Development Of New Chemical Semi-synthesis Platform For The Synthesis Of Ubiquitinated Proteins

Protein ubiquitination represents a widespread form of post-translational modification(PTM)that regulates diverse cellular processes in eukaryotes such as proteasomal degradation,signal transduction and DNA damage response,and the aberration of the ubiquitin system is associated with many serious illnesses including cancers and neurodegenerative diseases.Understanding the biochemical and biophysical mechanisms of protein ubiquitination involved in regulating the diverse cellular functions is of great significance for unraveling the mystery of life as well as the diagnosis and treatment of diseases.To achieve this goal,an important requirement is to obtain the homogeneous ubiquitinated proteins and the functional Ub-based probe tools.Protein chemical synthesis enables us to build the desired proteins at atomic level and provides the powerful technical platform for preparing the ubiquitinated proteins and tool molecules for studying protein ubiquitination,which plays an irreplaceable role in deciphering the complex Ub signaling.In this thesis,we focus on developing new technique for the facile synthesis of ubiquitinated proteins.We wish to explore the semi-synthetic strategies for preparing the important Ub-modified protein targets form the readily available recombinant protein materials through the functional handle-assisted protein conjugation.Combining the the powers of cysteine-aminoethylation reaction and native chemical ligation,a novel strategy for the semi-synthesis of ubiquitinated proteins is established herein.Firstly,we developed a cysteine-aminoethylation assisted chemical ubiquitination strategy(CAACU-strategy)for the efficient synthesis of ubiquitinated histones.The key step of the strategy is the site-specific installation of the 2-bromoethylamine derivative bearing a removable auxiliary group onto the Cys residue of recombinant histones through cysteine-aminoethylation,then ubiquitination and even SUMOylation can be accomplished through auxiliary-mediated protein ligation with recombinant Ub or SUMO hydrazides.Because both histones and Ub(or SUMO)are readily acquired through recombinant expression,the use of CAACU enables multi-milligram quantities of homogeneous mono-and di-Ub,and SUMO-modified histones are rapidly obtained.The resulting thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure,compared to their native counterparts.The ubiquitinated histone prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins.The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases.Cryo-electron microscopy of the nucleosome containing H2BKc34Ub indicated that the obtained CAACU histones were of good quality for structural studies.Secondly,the CAACU strategy was further extended and improved to enable the efficient semi-synthesis of various atypical Ub chains including di-Ubs,homogeneous tri-Ub,branched tri-Ub,and the SUMOlated Ub.The crystallographic analysis of Kc27-diUb not only confirmed the high purity of the synthetic samples,but also confirmed that the thioether-containing isopeptide bond exhibits minor structural perturbation on the native structure.Moreover,by using this strategy,we developed a novel non-hydrolyzable di-Ub probe containing a thioether isopeptide bond and demonstrated it as a useful tool for the mechanistic studies of atypical Ub chains recognition by deubiquitinases.These results indicated that CAACU strategy can provide the useful tool molecules to facilitate our understanding of the roles and functions of atypical Ub chains.In addition,we provided a practical method for the one-pot synthesis of the bis-thio-acetone(BTA)linked ubiquitinated histones by using commercially available 1,3-dibromoacetone as cross-link reagent.The Ub mutant UbG76C was activated under an optimal acidic borate buffer,and then coupled to histones in one-pot form with greatly improved efficiency.This strategy requires no complicated chemical transform and special experimental settings,and can be easily conducted in the biochemical labs.Moreover,with the assistance of affinity purification scheme based on tandem orthogonal tags,this strategy is expected to prepare the ubiquitinated proteins under the mild undenatured conditions.