Label‐Free Detection of Protein Tyrosine Phosphatase 1B (PTP1B) by Using a Rationally Designed Förster Resonance Energy Transfer (FRET) Probe

ChemBioChem ◽  
2018 ◽  
Vol 19 (23) ◽  
pp. 2495-2501 ◽  
Author(s):  
Trishaladevi Durgannavar ◽  
Se Jeong Kwon ◽  
Amar B. T. Ghisaidoobe ◽  
Kyungmin Rho ◽  
Ju Hwan Kim ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
Protein Tyrosine Phosphatase 1B ◽  
Label Free ◽  
Protein Tyrosine ◽  
Label Free Detection

scholarly journals A High-Throughput Screen for Receptor Protein Tyrosine Phosphatase–γ Selective Inhibitors

2011 ◽  
Vol 16 (5) ◽  
pp. 476-485 ◽  
Author(s):  
Kingsley K. Appiah ◽  
Walter A. Kostich ◽  
Samuel W. Gerritz ◽  
Yanling Huang ◽  
Brian D. Hamman ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Protein ◽  
Protein Tyrosine ◽  
Compound Collection ◽  
Ptp 1B

Protein tyrosine phosphatase–γ (PTP-γ) is a receptor-like PTP whose biological function is poorly understood. A recent mouse PTP-γ genetic deletion model associated the loss of PTP-γ gene expression with a potential antidepressant phenotype. This led the authors to screen a subset of the Bristol-Myers Squibb (BMS) compound collection to identify selective small-molecule inhibitors of receptor-like PTP-γ (RPTP-γ) for use in evaluating enzyme function in vivo. Here, they report the design of a high-throughput fluorescence resonance energy transfer (FRET) assay based on the Z′-LYTE technology to screen for inhibitors of RPTP-γ. A subset of the BMS diverse compound collection was screened and several compounds identified as RPTP-γ inhibitors in the assay. After chemical triage and clustering, compounds were assessed for potency and selectivity by IC50 determination with RPTP-γ and two other phosphatases, PTP-1B and CD45. One hundred twenty-nine RPTP-γ selective (defined as IC50 value greater than 5- to 10-fold over PTP-1B and CD45) inhibitors were identified and prioritized for evaluation. One of these hits, 3-(3, 4-dichlorobenzylthio) thiophene-2-carboxylic acid, was the primary chemotype for the initiation of a medicinal chemistry program.

scholarly journals A New Highly Efficient Substrate-trapping Mutant of Protein Tyrosine Phosphatase 1B (PTP1B) Reveals Full Autoactivation of the Insulin Receptor Precursor

2011 ◽  
Vol 286 (22) ◽  
pp. 19373-19380 ◽  
Author(s):  
Samira Boubekeur ◽  
Nicolas Boute ◽  
Patrick Pagesy ◽  
Vladimir Zilberfarb ◽  
Névéna Christeff ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Insulin Receptor ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Insulin Binding ◽  
Trapping Efficiency ◽  
Protein Tyrosine ◽  
Highly Efficient

PTP1B is a protein tyrosine-phosphatase located on the cytosolic side of the endoplasmic reticulum that plays an important role in the regulation of the insulin receptor (IR). Replacement of the conserved Asp-181 by alanine is known to convert PTP1B into a substrate-trapping protein that binds to but cannot dephosphorylate its substrates. In this work, we have studied the effect of an additional mutation (Y46F) on the substrate-trapping efficiency of PTP1B-D181A. We observed that this mutation converts PTP1B-D181A into a highly efficient substrate-trapping mutant, resulting in much higher recovery of tyrosine-phosphorylated proteins coimmunoprecipitated with PTP1B. Bioluminescence resonance energy transfer (BRET) experiments were also performed to compare the dynamics of interaction of the IR with these mutants. Basal BRET, which mainly reflects the interaction of PTP1B with the IR precursor during its biosynthesis in the endoplasmic reticulum, was markedly increased with the PTP1B-D181A-Y46F mutant. In contrast, insulin-induced BRET was markedly reduced with PTP1B-D181A-Y46F. I125 insulin binding experiments indicated that PTP1B-D181-Y46F reduced the expression of IR at the plasma membrane. Reduced expression at the cell surface was associated with higher amounts of the uncleaved IR precursor in the cell. Moreover, we observed that substantial amounts of the uncleaved IR precursor reached the Tris-phosphorylated, fully activated form in an insulin independent fashion. These results support the notion that PTP1B plays a crucial role in the control of the activity of the IR precursor during its biosynthesis. In addition, this new substrate-trapping mutant may be a valuable tool for the identification of new PTP1B substrates.

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