Development Of Novel Strategies To Prepare Ubiquitin Toolboxes For Biochemical And Functional Studies

Proteins are biological macromolecules that play vital and diverse roles in the biological world.There are varieties of post-translational modifications(PTMs)on proteins,such as phosphorylation,acetylation,methylation,ubiquitination,etc.These modifications regulate the structure of proteins and can significantly alter the physiological functions of proteins.Among them,ubiquitin is a small molecule protein consisting of 76 amino acids.Unlike other post-translational modifications,it functions more complexly and is involved in the regulation of physiological processes,such as transcriptional regulation,protein degradation,apoptosis,and DNA damage repair.Ubiquitin(Ub)cascade is sequentially orchestrated by E1 activating enzymes(E1s),E2 conjugating enzymes(E2s),and E3 ligases(E3s).Currently,E3 ligases can be divided into three main types including RING(really interesting new gene),HECT(homologous to the E6AP carboxyl terminus)and RBR(RING-in-between-RING),depending on the presence of characteristic domains and the mechanism of Ub transfer to the substrate.The ubiquitin can also be removed by different functional deubiquitinating enzymes(DUB).The activity of related enzymes in the ubiquitination process is strictly regulated,while the genetic changes,abnormal expression or dysfunction is usually accompanied with some pathological diseases.For instance,mutation of E3 ligase Parkin causes early-onset autosomal recession heritability;DUB USP28 is overexpressed in colon and breast tumours.The ubiquitin possesses seven different sites of lysine(K6,K11,K27,K29,K33,K48,K63)and the N-terminal methionine residues(M1),each of which can form types of homogenous polyubiquitin chains as well as heterogeneous polyUb(mixed or branched chain).This feature adds significant complexity to intracellular Ub signaling networks because it allows for the assembly of Ub chains with different types of linkages and lengths with the potential to control distinct biological processes.At present,in order to explore the complex ubiquitin system,three aspects of research are flourishing:1.Preparation large amounts of pure samples of Ub chains used for biochemical,biophysical,and structural characterization to reveal the biochemical mechanisms of different ubiquitin chains regulating modified proteins.2.Development of ubiquitin-related active probes used for monitoring multiple enzyme activities in different cellular environments and studying the mechanism of action in cancer cells.3.Development of ubiquitin fluorescent reagents used for direct and quantitative detection of ubiquitinase activity and for screening related inhibitors.In the first part of the work,we have developed a practical method to prepare polyUb chains by using a Ub monomer modified by a premade auxiliary as the key building block.The advantage of this strategy is that all Ub monomers are readily available through bacterial recombinant expression,which allows isotopic labeling of Ub unit for NMR analysis.Using the building block,K48-linked tri-Ub and atypical K6-Iinked tri-Ub,even a mixed-linkage K6,K48-linked triUb could be readily synthesed through auxiliary-mediated NCL.The DUB hydrolysis experiments further validated the biological activity.We anticipate that this approach can be further extended to the synthesis of ubiquitinated proteins,laying the foundation for ubiquitin-related biochemical and biophysical studies.In the second part of our work,we developed a new method that combines the hydrazide-based NCL and sequential Dha formation to prepare E2-Ub-Dha probe.Benefit from its simple operation and commercial available materials,this strategy could be easier to implement in conventional biochemical laboratories.Using this probe in hand,we not only achieved efficient labeling of the HECT E3 enzyme expressed in vitro,but also the HECT E3 enzyme in a complex cell lysate environment.The development of this probe would help us understand the role and function of HECT E3 enzyme in different cellular environments,and further promote the development of related inhibitors.In the third part of the work,we developed an effective strategy to synthesize Ub-AMC.The synthesis can be processed through two-segement hydrazide-based NCL and one-pot ligation-desulfurization.Furthermore,the replacement of methionine(Met)by norleucine(Nle)facilitates the synthesis of higher purity peptide fragments without oxidation by-products.Bioactivity experiments further demonstrate that Ub-AMC afforded satisfied biological activity.We anticipate that this synthetic strategy can be further applied to the synthesis of other ubiquitin fluorescent reagents to provide raw materials for the screening of related deubiquitinating enzyme inhibitors.In summary,this paper focuses on the development of novel strategies for the preparation of ubiquitin chains,ubiquitin probes and ubiquitin fluorescent reagents(collectively referred to as ubiquitin toolboxes)for ubiquitin-related biochemical studies.We expect that the development of ubiquitin toolboxes provides a basis for the future study of ubiquitin signaling pathways and their physiological mechanisms.