Identification Of Proteins Containing Unsaturated Double Bonds By Chemical Probe Based On Michael Addition

Eliminylation refers to irreversible elimination of serine,threonine hydroxyl group or cysteine thiol group produce unsaturated amino acids by ?-elimination reaction.Eliminylation mediates by phosphothreonine lyase is irreversible "dephosphorylation" of p-MAPKs,which eliminates one molecule of phosphoric acid permanently.The eliminylation acts on the phosphothreonine C?-OP? bond and cleaves it to form an unsaturated C?=C? system,while the dephosphorylation reaction acts on P-O to generate a hydroxyl group,so eliminylation is irreversible.Phosphothreonine lyase is an effector protein of pathogenic bacterium,which suppress the host's immune response by irreversibly "dephosphorylating" p-MAPKs.There are no eukaryotic proteins that share the similar sequence with phosphothreonine lyase.Are there other eliminated proteins in eukaryotic cells? There is no reliable method to identify eliminated protein leading to that we have little knowledge about eliminylation.The researchers have identified just two proteins and about two hundred peptides until now.Crotonylation was firstly discovered on histones acting as a transcriptional activator,which distributes on transcription promoters and enhancers.Subsequent studies showed that numerous crotonylated proteins are involved in diverse cellular functions and signaling pathways,which expands our understanding of crotonylation.Different from other known short-chain acylations,it is similar to eliminylation in structure,crotonylation also contain unsaturated double bond,which can mediate the interaction between crotonylated protein and crotonylation protein "reader".Combined antibody enrichment and mass spectrometry detection is a conventional method for post-translational modification,there is no pan-antibody for eliminylation,therefor,since the concept of eliminylation was proposed in 2007,there is no ideal identification method so far.We noticed the characteristic structure of Dha or Dhbmodified proteins,carbon-carbon double bonds can be attacked with nucleophiles.Therefore,we developed a chemical probe for labeling unsaturated double bond of protein based on the Michael addition reaction.The target protein derived a biotin tag after labeled with chemical probe,using the inherent affinity of streptavidin and biotin to make the labeled target protein immobilized on the streptavidin beads.However,the binding force between streptavidin and biotin is irreversible under general conditions,which become a hindrance for downstream research.Therefore,we engineered a streptavidin mutant that caused a surprisingly decrease in streptavidin-biotin stability,which aim to elute the labeled target proteins with free biotin.Based on the above discussion,the following studies are carried out:Firstly,the mechanism of phosphothreonine lyase mediated eliminylation was used to perform biochemical reactions in vitro,so as to obtain eliminated protein.The proteins such as His-ERK,His-Adk-MEK1R4 F,and Osp F were purified to catalyze the phosphorylation of ERK,and Osp F mediated p-ERK to eliminate a molecule of phosphoric acid to generate eliminated ERK(?-ERK).Then,?-ERKwas used to study the labeling of eliminated proteins with the chemical probes.However,it was found that there were side reactions during the experiment.On the one hand,it might be caused by the by-products in the process of probe synthesis;on the other hand,it might be caused by disulfide bond,but the probe could specifically label the unsaturated double bond of protein after pretreatment the probe;Secondly,histone was used to explore the labeling of crotonyl unsaturated double bonds with the chemical probe;Finally,we purified the target protein with streptavidin system;According to the method established above,potential double bond proteins were labeled in the lysis.In conclusion,we established a method for labeling unsaturated double bonds of protein based on the theory of Michael addition reaction,this method especially provides a reasonable strategy for eliminylation that lack reliable identification methods now,and it is also a supplementary method for identifying crotonylated proteins.