Expression,purification and characterization of recombinant protein tyrosine phosphatase from Thermus thermophilus HB27

The low-molecular-weight protein tyrosine phospha- tases （PTPase） exist ubiquitously in prokaryotes and eukaryotes and play important roles in the regulation of physiological activities. We report here the expression, purification and characterization of an active and soluble PTPase from Thermus thermophilus HB27 in Escherichia coli. This PTPase has an optimum pH range of 2.8-4.8 when using p-nitrophenyl phosphate as the substrate. The thermal inactivation results indicate a high thermal stability of this enzyme, with the optimum temperature of 75℃ for activity. It can be activated by Mn^2＋, Mg^2＋, Ca^2＋, Ba^2＋, and Ni^2＋, but inhibited by Zn^2＋, Cu^2＋, Cl^-, and SO^2-. These results suggest that this heat-resistant PTPase may play important roles in vivo in the adaptation of the microorganism to extreme temperatures and specific nutritional conditions.     

Differential effects of flavonoids on bovine kidney low molecular mass protein tyrosine phosphatase

Among the structurally related flavonoids tested on the bovine kidney low molecular weight protein tyrosine phosphatase (LMrPTP) activity, quercetin activated by about 2.6-fold the p-nitrophenyl-phosphate (p-NPP)-directed reaction, in contrast to morin that acted as a competitive inhibitor, with Ki values of 87, 73 and 50 μM for p-NPP, FMN, and tyrosine-phosphate, respectively. Other related flavonoids, such as rutin, kaempferol, catechin, narigin, phloretin and taxifolin did not significantly affect the LMrPTP activity.

The positions of the hydroxyl groups in the structures of the flavonoids were important for their distinct effects on LMrPTP activity. The hydroxyl groups at C3′ and C4′ and the presence of a double bond at C2 and C3 were essential for the activating effect of quercetin. The absence of the 3′-OH (kaempferol), absence of the double bond (taxifolin) and the presence of the sugar rutinose at the 3-OH (rutin) suppressed the effect of quercetin. The C2′- and C4′-hydroxyl groups, the presence of the double bond, and a C4-ketone group were important requirements for the inhibitory effects of morin.     

Expression,purification, and characterization of a novel acid phosphatase that displays protein tyrosine phosphatases activity from Metarhizium anisopliae strain CQMa102

The protein tyrosine phosphatase (PTPase) plays an important role in insect immune system. Our group has purified a type of acid phosphatase that could specifically dephosphorylate trans-Golgi p230 in vitro. In order to study this phosphatase further, we have identified and cloned the phosphatase gene from a locust specific Metarhizium anisopliae Strain CQMa102. The CQMa102 phosphatase was expressed in Pichia pastoris to verify its protease activity. The molecular weight (M_W) and the isoelectric point (pI) of the phosphatase were about 85 kDa and 6.15, respectively. Substrate specificity evaluation showed that the purified enzyme exhibited high activity on O-phospho-L-tyrosine. At its optimal pH of 6.5 and optimum temperature of 70 °C, the protein showed the highest activity respectively. It can be activated by Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺, Co²⁺ and phosphate analogs, but inhibited by Zn²⁺, Cu²⁺, fluoride, dithiothreitol, β-mercaptoethanol and N-ethylmaleimide.     

Molecular cloning,nucleotide sequence and expression of a cDNA encoding an intracellular protein tyrosine phosphatase,PTPase-2, from mouse testis and T-cells

The PTP-2 cDNA encoding an intracellular protein tyrosine phosphatase (PTPase-2) was isolated and sequenced from mouse testis and T-cell cDNA libraries. This PTP-2 cDNA was found to be homologous to human PTP-TC and rat PTP-S, and contained 1,551 nucleotides, including 1,146 nucleotides encoding 382 amino acids as well as 5 (61 nucleotides) and 3 (344 nucleotides) non-coding regions. Northern blot analysis indicated that PTP-2 mRNA of 1.9 Kb was most abundant in testis and kidney, although it was also present in spleen, muscle, liver, heart and brain.Abbreviations PTPase Protein Tyrosine Phosphatase (EC3.1.3.48) - PTKase Protein Tyrosine Kinase (EC2.7.1.112)     

The receptor protein tyrosine phosphatase (RPTP)beta/zeta is expressed in different subtypes of human breast cancer

Increasing evidence suggests mutations in human breast cancer cells that induce inappropriate expression of the 18-kDa cytokine pleiotrophin (PTN, Ptn) initiate progression of breast cancers to a more malignant phenotype. Pleiotrophin signals through inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP)beta/zeta, leading to increased tyrosine phosphorylation of different substrate proteins of RPTPbeta/zeta, including beta-catenin, beta-adducin, Fyn, GIT1/Cat-1, and P190RhoGAP. PTN signaling thus has wide impact on different important cellular systems. Recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTPbeta/zeta signaling pathway; this discovery potentially is very important, since constitutive ALK activity of nucleophosmin (NPM)-ALK fusion protein is causative of anaplastic large cell lymphomas, and, activated ALK is found in other malignant cancers. Recently ALK was identified in each of 63 human breast cancers from 22 subjects. We now demonstrate that RPTPbeta/zeta is expressed in each of these same 63 human breast cancers that previously were found to express ALK and in 10 additional samples of human breast cancer. RPTPbeta/zeta furthermore was localized not only in its normal association with the cell membrane but also scattered in cytoplasm and in nuclei in different breast cancer cells and, in the case of infiltrating ductal carcinomas, the distribution of RPTPbeta/zeta changes as the breast cancer become more malignant. The data suggest that the PTN/RPTPbeta/zeta signaling pathway may be constitutively activated and potentially function to constitutively activate ALK in human breast cancer.     

Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.     

Molecular mechanism of T-cell protein tyrosine phosphatase (TCPTP) activation by mitoxantrone

T-cell protein tyrosine phosphatase (TCPTP) is a ubiquitously expressed non-receptor protein tyrosine phosphatase. It is involved in the negative regulation of many cellular signaling pathways. Thus, activation of TCPTP could have important therapeutic applications in diseases such as cancer and inflammation. We have previously shown that the α-cytoplasmic tail of integrin α₁β₁ directly binds and activates TCPTP. In addition, we have identified in a large-scale high-throughput screen six small molecules that activate TCPTP. These small molecule activators include mitoxantrone and spermidine. In this study, we have investigated the molecular mechanism behind agonist-induced TCPTP activation. By combining several molecular modeling and biochemical techniques, we demonstrate that α₁-peptide and mitoxantrone activate TCPTP via direct binding to the catalytic domain, whereas spermidine does not interact with the catalytic domain of TCPTP in vitro. Furthermore, we have identified a hydrophobic groove surrounded by negatively charged residues on the surface of TCPTP as a putative binding site for the α₁-peptide and mitoxantrone. Importantly, these data have allowed us to identify a new molecule that binds to TCPTP, but interestingly cannot activate its phosphatase activity. Accordingly, we describe here mechanism of TCPTP activation by mitoxantrone, the cytoplasmic tail of α₁-integrin, and a mitoxantrone-like molecule at the atomic level. These data provide invaluable insight into the development of novel TCPTP activators, and may facilitate the rational discovery of small-molecule cancer therapeutics.     

Biochemical characterization of a protein tyrosine phosphatase from Trypanosoma cruzi involved in metacyclogenesis and cell invasion

Protein tyrosine phosphatases (PTPs) form a large family of enzymes involved in the regulation of numerous cellular functions in eukaryotes. Several protein tyrosine phosphatases have been recently identified in trypanosomatides. Here we report the purification and biochemical characterization of TcPTP1, a protein tyrosine phosphatase from Trypanosoma cruzi, the causing agent of Chagas’ disease. The enzyme was cloned and expressed recombinantly in Escherichia coli and purified by Ni-affinity chromatography. Biochemical characterization of recombinant TcPTP1 with the PTP pseudo-substrate pNPP allowed the estimation of a Michaelis–Menten constant K_m of 4.5 mM and a k_cat of 2.8 s⁻¹. We were able to demonstrate inhibition of the enzyme by the PTP1b inhibitor BZ3, which on its turn was able to accelerate the differentiation of epimastigotes into metacyclic forms of T. cruzi induced by nutritional stress. Additionally, this compound was able to inhibit by 50% the infectivity of T. cruzi trypomastigotes in a separate cellular assay. In conclusion our results indicate that TcPTP1 is of importance for cellular differentiation and invasivity of this parasite and thus is a valid target for the rational drug design of potential antibiotics directed against T. cruzi.     

Atomic resolution crystal structure of VcLMWPTP-1 from Vibrio cholerae O395: Insights into a novel mode of dimerization in the low molecular weight protein tyrosine phosphatase family

Low molecular weight protein tyrosine phosphatase (LMWPTP) is a group of phosphotyrosine phosphatase ubiquitously found in a wide range of organisms ranging from bacteria to mammals. Dimerization in the LMWPTP family has been reported earlier which follows a common mechanism involving active site residues leading to an enzymatically inactive species. Here we report a novel form of dimerization in a LMWPTP from Vibrio cholera 0395 (VcLMWPTP-1). Studies in solution reveal the existence of the dimer in solution while kinetic study depicts the active form of the enzyme. This indicates that the mode of dimerization in VcLMWPTP-1 is different from others where active site residues are not involved in the process. A high resolution (1.45 Å) crystal structure of VcLMWPTP-1 confirms a different mode of dimerization where the active site is catalytically accessible as evident by a tightly bound substrate mimicking ligand, MOPS at the active site pocket. Although being a member of a prokaryotic protein family, VcLMWPTP-1 structure resembles very closely to LMWPTP from a eukaryote, Entamoeba histolytica. It also delineates the diverse surface properties around the active site of the enzyme.     

cDNA cloning,genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP) gene from Japanese larch (Larix leptolepis)

A full-length cDNA and genomic sequences of a translationally controlled tumor protein (TCTP) gene were isolated from Japanese larch (Larix leptolepis) and designated LaTCTP. The length of the cDNA was 1043 bp and contained a 504 bp open reading frame that encodes a predicted protein of 167 amino acids, characterized by two signature sequences of the TCTP protein family. Analysis of the LaTCTP gene structure indicated four introns and five exons, and it is the largest of all currently known TCTP genes in plants. The 5′-flanking promoter region of LaTCTP was cloned using an improved TAIL-PCR technique. In this region we identified many important potential cis-acting elements, such as a Box-W1 (fungal elicitor responsive element), a CAT-box (cis-acting regulatory element related to meristem expression), a CGTCA-motif (cis-acting regulatory element involved in MeJA-responsiveness), a GT1-motif (light responsive element), a Skn-1-motif (cis-acting regulatory element required for endosperm expression) and a TGA-element (auxin-responsive element), suggesting that expression of LaTCTP is highly regulated. Expression analysis demonstrated ubiquitous localization of LaTCTP mRNA in the roots, stems and needles, high mRNA levels in the embryonal-suspensor mass (ESM), browning embryogenic cultures and mature somatic embryos, and low levels of mRNA at day five during somatic embryogenesis. We suggest that LaTCTP might participate in the regulation of somatic embryo development. These results provide a theoretical basis for understanding the molecular regulatory mechanism of LaTCTP and lay the foundation for artificial regulation of somatic embryogenesis.