| Objective: To investigate the topological structureproperties of cellular signaling network based on constructed network model, andto analysis the functional status of different nodes based on experiments andcomputer simulation technologies for recognizing the tolerance ability of cellularsignaling network against random nodes errors, then providing assistance forfurther research on the complexity of cellular signaling system. Methods:①Signaling network construction and topological structure analysis: a complexcellular signaling transduction network was constructed in terms of available cellsignaling transduction pathways and research data of the mechanism ofbiochemical action among signal molecules. Three major steps were included inconstruction: Firstly, linear pathways which could produce output effects bystimulus were defined. Next, the connection relations of nodes that haveinteractions among pathways were analyzed. Finally, functional modules wereorganized. The topological structure properties of cellular signaling network wereanalyzed at several levels, such as the connectivity of nodes, the organizingfeatures of nodes, crosstalks among pathways and the effect of random nodedeletion on network, according to the network model.②Computer simulationanalysis: computer simulation soft SIMTRAN2.0 which was complied byourselves was utilized to perform dynamic simulation analysis of cellularsignaling network with various node suffered disturbances. Simulation time was120min, by step of 0.2min. The dynamic parameters of corresponding reactionequations were changed to imitate stimulating or inhibitory attacks on nodes. Thehigher or lower connectivities of nodes were included in the targets, andstimulating or inhibitory attacks were exerted on nodes.③The effects of nodeinhibition on neuronal signaling network of mice: after interfering, culturedneurons of mice were divided into 4 groups, including 1 controlled group and 3experiment groups. Experiment groups were added with the specific inhibitor ofRas (Manumycin A), Jak (420099), or PDE(R1096) respectively, and thecontrolled group was not added any inhibitor. Neurons were incubated at 38℃ inthe presence of 5%CO2 overnight. The experiment and controlled groups were alladded EGF (20ng/μ1), and stimulated for 0min,10min,20min,60min,120minrespectively , then fluid nitrogen was used to end the stimulations of EGF.Neurons were lysed on ice with agitation for half an hour. The cell lysates werecollected with E.P. tubes, and centrifuged at 12000rpm for 30min at 4℃. At last,the protein concentrations were determined by Bradford method. AfterSDS-PAGE, the separated proteins in the polyacrylamide gels wereelectrotransfered to PVDF membranes. The relative intensities of Phospho-Aktand Phospho-MEK1/2 were determined by Phospho-Akt and Phospho-MEK1/2Rabbit mAB. Results:①A model of complex cellular signaling network wasachieved by construction and the network model obviously shows inhomogeneoustopological structure. In this network, a few nodes were connected to many othernodes, and the majority of nodes were connected to only a few other nodes.②Computer simulation results indicated that the alteration intensity and range ofsignaling network outputs caused by sustained stimulating of Ras node weremuch more obviously than those of transient excess stimulating of Ras node. Thedisturbance effects of important nodes (Ras, PI3K, PKA) on the network weremore serious than those of common nodes(PDE, PAK, PLA2).③The alterationsof network dynamic behavior were intricate when multi-nodes suffered attacks.④It was shown that the decreases of phosphorylated MEK1/2 and Akt wereprominent when Ras node was inhibited by the specific inhibitor ManumycinA .But the changes were not obvious when Jak or PDE node was blocked by itsspecific inhibitor respectively. More clearly, the changes could be observed in thetime courses of phosphorylated MEK1/2 and Akt by relative intensity versus time.It was noticeable that the decreases of relative intensity of phosphorylatedMEK1/2 and Akt were very obvious when Ras node was inhibited. Themaximally relative intensity of phosphorylated MEK1/2 prominently decreasedfrom 62.81 to 8.86, and that of phosphorylated Akt decreased from 46.13 to 15.32.On the contrary, the changes of time courses of phosphorylated MEK1/2 and Aktwere slight when Jak or PDE node was inhibited respectively. Conclusion:①Cellular signaling network belong to scale-free networks, and key nodes play apivotal role in it. Cellular signaling network displays a surprising degree oftolerance against random nodes errors.②The dynamic behaviors of cellularsignaling network present nonlinear features obviously. ③ The topologicaldifferences between important nodes and common nodes in signaling networksresult in the distinguish of their network function. Important nodes dominatemany other nodes through direct or indirect interactions, and common nodes onlyare related to a few of other nodes. The errors of important nodes play animportant role in the turbulence of signaling network.④The numerous complexcrosstalks existed in signaling network do not induce the diffusibility of signaltransduction, and there are complex mechanisms for the specificity and fidelity incellular signaling.
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