Computational Biological Research Of The Phosphorylation Modification Of Tyrosines In Signal Pathways And The Interactions Of SHC1 And SHC3 With Related Signal Molecules

To study the molecular mechanism of cell cycle exit of mature neuron is very important for the understanding of the degeneration diseases of central nervous system (CNS). SHC1 and SHC3 are homology molecules with the same PTB-CH1-SH2 motif architecture and both are initial molecules in RAS pathways downstream receptor tyrosine kinases (RTKs). However, they can function differently in signal transduction and consequently mediate different signal pathways. In CNS, SHC1 was only expressed in neural stem cells, while SHC3 in mature neurons. This provides us a good cut point from which to study the molecular mechanism of cell cycle exit of mature neuron by comparing the signal transduction differences between SHC molecules. However, SHC molecules can bind many molecules upstream and downstream; it's hard to choose specific molecules for immediate experimental study. In this study, we planned to use computational biological methods to simulate and compare the interactions between SHC molecules and other related signal molecules, and consequently find the signal transduction differences between SHC1 and SHC3 molecules, and then verify some computational results by experiments.For this purpose, a high efficiency structural biology simulation platform is needed badly. We have used super blade computer system as hardware, adopted AMBER, CHARMM VMD and other notable software, and consequently constructed a high efficiency parallel computational structural biology simulation platform. In this platform, we have confirmed the most proper force filed (AMBER) for phosphorylated amino acids; compiled molecular dynamics, steered molecular dynamics, umbrella sampling molecular dynamics, constrained dynamics and other rapid computation methods; realized the method to build a protein-membrane system; constructed a method to dock protein and phosphorylated peptide; constructed a step-by-step steered molecular dynamics method to detach bound protein and not lead to much structural disturbance; constructed a method to retrieve parameter values for certain time points and amino acids from the AMBER trajectory and to view its variation and contribution during the whole trajectory. The construction of this platform laid a strong tool foundation for further simulation analysis.In the preliminary simulations, we have chosen the complex of the SHC1 SH2 domain and the T cell receptor zeta chain CD247-zeta as the object to simulate their structural and binding energy variations with or without phosphorylation modification of CD247-zeta. The result showed that when unphosphorylated their binding force declined to one third of that when phosphorylated. And the CD247-zeta tended to leave the interaction center. This demonstrated the importance of the phosphorylation modification of tyrosine in their binding, which was verified by pull-down experiment. Detailed analysis of the phosphorylated tyrosine showed that it can not only form hydrophobic interactions with hydrophobic residues in self peptide through benzene ring, but also form hydrophilic interactions with hydrophilic residues in binding partner peptide through the phosphate group. This kind of unification of contrary characterization make tyrosine an excellent regulon to mediate signal transduction by its reversible phosphorylation. Thus, in the following studies, we pay much attention on the role of phosphorylation of tyrosine.To simulation the interactions of SHC molecules with RTKs, the RTKs were split into many peptides containing tyrosines that can be phosphorylated, and the SHC molecules were split into peptides corresponding PTB,CH1 and SH2 domains respectively. The structures of these peptides were homology modeled and the interactions of RTK peptides with whole SHC molecules and its domains were simulated using the improved phosphotyrosine suitable AMBER force field. The results showed that the binding of SHC3 with IR999, TRKA490 and CD247-zeta were stronger than that of SHC1; among these RTKs, SHC3 bind TRKA490 strongest, while SHC1 bind EGFR and TRKA490 strongest; among these SHC domains, the PTB domain of SHC3 bind TRKA490 strongest, next came the SH2 domain of SHC3 with TRKA490. As for the downstream signal molecules, we simulated the interaction of the SHC CH1 domains with GRB2 and found that the binding of SHC3 with GRB2 is weaker than that of SHC1 obviously. When double phosphorylated at Y341/Y342 and Y379/Y380 in SHC3 CH1 domain, its binding with GRB2 declined, whereas when double phosphorylated at corresponding tyrosines(Y239/Y240) in SHC1 CH1 domain, the binding of SHC1 with GRB2 was enhanced obviously. All these implied that SHC1 and SHC3 should select different downstream signal molecules. Finally, the mechanism which is involved in regulating the phosphorylation of SER/THR in SHC1-PTB domain is successfully imitated. The binding force between SHC1 and IR99 which ultimately influences cell proliferatioon descreases because of the phosphorylation of SER29. The similiar SER/THR phosphorylation sites also exist in SHC3-PTB domain. Thus, further study needs the understanding of SER/THR phosphorylation sites in SHC3.In general, although similar in structure architecture, there are many differences in upstream RTKs and downstream signal molecules mediated by SHC1 and SHC3. This study laid a useful theoretical computational foundation for further experimental studies on the these differences to determine their possible roles in the cell cycle exit of mature neurons.