A chemical model for redox regulation of protein tyrosine phosphatase 1B (PTP1B) activity

Growing evidence indicates that endogenously produced hydrogen peroxide acts as a cellular signaling molecule that (among other things) can regulate the activity of some protein phosphatases. Recent X-ray crystallographic studies revealed an unexpected chemical transformation underlying the redox regulation of protein tyrosine phosphatase 1B, in which oxidative inactivation of the enzyme yields an intrastrand protein cross-link between the catalytic cysteine residue and its neighboring amide nitrogen. This work describes a small organic molecule that serves as an effective model for the redox-sensing assembly of functional groups at the active site of PTP1B. Findings obtained using this model system suggest that the oxidative transformation of PTP1B to its "crosslinked" inactive form can proceed directly via oxidation of the active-site cysteine to a sulfenic acid (RSOH). The remarkably facile nature of this protein cross-link-forming reaction, along with the widespread cellular occurrence of protein sulfenic acids generated via oxidation of cysteine residues, suggests that the type of oxidative protein cross-link formation first seen in the context of PTP1B represents a potentially general mechanism for redox "switching" of protein function. Thus, the chemistry characterized here could have broad relevance to both redox-regulated signal transduction and the toxic effects of oxidative stress.     

Regulation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B activity by casein kinase II- and p56lck-mediated phosphorylation

Protein tyrosine phosphorylation and dephosphorylation are important in the regulation of cell proliferation and signaling cascade. In order to examine whether phosphatase activity of CPTP1 and HPTP1B, typical nontransmembrane protein tyrosine phosphatase, could be controlled by phosphorylation, affinity-purified PTPs were phosphorylated by CKII and p56lck in vitro. Phosphoamino acid analysis revealed that CPTP1 was phosphorylated on both serine and threonine residues by CKII, and tyrosine residue by p56lck. Phosphatase activity of CPTP1 was gradually increased by three-fold concomitant with phosporylation by CKII. Phosphorylation of HPTP1B by CKII resulted in quick two-fold enhancement of its phosphatase activity within 5 min of incubation and remained in that state. In the presence of CKII inhibitor, heparin or poly(Glu.Tyr), both phosphorylation and enhancement of phosphatase activity of CPTP1 and HPTP1B were mostly blocked. p56lck catalyzed tyrosine phosphorylation of CPTP1 and HPTP1B was only observed by inhibiting the intrinsic tyrosine phosphatase activity. Taken together, these results indicate that CPTP1 or HPTP1B possesses a capability to regulate its phosphatase activity through phosphorylation processes and may participate in the cellular signal cascades.     

Negative regulation of a protein tyrosine phosphatase by tyrosine phosphorylation

The low molecular weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitously expressed enzyme with several proposed roles in cell signaling. Previously, two tyrosine phosphorylation modifications of LMW-PTP at sites Tyr-131 and Tyr-132 in response to growth factor stimulation have been mapped and suggested to stimulate LMW-PTP phosphatase activity. Biochemical analysis of tyrosine phosphorylation of a tyrosine phosphatase is challenging because of the intrinsic instability of these modifications. Here we used expressed protein ligation to site-specifically incorporate a phosphotyrosine mimic (phosphonomethylenephenylalanine, Pmp) at the Tyr-131 and Tyr-132 positions and measured the catalytic activity of these semisynthetic LMW-PTPs. The phosphonate-modified LMW-PTPs were 10- to 23-fold less active in dephosphorylating phosphotyrosine peptides derived from the PDGF receptor and p190RhoGap, two putative cellular substrates. These findings suggest the first example of a tyrosine phosphatase that is inhibited by tyrosine phosphorylation and provide a new model for the regulation of LMW-PTP and its role in cell adhesion.     

Synthesis of mangiferin derivatives as protein tyrosine phosphatase 1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) has received much attention due to its pivotal role in type 2 diabetes and obesity as a negative regulator of the insulin signaling pathway. Mangiferin, a xanthone glucoside, has been reported to possess significant antidiabetic activity. In the present study, a series of mangiferin derivatives was synthesized and evaluated for their PTP1B inhibitory activity. Some of the screened compounds displayed good PTP1B inhibitory activity. Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 549–551, November–December, 2007.     

