Chemical Synthesis And Application Of Ubiquitin-based Probes

In cells,especially in eukaryotic cells,proteins are often associated with a large number of post-translational modifications,such as phosphorylation,methylation,acetylation,glycosylation,ubiquitination et al.Those post-translational modifications greatly enrich the type and amount of intracellar proteins.The post-tanslational modifications usually occur on the side chain residue or N-terminal amino group of the proteins,which can affect structure,stability and cell localization.Howerve,the investigation of ubiquitin signaling was largely precluded.One of the main reasons is that it is unable to obtain a sufficient amount of proteins with homogeneous post-translational modification.For the acquisition of post-translational modified proteins,traditional biological methods,such as tissue extraction,recombinant expression and enzymatic methods,are difficult.Although the strategy of genetically encoded non-canonical amino acids has been successfully utilized,but the yeild is low and incorporation of multiple modifications simultaneous is difficult.On the other hand,the strategy of chemical synthesis can be a very flexible way to obtain post-translational modified proteins,which promote the analysis of post-translational modification signaling significantly.In this paper,we focus on ubiquitination and emphasize the chemical synthesis strateges on the analysis of ubiquitin signals.Ubiquitination is a commonly used post-translational modification,involving in the regulation of many basic physiological processes,such as protein degradation,transcription,and DNA damage repair.Ubiquitination is generally achieved by the synergistic effect of three enzymes(E1,E2,E3),which link the C-terminal glycine of ubiquitin to the side chain amino group of the lysine in the substrate protein through the isopeptide bond.Ubiquitin can also be used as a substrate protein,using its N-terminal Met or seven lysines(Lys6,Lys11,Lys27,Lys29,Lys33,Lys48 or Lys63)to form different lengths,homogeneous or non-homogeneous polyubiquitin chains.The differences in the connection of polyubiquitin chains tend to produce different molecular signals,Lys48-linked ubiquitin chain mainly functions as a protein degradation signal,while Lys63-linked ubiquitin chain appears to be involved in non-protein degradation process.Ubiquitination is a reversible process,the ubiquitin tag can be removed from the target protein by a class of enzymes known as deubiquitinating enzymes(DUBs).The disordered DUBs are associated with many diseases,such as cancer and neur-degenerative diseases,and some of them have been identified as important drug targets.In order to monitor the activity of DUBs and screen bioactive molecules inhibitors against DUBs,biochemical tools,such as Ub-AMC,have been developed.Ub-AMC can be hydrolyzed by DUBs,and then produces DUBs-activity-associated fluorescent signals,which have been widely used in high-throughput screening of small molecules inhibitors against DUBs.The preparation of Ub-AMC is usually performed by aminolysis of ubiquitin thioester,but the aminolysis reaction always suffers from heavy hydrolysis and low yield.To solve this problem,we report two alternative approaches to prepare Ub-AMC,and confirm the bioactivity of the prepared Ub-AMC through the measurement of its DUBs activity.Ubiquitination is involved in regulating many cellular processes,which is precisely controlled by ubiquitin-binding proteins.Therefore,the ability to identify ubiquitin binding proteins,especially those can recognize ubiquitin chains of specific length and linkage,is important to understand the functions of ubiquitin in cellular processes.The traditional methods of identification include yeast two-hybrid technology,bioinformatics and affinity enrichment strategy,but proteome-wide profiling of ubiquitin signaling interactors have not been conducted.To address the aforementioned issues,we report a photo-affinity covalent capture approach,which enables proteome-wide profiling of ubiquitin-binding proteins from cell lysates.One of our central findings is that the probes containing just one ubiquitin unit is insufficient to capture ubiquitin binding proteins,therefore the probes with at least two ubiquitin units is necessary.In addition,we also find that the probes with diubiquitin chain of various linkage types are of different capturing abilities,it is necessary to develop probes with polyubiquitin chain with various linkage types for screening the steady-state and non steady-state ubiquitin binding proteins in physiology and pathology.The probes containing polyubiquitin chain of various lengths and linkage types cannot be readily obtained through biological expression.This highlights the value of modern chemical protein synthesis as powerful tools to understand the role of ubiquitination.