Development of activity-based chemical probes for the analysis of protein tyrosine phosphatases in vivo.

• Main Authors: Chun-Ping Chang, 張君平
• Other Authors: Jing-Jer Lin
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/66873815837195151471

碩士 === 國立陽明大學 === 生物藥學研究所 === 99 === PART I Protein tyrosine (pTyr) phosphorylation is a common post-translational modification which can create novel recognition motifs for protein interactions and cellular localization, affect protein stability, and regulate enzyme activity. Protein tyrosine phosphatase (PTP) is an important enzyme family that remove phosphate group from tyrosine residues of protein. Previously we have adopted a mechanism-based approach and developed several activity probes for PTPs. However, these cannot pass cell membrane due to their charge and/or polarity properties. Thus, application of these chemical probes was limited to in vitro analysis. Here, we report the design, synthesize, and characterization of new chemical probes that can be used for in vivo labeling of cellular PTPs. The new designed probe has a 1,4-foluorine reactive group that was modified to neutralize the negative charges on phosphate. A bodipy was also used as the reported group to help the passage through cell membrane. The newly designed PTP probes effectively labeled PTPs including PTP1B, SHP2, PEST, TCPTP, and VHR. It also entered mammalian cells efficiently. Moreover, it readily labeled cellular proteins. Thus, the newly designed chemical probes could be used as a tool in detecting the cellular activities of PTPs in vivo. PART II Protein phosphorylation is play an important regulation rule in mammalian signal transduction. Src-homology 2 (SH2) domains are modulators of a lot of functionally diverse proteins involved in mammalian signal transduction including enzymes, adaptors, regulators and transcription factors. SH2 domains pass through the excellular signal by interaction with phosphotyrosine. SH2 domain is a high conserve domain, which can specific interaction of short tyrosine phosphorylated peptide motifs. SH2 domain-containing proteins are play an important regulation rule in protein tyrosine kinase (PTK) signaling pathways and related with many diseases. Blocking the interaction between SH2 domain and phosphotyrosine-containing protein. Can inhibition the overexpression signal pathway in cancer cells. Generally, the inhibitors of SH2 somain-containing proteins are tyrosine phosphorylated peptide mimic small molecular. In our study, design three series (A,B,C) of SH2 somain-containing proteins inhibitors for library. And establish ELISA assay to screen the inhibitors of Grb2 and SAP. Now, I already screen some inhibitors which can inhibition SH2 somain-containing proteins (Grb2 and SAP) interaction with the phosphotyrosine-containing substrate. Grb2`s inhibitor is C20P. SAP`s inhibitor are B8, B9, B11P, and C20.