumed in the caspase reaction, the DNA fragmentation becomes independent of the Fas ligand concentration, which disagrees with the experimental results. Therefore, we can guess that autocatalytic processes must be slow if they are at present. To examine the effect of autocatalytic processes of caspases on the apoptosis by Fas ligand, DNA fragmentation is simulated when the stimulation by Fas ligand stopped after a short period. Table 2 shows a simulation result that the apoptosis proceeds more with the increase of the autocatalytic rate of caspases even for a short period stimulation.

Table 2: DNA fragmentation at four autocatalytic rates of caspases (rate0=0, rate1=mA*mB/8000, rate2=mA*mB/4000, and rate3=mA*mB/2500), which are assigned to the transition TA in Figure 4. The stop time represents the period after that Fas ligand stimulation is stopped. The initial Fas ligand concentration is set to be n = 200. Variables mA and mB represent the contents of the continuous places going into TA.

DNA Fragmentation
Stop time rate0 rate1 rate2 rate3
10 0 0 0 779
15 0 405 715 1824
20 402 567 869 2025

Figure 5 shows simulated time courses of the HFPN in Figure 2 with Cell Illustrator. Some intermediates during apoptosis at three levels of Fas ligand concentrations are measured. These time courses might be useful to plan new experiments such as addition of inhibitors to some step. However, it is necessary to estimate the realistic rates of each reaction by the comparison with the experimental data. It is also necessary to add other pathways through Bcl-2 family [1] or p53 to describe the real apoptosis held in various cells and by various inducers. Cell Illustrator is a very useful tool for biochemists to describe the complex biological pathways semi-quantitatively on a figure.

Figure 5: Simulated time courses of some intermediates during apoptosis for the Fas ligand concentration n=70, 150, 200.

Publication

  • Matsuno H, Tanaka Y, Aoshima H, Doi A, Matsui M, Miyano S, Biopathways Representation and Simulation on Hybrid Functional Petri Net, In Silico Biology, 3(3): 389-404 (2003).(PubMed_12954096)(.html)

References

[1] Harada, H. (1999) Regulation of apoptosis by BH3 domain only proteins. Jikkenigaku, 17, 1603-1606.

[2] Hugunin, M., Quintal, L.J., Mankovich, J.A. and Ghayur, T. (1996) Protease activity of in vitro transcribed and translated Caenorhabditis elegans cell death gene (ced-3) product. Journal of Biological Chemistry, 271, 3517-3522.

[3] Kuwana, T., Smith, J.J., Muzio, M., Dixit, V., Newmeyer, D.D., and Kornbluth, S. (1998) Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c. Journal of Biological Chemistry, 273, 16589-16594.

[4] Nijhawan, D., Honarpour, N. and Wang, X. (2000) Apoptosis in neural development and disease. Annual Reviews of Neuroscience, 23, 73-87.