Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer''s disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC₅₀ of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and in vivo in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.     

Induction of a Tumor-associated Activating Mutation in Protein Tyrosine Phosphatase Ptpn11 (Shp2) Enhances Mitochondrial Metabolism,Leading to Oxidative Stress and Senescence

Activating mutations in Ptpn11 (Shp2), a protein tyrosine phosphatase involved in diverse cell signaling pathways, are associated with pediatric leukemias and solid tumors. However, the pathogenic effects of these mutations have not been fully characterized. Here, we report that induction of the Ptpn11^E76K/+ mutation, the most common and active Ptpn11 mutation found in leukemias and solid tumors, in primary mouse embryonic fibroblasts resulted in proliferative arrest and premature senescence. As a result, apoptosis was markedly increased. These cellular responses were accompanied and mediated by up-regulation of p53 and p21. Moreover, intracellular levels of reactive oxygen species (ROS), byproducts of mitochondrial oxidative phosphorylation, were elevated in Ptpn11^E76K/+ cells. Since Shp2 is also distributed to the mitochondria (in addition to the cytosol), the impact of the Ptpn11^E76K/+ mutation on mitochondrial function was analyzed. These analyses revealed that oxygen consumption of Ptpn11^E76K/+ cells and the respiratory function of Ptpn11^E76K/+ mitochondria were significantly increased. Furthermore, we found that phosphorylation of mitochondrial Stat3, one of the substrates of Shp2 phosphatase, was greatly decreased in the mutant cells with the activating mutation Ptpn11^E76K/+. This study provides novel insights into the initial effects of tumor-associated Ptpn11 mutations.     

人源蛋白酪氨酸磷酸酶SHP-2基因克隆与原核表达

目的:构建蛋白酪氨酸磷酸酶SHP-2的原核表达载体并在大肠杆菌中表达。方法:以人脑组织mRNA为模板,通过RT-PCR扩增出目标cDNA,构建蛋白酪氨酸磷酸酶SHP-2-pEASY-E1重组质粒。将重组质粒转化进E.coli TOP10感受态细胞中,通过菌落PCR和测序进行阳性克隆的筛选和验证,将正确的质粒转化E.coli Transetta感受态细胞中,通过SDS-PAGE和western-blot进行蛋白检测和验证,酶促动力学分析SHP-2可溶性蛋白的活性。结果:成功克隆SHP-2功能域,构建SHP-2-pEASY-E1原核表达载体,完成可溶性蛋白的表达;酶促动力学分析结果为:米氏常数Km=0.97mmol/L,Vmax为13.57mmol/L/s。结论:本研究成功构建SHP-2的原核表达载体,重组表达的SHP-2蛋白具有较高的磷酸酶活性。     

一种新的蛋白酪氨酸磷酸酶基因的克隆、定位和组织表达谱研究

从人胎肝cDNA库分离出一长度为5248bp的cDNA克隆,该基因包含26个外显子和25个内含子,染色体定位于在某些肿瘤细胞中易缺失的3p21.1-21.33。其可读框编码1636个氨基酸,该蛋白属于蛋白酪氨酸磷酸酶（PTP）家族,其C端有一个典型的PTP结构域,N端含有约800氨基酸残基的BRO1样结构域及随后2个可能的SH3结构域结合位点,在这两个结构域之间及C末端还各有一个脯氨酸富集区。Northern杂交和点杂交分析显示,该基因以大约5.4kb的单一转录物广泛表达于人体各种组织,而且在人部分肿瘤细胞中高表达。结果提示,人源PTP－TD14是一个新的蛋白酪氨酸磷酸酶。     

