PTP1B inhibition, catalysis, and physiological substrate

dc.contributor.authorGuo, Xiaoling
dc.date.accessioned2018-07-12T17:35:00Z
dc.date.available2018-07-12T17:35:00Z
dc.date.issued2008
dc.description.abstractProtein-tyrosine phosphatases (PTPs) consist of over a hundred of enzymes that are important for the control of proper cellular tyrosine phosphorylation. Despite the emerging roles played by PTPs in human diseases, a detailed understanding of the role played by PTPs in normal physiology and under pathogenic conditions has been hampered by the absence of PTP-specific inhibitors. Such inhibitors could serve as useful tools for determining the physiological functions of PTPs and may constitute valuable therapeutics in the treatment of several human diseases. However, because of the highly conserved nature of the active site, it has been difficult to develop selective PTP inhibitors. By taking an approach to tether together two small ligands that can interact simultaneously with the active site and a unique proximal noncatalytic site, we have recently acquired compound 2, whose chemical structure is depicted in Figure 3 chapter 2 (page 42). Compound 2 is the most potent and selective PTP1B inhibitor identified to date, which exhibits several orders of magnitude selectivity in favor of PTP1B against a panel of PTPs. We describe an evaluation of the interaction between 2 and PTP1B. We established the binding mode of Compound 2. Hydrogen/deuterium exchange of PTP1B backbone amides in the presence and absence of 2 gave a big picture of where compound 2 binds to PTP1B. Site-directed mutagenesis identified 12 PTP1B residues that are important for the potency and selectivity of Compound 2. Although many of the residues important for Compound 2 binding are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which suggest that the binding surface defined by these residues in individual PTPs determines inhibitor selectivity. Our results provide structural information for understanding of the molecular basis for potent and selective PTP1B inhibition and further establish the feasibility of acquiring potent, yet highly selective, PTP inhibitory agents.;Previous kinetic and structural studies have revealed a critical role for Asp181 in PTP-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. The neighbouring residue 182 is a phenylalanine in PTP1B, TCPTP, PTPalpha, PTP&egr; and a glutamine in Yersinia PTP. Little attention has been paid to the fact that this residue is a histidine in most other PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison to the Phe182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/K m values. X-ray crystallographic study indicates that the effect of His182 is due to interactions with Asp181 and with Gln262. We conclude that residue 182 affects the E- P hydrolysis of PTP-mediated catalysis by modulating the functionality of Asp181 and Gln262.;Understanding of the functions of PTPs requires thorough studies using the physiological substrates in addition to small molecule artificial substrates that are commonly used in vitro. As the cellular counterpart of transforming v-Src, c-Src shows elevated activity in mammary cancer cells and colonic polyps. The activity of c-Src is tightly regulated by phosphorylation on two tyrosines -416 and 527. Phosphorylated Tyr527 binds to its own SH2 domain and produces a closed conformation. During the activation process, the intramolecular interactions are released and Src autophosphorylates on Tyr416 to become fully activated. Several PTPs have been proposed to play a role in the dephosphorylation of c-Src. For example, recent studies with PTP1B specific inhibitors or in PTP1B knockout cells suggest PTP1B plays a positive role downstream of integrins by dephosphorylating Src pTyr527.;To elucidate the molecular basis of Src dephosphorylation, we carried out detailed analysis of Src dephosphorylation by PTPs using purified proteins. PTP1B dephosphorylated Src pTyr527 much more slowly than Src pTyr416. When we removed SH3 and SH2 domains of Src, the dephosphorylation of pTyr527 in the catalytic domain of Src by PTP1B showed a similar rate as dephosphorylation of pTyr416, suggesting that pTyr527 is protected by the intramolecular interaction. Disruption of c-Src intramolecular interactions by mutations facilitated PTP1B's dephosphorylation of Src pTyr527. This conformational protection mechanism applies to other candidate PTPs tested---SHP1, FAP-1, DEP-1, CD45, and PTPalpha. Our data has demonstrated PTPs' ability to be involved in both the activation and inactivation process of c-Src. In the complex signaling process, additional modulators are required to regulate the dephosphorylation of c-Src by PTPs.
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 69-06, Section: B, page: 3565.;Advisors: Zhong-Yin Zhang.
dc.identifier.urihttps://ezproxy.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:3318743
dc.identifier.urihttps://hdl.handle.net/20.500.12202/973
dc.publisherProQuest Dissertations & Theses
dc.subjectBiochemistry.
dc.titlePTP1B inhibition, catalysis, and physiological substrate
dc.typeDissertation

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