Identification And Exploration Of New Protein Post-Translational Modifications

Protein post-translational modifications（PTM）are vital process to regulate protein properties and functions,after amino acids are assembled into polypeptide chains in a sequence determined by DNA sequence of gene through translation.Chemical biology tools coupled with mass spectrometry methods are promising approaches for PTM enrichment and function research.We identified O-Glc NAcylation occurs on YTHDF1 and YTHDF3,but not on YTHDF2,with mass spectrometry.We used Gal-T1 chemoenzymatic labeling method to validate that S157 and S196 are the modification sites of YTHDF1 and T205 and S229 are the modification sites of YTHDF3.The modification sites are on the effector domain of YTHDF1/3 and the modification represses the recruitment of translation related proteins to YTHDF1/3 which directly bring to the inhibition of translation promotion function of YTHDF1/3.Furthermore,we found that YTHDF1/3 contains high O-Glc NAcylation level in HEK293T cells（～50%）but low modification level in He La cells（～15%and～25%）.Additionally,the modification level was also regulated at different stages of cell cycle.The modification level difference between cells leads to different functions of YTHDF1/3 in different cells.We used metabolic labeling method to label O-Glc NAcylated proteins by Ac₄Glc NAz.Cells intake Ac4Glc NAz and hydrolyze it to Glc NAz,the mimic of Glc NAc.Glc NAz can be recognized by OGT and modified on hydroxy group of serine and threonine.O-Glc NAcylation in the oocytes and embryos of mice was detected and when the modification was inhibited by OGT inhibitor,oocyte development from GV to MII was arrested.With mass spectrometry and Gal-T1 chemoenzymatic labeling methods,T114site of CNOT2 was identified as the major O-Glc NAcylation sites in oocyte development.The modification will change the conformation of CNOT2 structure and promotes the assembly of CCR4-NOT complex.The dysfunction of CNOT2 caused by loss of modification arrests oocyte and embryo development and leads to lethal.We used genetic code expansion and SORT technology to incorporate Met K（methionyl lysine）and Pra K（mimic of aminoacyl lysine）into proteome.One new internalα-amine is introduced by lysine aminoacylation.The internalα-amine with lower p Ka thanε-amine can be subjected to uniquitination,which named as aminoacylated lysine ubiquitination（K-XUb）.We identified 2205 modification sites on 1217 substrates by ubiquitination dataset enriched by Ubi Site antibody.The modified substrates are enriched in pathways of cell cycle,protein catabolic process and so on.Finally,K-XUb modification will promote the degradation of modified substrates and this type of degradation is proteasome dependent which can be rescued by proteasome inhibitors.This project used chemical biology tools coupled with mass spectrometry and identified new sites and new types of protein post-translational modifications.Chemical biology tools can be used for high quality of modification enrichment and mass spectrometry is able to be used for high resolution and sensitivity of modification identification.The result of this project is highly encouraged for protein modifications research.