种新的蛋白酪氨酸磷酸酶基因的克隆、定位和组织表达谱研究

从人胎肝cDNA文库分离出一长度为5248bp的cDNA克隆,该基因包含26个外显子和25个内含子,染色体定位于在某些肿瘤细胞中易缺失的3p21.1-21.33.其可读框编码1636个氨基酸,该蛋白属于蛋白酪氨酸磷酸酶(PTP)家族,其C端有一个典型的PTP结构域,N端含有约800氨基酸残基的BRO1样结构域及随后2个可能的SH3结构域结合位点,在这两个结构域之间及C末端还各有一个脯氨酸富集区.Northern杂交和点杂交分析显示,该基因以大约5.4kb的单一转录物广泛表达于人体各种组织,而且在人部分肿瘤细胞中高表达.结果提示,人源PTP-TD14是一个新的蛋白酪氨酸磷酸酶。 Abstract:A human cDNA of 5248bp encoding a novel protein tyrosine phosphatase PTP-TD14(1636aa) has been isolated from fetal liver.The gene is located at chromosome 3p21.3,an area frequently deleted in many types of cancer,and composed of at least 26 exons and 25 introns.The phosphatase has unique features in its domain structure:a tyrosine phosphatase domain,a C-terminal PEST motif,two SH3-binding motifs,two proline-rich region and an N-terminal domain similar to yeast BRO1 (a yeast protein that is involved in the mitogen-activated protein kinase signaling pathway).Northern blot and dot blot hybridizations indicate that it is expressed ubiquitously in human 50 tissues and 7 cancer cell lines.Thus,it is a novel protein tyrosine phosphatase gene located on 3p21.3.     

Hypoxia-Ischemia in Perinatal Rat Brain Induces the Formation of a Low Molecular Weight Isoform of Striatal Enriched Tyrosine Phosphatase (STEP)

Protein tyrosine phosphatases play a critical role in controlling tyrosine phosphorylation levels of proteins. Ischemia induces changes in tyrosine phosphorylation. As part of our investigations of the mechanisms responsible for these changes, we studied the effects of cerebral hypoxia-ischemia in 7-day-old (P7) and P21 rat brains on expression of the STEP (striatal enriched phosphatase) family of protein tyrosine phosphatases. P7 and P21 rats were subjected to unilateral hypoxia-ischemia, and brains were analyzed at various intervals of recovery for the presence of STEP. Hypoxia-ischemia induced the formation of a low Mr isoform of STEP, STEP33, in the ipsilateral (damaged) hemisphere but not in the contralateral (undamaged) side. STEP33 produced as a result of ischemia was located exclusively in the cell soluble fraction. In P21 rats, the ischemia-induced elevation in STEP33 was delayed relative to P7 rats. STEP33 was produced by digestion of postsynaptic densities with calpain I and by exposure of NT2/D1 cells expressing STEP to the calcium ionophore A23187. The results suggest that ischemia-induced calcium influx results in the calcium-dependent proteolysis of membrane-associated STEP61 and the concomitant release of STEP33 into the cytoplasm.     

Src Homology-2 Domain-Containing Protein Tyrosine Phosphatase (SHP) 2 and p38 Regulate the Expression of Chemokine CXCL8 in Human Astrocytes

CXCL8, one of the first chemokines found in the brain, is upregulated in the brains and cerebrospinal fluid of HIV-1 infected individuals suggesting its potential role in human immune deficiency virus (HIV)-associated neuroinflammation. Astrocytes are known to be the major contributors to the CXCL8 pool. Interleukin (IL)-1β activated astrocytes exhibit significant upregulation of CXCL8. In order to determine the signaling pathways involved in CXCL8 regulation in astrocytes, we employed pharmacological inhibitors for non-receptor Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and mitogen-activated protein kinases (MAPK) pathway and observed reduced expression of CXCL8 following IL-1β stimulation. Overexpression of SHP2 and p38 enzymes in astrocytes led to elevated CXCL8 expression; however, inactivating SHP2 and p38 with dominant negative mutants abrogated CXCL8 induction. Furthermore, SHP2 overexpression resulted in higher SHP2 and p38 enzyme activity whereas p38 overexpression resulted in higher p38 but not SHP2 enzyme activity. Phosphorylation of SHP2 was important for phosphorylation of p38, which in turn was critical for phosphorylation of extracellular signal regulated kinase (ERK). Thus, our findings suggest an important role for SHP2 in CXCL8 expression in astrocytes during inflammation, as SHP2, directly or indirectly, modulates p38 and ERK MAPK in the signaling cascade leading to CXCL8 production. This study provides detailed understanding of the mechanisms involved in CXCL8 production during neuroinflammation.     

