Chemical Synthesis And Application Of Ubiquitin Protein Tools

Chemical transformation and modification of biomass molecules can improve their added values.At present,researches on chemical transformation of biomass mainly focus on components containing C,O and H(lignin,cellulose,etc.),and less attention is paid to components containing N in biomass(protein).Ubiquitin,as an important protein resource,exists widely in eukaryotic cells and has a high abundance.However,the complexity of ubiquitin modification and physiological functions has hindered their cognition and chemical transformation to a large extent.Chemical modification of ubiquitin to obtain ubiquitin tools carrying reactive groups,and subsequent in-depth research on ubiquitination and deubiquitination,which is helpful to understand ubiquitin function and of great significance for their chemical transformation.Ubiquitination is a widespread post-translational modification in eukaryotes.The carboxyl terminus of ubiquitin is covalently linked to the amino group of lysine side chain of the substrate protein through a E1/E2/E3 enzyme cascade.Deubiquitinating enzyme(DUB)can remove ubiquitin from the substrate protein to maintain the balance of ubiquitination in cell.The above four types of enzymes coordinate with each other to form a complex and dynamic ubiquitin-related enzymatic system.This system regulates almost all cellular processes in eukaryotes,and its activity disorder can lead to many diseases such as cancer and neurological diseases.It makes sense for disease diagnosis and treatment to deeply understand the catalytic mechanism of ubiquitin enzymatic system in living cells.An important requirement to achieve this goal is to obtain cell-penetrating ubiquitin tools that can be activated on demand.In view of this,this paper developed a new method for the efficient synthesis of photocaging ubiquitin protein tools through backbone modification.Using this tool,we carried out profiling and regulating of ubiquitin-related enzymatic system in living cells.The main work is as follows:Firstly we developed a new method for one-pot efficient semi-synthesis of biotinbearing ubiquitin protein tools.Starting from recombinant expressed ubiquitin variant,a series of ubiquitin protein tools with biotin tag were efficiently prepared in one pot by combining chemical enzymatic method,N-S acyl transfer and ammonolysis reaction.Subsequently,the activity of ubiquitin-related enzymatic system was preliminarily studied in vitro using these probe tools.Then we developed photocaging ubiquitin tools which can be activated on demand through backbone modification,and realized time-resolved monitoring of DUB activity in living cells.The key of this strategy is to install a photosensitive group onto the amide backbone at the C-terminus of ubiquitin,which prevents ubiquitin from entering the catalytic center of DUB,thereby temporarily masking the activity of ubiquitin probes.After light activation,the photosensitive group was removed and the probe returned to the native structure,thereby realizing time-resolved labeling of DUB.Combined with proteomics,we captured more than 30 DUB from four families in living cells.We also analyzed the changes of DUB activities during oxidative stress,and found that DUB activity first decreased and then increased at different time points.In addition,we synthesized cell-penetrating ubiquitin chain dehydroalanine probes and realized timeresolved labeling of chain-specific DUB for the first time.Different from deubiquitination,E1/E2/E3 enzyme cascade system contains multiple enzymes with synergistic catalytic processes,making it more challenging to cage-activate and profile the newly formed ubiquitination events in living cells.We replaced G76 of ubiquitin with nitrobenzyl-modified glycine and successfully prepared caged ubiquitin by protein chemical synthesis,which temporarily hindered its utilization by E1 enzyme,thereby achieving the masking and on demand activation of the ubiquitination process in living cells.Combined with proteomics,we explored new formed ubiquitinated proteins during oxidative stress.These proteins are mainly involved in RNA splicing,protein transport,and protein translation.The above studies show that the backbone modification strategy we developed provides a powerful tool for drawing time-resolved maps of ubiquitination and deubiquitination events in different states,and also provides a sight for the time-resolved monitoring of ubiquitinlike enzymatic system.