Chemical constituents of Blumea balsamifera of Indonesia and their protein tyrosine phosphatase 1B inhibitory activity

A methanol extract of the leaves of Blumea balsamifera (L.) DC. (Asteraceae) afforded a new guaian-type sesquiterpene, epiblumeaene K (1), together with four known guaian-type sesquiterpenes (2-5), three known sesquiterpenes (6-8), and nine known flavonoids (9-17) by a combination of chromatography and preparative TLC techniques. Their structures were elucidated by extensive spectroscopic methods and comparison with the literature data. Among the isolated compounds, a known sesquiterpene, beta-caryophyllene 8R,9R-oxide (6), exhibited a significant PTP1B inhibitory activity in a dose-dependent manner, with an IC50 value of 25.8 microM (5.62 microg/mL).     

Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

Background  

The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro.     

Redox regulation of protein tyrosine phosphatase 1B (PTP1B): a biomimetic study on the unexpected formation of a sulfenyl amide intermediate

The effect of steric and electronic environments around the sulfur and nitrogen atoms and the role of nonbonded S...O/N interactions on the cyclization reactions of amide substituted benzene sulfenic acids are described. The reaction profiles and the role of different substituents on the cyclization are investigated in detail by theoretical calculations. It is shown that the synthetic thiols having ortho-amide substituents may serve as good models for the enforced proximity of the amide and cysteine thiol groups at the active site of protein tyrosine phosphatase 1B (PTP1B). However, some of the sulfenic acids derived from such models do not effectively mimic the cyclization of protein sulfenic acids. This is mainly due to the requirement of very high energy for breaking the S-O bond to form a planar five-membered ring of isothiazolidinone. It is shown that the sulfenic acid having two substituents-an amide moiety and a heterocyclic group-in the ortho-positions undergoes a rapid cyclization reaction to produce the corresponding sulfenyl amide species. These studies reveal that the introduction of a substituent at the 6-position of the benzene ring enhances the cyclization process not only by facilitating a closer approach of the -OH group and the backbone -NH moiety but also by increasing the electrophilicity of the sulfur atom in the sulfenic acid.     

Reactions of an organoruthenium anticancer complex with 2-mercaptobenzanilide--a model for the active-site cysteine of protein tyrosine phosphatase 1B

The organometallic anticancer complex [(η(6)-p-cymene)Ru(en)Cl]PF(6) (1, en = ethylenediamine) readily reacts with thiols and forms stable sulfenate/sulfinate adducts which may be important for its biological activity. Protein tyrosine phosphatase 1B (PTP1B), a therapeutic target, contains a catalytic cysteinyl thiol and is involved in the regulation of insulin signaling and the balance of protein tyrosine kinase activity. On oxidation, the catalytic Cys215 can form an unusual sulfenyl-amide intermediate which can subsequently be reduced by glutathione. Here we study reactions of 1 with 2-mercaptobenzanilide, 2, a recognized model for the active site of PTP1B. We have characterized crystallographically compound 2 and its oxidized sulfenyl-amide derivative 2-phenyl-1,2-benzisothiazol-3(2H)-one (4), which shows a close structural similarity to the sulfenyl-amide in oxidized PTP1B. At pH 7.4 and 5.3, 1 reacted with 2, affording a mono-ruthenium thiolato complex [(η(6)-cym)Ru(en)(S-RS)](+) (7(+), R = (C(6)H(4))CONH(C(6)H(5))) and a triply-S-bridged thiolato complex [((η(6)-cym)Ru)(2)(μ-S-RS)(3)](+) (8(+)), respectively. Coordination of Ru to the S atom in 7 allows formation of a strong H-bond (2.02 ?) between the en-NH and the carbonyl oxygen. To assess the possible effect of ruthenium coordination on the redox regulation of PTP1B, reactions of these thiolato products with H(2)O(2) and/or GSH were then investigated, demonstrating that coordination to Ru largely retards both the oxidation (deactivation) of the thiol in compound 2 by H(2)O(2) and the subsequent reduction (reactivation) of the sulfenyl-amide by GSH, implying that the inhibition of complex 1 on PTP1B (IC(50) of 19 μM) may be attributed to coordination to its catalytic cysteine.     

