טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMeledin Roman
SubjectChemical and Semisynthesis of Ubiquitinated Proteins for
Functional Analyses
DepartmentDepartment of Chemistry
Supervisor Professor Ashraf Brik
Full Thesis textFull thesis text - English Version


Abstract

The development of ubiquitin (Ub) - based reagents to study and monitor deubiquitinase’s activity is a highly desired goal for many laboratories in academia and industry due to the importance of these enzymes in many pathological states. However, due to the limitations of total chemical synthesis and a shortage of suitable semisynthetic tools for the preparation of large protein-Ub conjugates, this field is essentially restricted. In course of my PhD, we have developed a strategy for the site-specific protein ubiquitination using dehydroalanine (Dha) chemistry for the preparation of Ub conjugates and ubiquitinated protein activity-based probes bearing a very close mimic of the native isopeptide bond. Our approach relies on the selective formation of Dha followed by conjugation with a single amino acid or peptide bearing a thiol handle derived from the C-terminus of Ub. Subsequently, the resulting synthetic intermediate undergoes native chemical ligation with the complementary part of the Ub polypeptide to achieve ubiquitinated protein product. Optionally, second Dha formation step can be performed to install the electrophilic warhead, which is one of the main elements of the activity-based probes. It has been proposed that the Michael addition step could result in the formation of a diastereomeric mixture as a result of unselective protonation of the enolate intermediate. It has also been proposed that the chiral protein environment may influence such an addition step. In the protein context, these questions remain open and no experimental evidence was provided as to how such a protein environment affects the diastereoselectivity of the addition step. As was previously proposed for the conjugation step on protein bearing Dha, the isopeptide bond formation step in our study resulted in the construction of two protein diastereomers. To assign the ratio of these diastereomers, trypsinization coupled with high-pressure liquid chromatography analyses were performed. Moreover, the obtained peptide diastereomers were compared with identical synthetic peptide standards having defined stereogenic centers, which enabled the determination of the configuration of the isopeptide mimic in each diastereomer. In addition, we applied this approach to construct physiologically and therapeutically relevant ubiquitinated α-globin probe, which was used for the enrichment and proteomic identification of α-globin modulating deubiquitinases. We found USP15 as a potential deubiquitinase, modulating α-globin, which its excess leads to beta-thalassemia symptoms. Our study, which offers a new method for isopeptide bond formation and protein ubiquitination, gives insights into the parameters that affect the stereoselectivity of the addition step to Dha for chemical protein modifications. Finally, this development opens new opportunities for activity based probe design to shed light on the important aspects underlining ubiquitination and deubiquitination in health and disease.