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Biochemical function and biomedical application of protein tyrosine O-sulfation
Protein tyrosine O-sulfation (PTS) is a type of post-translational modification that occurs on many secretory and transmembrane proteins. This modification plays crucial roles in either normal physiological or pathological processes. Hence, PTS could emerge as an important therapeutic target for the treatment of human diseases. However, the lack of understanding on the role and extent of PTS in mammalian cell biology severely hinders the protein sulfation-associated therapeutic interventions. The study of PTS is largely handicapped by a chronic lack of suitable chemical and biochemical tools. One major part of this Ph.D. thesis is dedicated to the directed evolution of Src Homology2 (SH2) domain to recognize sulfotyrosine (sTyr). The evolved SH2 domain mutants can be potentially used as “anti-sulfotyrosine antibody-mimetic” with the advantages of smaller size, higher affinity, and better availability. As is the case for other post-translational modifications, a good sTyr-recognition reagent will likely open doors to new studies and/or applications associated with PTS. In the first chapter, a short review is provided on the molecular basis and biological significance of PTS, as well as on the available methods and molecular tools to study PTS. In Chapter 2, the molecular recognition of phosphotyrosine (pTyr) and sulfotyrosine (sTyr) were examined. In Chapter 3, I successfully identified SH2 domain mutants with significantly enhanced affinity towards sTyr by using phage display technology. The best mutant, SH2-60.1, displayed more than 1700-fold improvement in KD value over the wild-type SH2 domain. Cell surface labeling of sulfoproteins with the evolved SH2 domain mutants was also demonstrated. Since sTyr is an essential recognition element for the binding of CCR5 co-receptor by envelope glycoprotein gp120 in the HIV-entry process, I sought to develop sTyr-based HIV-entry inhibitors through both rational design (Chapter 4) and directed evolution (Chapter 5). I developed an efficient synthesis of sTyr-dipeptide and evaluated its inhibitory effect on HIV-entry. Furthermore, I optimized a phage display system, which was coupled with the unnatural amino acid mutagenesis methodology, and screened a sTyr-containing peptide library. By using this approach, a much larger chemical and structural space of sTyr-containing peptide can be explored.
Ju, Tong, "Biochemical function and biomedical application of protein tyrosine O-sulfation" (2016). ETD collection for University of Nebraska - Lincoln. AAI10100907.