Discovery of a potent,selective protein tyrosine phosphatase 1B inhibitor using a linked-fragment strategy

Protein tyrosine phosphatase 1B (PTP1B) is an enzyme that downregulates the insulin receptor. Inhibition of PTP1B is expected to improve insulin action, and the design of small molecule PTP1B inhibitors to treat type II diabetes has received considerable attention. In this work, NMR-based screening identified a nonselective competitive inhibitor of PTP1B. A second site ligand was also identified by NMR-based screening and then linked to the catalytic site ligand by rational design. X-ray data confirmed that the inhibitor bound with the catalytic site in the native, "open" conformation. The final compound displayed excellent potency and good selectivity over many other phosphatases. The modular approach to drug design described in this work should be applicable for the design of potent and selective inhibitors of other therapeutically relevant protein tyrosine phosphatases.     

Polydopamine nanodots are viable probes for fluorometric determination of the activity of alkaline phosphatase via the in situ regulation of a redox reaction triggered by the enzyme

The authors describe an environmentally friendly and fast (~14 min) method for the synthesis of homogeneously distributed fluorescent polydopamine nanodots (PDA-NDs) using KMnO₄ as the oxidant. Alkaline phosphatase (ALP) catalyzes the hydrolysis of ascorbic acid 2-phosphate to release free ascorbic acid which undergoes an in-situ redox reaction with KMnO₄. Depending on the activity of ALP, more or less KMnO₄ is consumed, and this affects the formation of the PDA-NDs. Based on this finding, a sensitive method was worked out to quantify the activity of ALP via real-time formation of fluorescent PDA-NDs. The fluorometric signal (best measured at excitation/emission peaks of 390/500 nm) is linear in the 1 to 50 mU·mL^?1 ALP activity range, and the limit of the detection is as low as 0.94 mU·mL^?1 (based on 3 σ/m). The method was successfully applied to the determination of ALP activity in spiked human serum and in MCF-7 cell lysates. It was also applied in a method to screen for inhibitors of ALP.

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Graphical abstract Schematic of a fluorometric method for the determination of alkaline phosphatase (ALP) activity. The method is based on the in-situ regulation of the formation of fluorescent polydopamine nanodots (PDA-NDs) through the competition between the KMnO₄-induced polymerization of dopamine and ALP-directed ascorbic acid 2-phosphate (Asc-2P) hydrolysis. AA: Ascorbic acid.

     

Computational design, synthesis and biological evaluation of para-quinone-based inhibitors for redox regulation of the dual-specificity phosphatase Cdc25B

Quinoid inhibitors of Cdc25B were designed based on the Linear Combination of Atomic Potentials (LCAP) methodology. In contrast to a published hypothesis, the biological activities and hydrogen peroxide generation in reducing media of three synthetic models did not correlate with the quinone half-wave potential, E(1/2).     

The influence of lysis buffer composition on the expression and activity of protein tyrosine phosphatase

Lysis conditions are crucial in the extraction and solubilization of phosphotyrosine-containing proteins in a form that is immunoreactive, undegraded and enzymatically active. To establish optimal experimental conditions, we evaluated protein tyrosine phosphatase enzyme activity and the detection of PTP-1 B protein in human acute promyelocytic leukaemic cells, both before and after retinoic acid treatment. We found that the composition of the lysing buffer greatly influenced the efficiency of solubilization, resulting in major alterations in the activity of protein tyrosine phosphatases and on the mobility of PTP-1 B protein.     

