Towards Protein N-phosphorylation:Research Tool Development And Phosphosite Identification

Protein phosphorylation is one of the most common and significant post-translational modifications in organisms.Phosphorylation plays an important role in the regulation of signal transduction,gene expression,cell division and other biological processes by changing the conformation,activity of proteins and protein-protein interactions,and is closely related to the occurrence and development of tumors.However,in general,researches on phosphorylation mainly focused on O-phosphorylation.Several studies suggest that N-phosphorylation also plays an important role in life processes,for example,the histidine phosphorylation plays a key role in the two-component system for response to external stimuli,and the arginine phosphorylation serves as a mark for protein degradation in prokaryotes.However,comparing to O-phosphorylation,the study of N-phosphorylation was greatly underestimated,which is due to the acid and heat lability of N-phosphorylation.This instability property,which is different from O-phosphorylation,on the one hand is incompatible with the existing protein analysis procedures and on the other hand makes it difficult to develop specific enrichment materials.The lack of research methods hindered the comprehensive understanding of the number,classification,and spatiotemporal distribution of intracellular N-phosphorylated proteins.The related kinases and phosphatases are rarely found,and functions of N-phosphorylation in regulating protein are still not clear.Therefore,the development of research tools for N-phosphorylation is a basic and urgent scientific task.In fact,almost all breakthroughs in this area in recent years derived from the successful development of tools and the optimization or innovation of methods.The work discussed in this thesis is to follow this direction,including:1.Synthesis and Characterization of Stable Analogs.The stable analogues of pHis and pArg were synthesized and characterized.Then the charge properties of the compounds were explored.The mass spectrometry quantitatively proved that the phosphorylated compounds could be taken up by cells,which laid the foundation for the subsequent biological experiments.2.Development of N-phosphorylation antibodies.A complete set of methods for preparation,characterization,and application of N-phosphorylation antibodies was established.Monoclonal and polyclonal antibodies of high-affinity and high-specificity were successfully developed,and applied to various biochemical characterization experiments,revealing that pHis and pArg are widespread presented in eukaryotic cells.3.Evaluating the inhibitory effect of stable analogs on histidine phosphatase.A method for detecting the activity of histidine phosphatase(PHPT1)was developed based on the above-mentioned pHis antibody,which was used to characterize the in vitro inhibitory ability of the analogs on PHPT1;the effect of the analogs on the proliferation and migration of lung cancer cells was examined and preliminary revealed the relationship between this effect and PHPT1 activity.4.Identification of N-phosphorylated proteins and sites in human cells.Based on an optimized N-phosphorylationomics protocol,the widespread presence of N-phosphorylation was identified in the Jurkat cell line,revealing that the N-phosphorylated protein may play an important role in nucleic acid-related functions,and the N-phosphorylated sequence was found sharing several specific motifs.Furthermore,the above-mentioned antibodies were successfully applied to the verification of a protein identified by mass spectrometry.In short,focusing on the instability of N-phosphorylation,this paper developed efficient research tools and research procedures,and initially identified a large number of N-phosphorylation sites in eukaryotic cells,laying the foundation for subsequent large-scale identification experiments and biological function studies..