Studies of the mechanism of selectivity of protein tyrosine phosphatase 1B (PTP1B) bidentate inhibitors using molecular dynamics simulations and free energy calculations

Bidentate inhibitors of protein tyrosine phosphatase 1B (PTP1B) are considered as a group of ideal inhibitors with high binding potential and high selectivity in treating type II diabetes. In this paper, the binding models of five bidentate inhibitors to PTP1B, TCPTP, and SHP-2 were investigated and compared by using molecular dynamics (MD) simulations and free energy calculations. The binding free energies were computed using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) methodology. The calculation results show that the predicted free energies of the complexes are well consistent with the experimental data. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition analysis indicates that the residues ARG24, ARG254, and GLN262 in the second binding site of PTP1B are essential for the high selectivity of inhibitors. Furthermore, the residue PHE182 close to the active site is also important for the selectivity and the binding affinity of the inhibitors. According to our analysis, it can be concluded that in most cases the polarity of the portion of the inhibitor that binds to the second binding site of the protein is positive to the affinity of the inhibitors while negative to the selectivity of the inhibitors. We expect that the information we obtained here can help to develop potential PTP1B inhibitors with more promising specificity.     

Expression of the small tyrosine phosphatase (Stp1) in Saccharomyces cerevisiae: a study on protein tyrosine phosphorylation

Small tyrosine phoshatase 1 (Stp1) is a Schizosaccharomyces pombe low-molecular-mass phosphotyrosine-phosphatase 50% identical to Saccharomyces cerevisiae Ltp1. In order to investigate the role of Stp1 in yeast, a mutant was generated having the characteristic of a dominant negative molecule. Changes in protein tyrosine phosphorylation in S. cerevisiae proteome in response to Stp1 or its dominant negative mutant expression were analyzed by high-resolution two-dimensional (2-D) electrophoresis. The most remarkable result is the modification by phosphorylation on tyrosine of several proteins involved in carbohydrate metabolism. Twelve proteins were identified on the basis of their positions in the anti-phosphotyrosine immunoblot of the 2-D electrophoresis. Ten of these present tyrosyl residues that are within the consensus sequence for protein kinase CK2 (casein kinase-2). These data open the possibility for the identification of Stp1 substrates in yeast and provide hints about the nature of tyrosine phosphorylating agents in yeast and in other organisms where bona fide tyrosine kinases are lacking.     

A new fatty acid ester from Nigella sativa var. hispidula Boiss showing potent anti-protein tyrosine phosphatase 1B activity

A new fatty acid ester (1) and seven known phenolic compounds, i.e. salfredin B₁₁ (2), nigephenol C (3), nigephenol B (4), acetovanillion (5), p-hydroxybenzoic acid (6), p-hydroxy-acetophenone (7) and p-hydroxybenzaldehyde (8), were isolated from the seeds of Nigella sativa var. hispidula. Among them, compounds 5, 7 and 8 were isolated from Nigella for the first time. Their structures were elucidated with HR-ESI-MS, 1D and 2D NMR spectra. Evaluation of the isolated compounds on protein tyrosine phosphatase (PTP1B) assay indicated that although compounds 2–8 show no promising anti-PTP1B activities, compound 1 possess anti-PTP1B activity with an IC₅₀ value of 7.38 ± 0.14 μM in vitro.     

Development of activity-based probes with tunable specificity for protein tyrosine phosphatase subfamilies

Herein we describe the development of activity-based probes toward protein tyrosine phosphatase (PTP) subfamilies. A novel phosphotyrosine analog serving as the latent trapping unit has been designed and explored. It allows addition of various amino acid residues to its C- and N-termini to extend the recognition element. As a proof-of-concept, we have synthesized three tripeptide probes, which carry the phosphotyrosine analog in the middle position and a leucinamide residue at the C-terminus. The three tripeptide probes differed only in their N-terminal amino acid (Glu, Phe, and Lys). The labeling properties of these probes were determined and the results showed the newly synthesized probes could selectively label PTPs in an activity-dependent manner. In addition, the probes’ target specificity was also shown to be influenced by the amino acid residues flanking the phosphotyrosine analog.