Identification of Protein Tyrosine Phosphatase Receptor Gamma Extracellular Domain (sPTPRG) as a Natural Soluble Protein in Plasma

Background

PTPRG is a widely expressed protein tyrosine phosphatase present in various isoforms. Peptides from its extracellular domain have been detected in plasma by proteomic techniques. We aim at characterizing the plasmatic PTPRG (sPTPRG) form and to identify its source.

Methodology/Principal Findings

The expression of sPTPRG was evaluated in human plasma and murine plasma and tissues by immunoprecipitation and Western blotting. The polypeptides identified have an apparent Mr of about 120 kDa (major band) and 90 kDa (minor band) respectively. Full length PTPRG was identified in the 100.000×g pelleted plasma fraction, suggesting that it was present associated to cell-derived vesicles (exosomes). The release of sPTPRG by HepG2 human hepatocellular carcinoma cell line was induced by ethanol and sensitive to metalloproteinase and not to Furin inhibitors. Finally, increased levels of the plasmatic ∼120 kDa isoform were associated with the occurrence of liver damage.

Conclusions

These results demonstrate that sPTPRG represent a novel candidate protein biomarker in plasma whose increased expression is associated to hepatocyte damage. This observation could open a new avenue of investigation in this challenging field.     

Discovery of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B (PtpB) Inhibitors from Natural Products

Protein tyrosine phosphatase B (PtpB) is one of the virulence factors secreted into the host cell by Mycobacterium tuberculosis. PtpB attenuates host immune defenses by interfering with signal transduction pathways in macrophages and, therefore, it is considered a promising target for the development of novel anti-tuberculosis drugs. Here we report the discovery of natural compound inhibitors of PtpB among an in house library of more than 800 natural substances by means of a multidisciplinary approach, mixing in silico screening with enzymatic and kinetics studies and MS assays. Six natural compounds proved to inhibit PtpB at low micromolar concentrations (< 30 µM) with Kuwanol E being the most potent with K _i = 1.6 ± 0.1 µM. To the best of our knowledge, Kuwanol E is the most potent natural compound PtpB inhibitor reported so far, as well as it is the first non-peptidic PtpB inhibitor discovered from natural sources. Compounds herein identified may inspire the design of novel specific PtpB inhibitors.     

Human Multipotent Stromal Cells (MSCs) Increase Neurogenesis and Decrease Atrophy of the Striatum in a Transgenic Mouse Model for Huntington's Disease

Background

Implantation of human multipotent stromal cells from bone marrow (hMSCs) into the dentate gyrus of the hippocampus of mice was previously shown to stimulate proliferation, migration and neural differentiation of endogenous neural stem cells. We hypothesized that hMSCs would be beneficial in a mouse model of Huntington disease (HD) due to these neurogenic effects.

Results

We implanted hMSCs into the striatum of transgenic mice (N171-82Q) that are a model for HD. The implanted hMSCs rapidly disappeared over 3 to 15 days. However, they increased proliferation and neural differentiation of endogenous neural stem cells for up to 30 days. They also increased neurotrophic signaling and decreased atrophy of the striatum in 3-month old HD mice implanted with hMSCs one month earlier.

Conclusions

The results therefore suggested that neural implantation of hMSCs may be of benefit in HD but a number of parameters of dose, treatment schedule, and route of administration need to be optimized.     

Mouse Model for Protein Tyrosine Phosphatase D (PTPRD) Associations with Restless Leg Syndrome or Willis-Ekbom Disease and Addiction: Reduced Expression Alters Locomotion,Sleep Behaviors and Cocaine-Conditioned Place Preference

The receptor type protein tyrosine phosphatase D (PTPRD) gene encodes a cell adhesion molecule likely to influence development and connections of addiction-, locomotion- and sleep-related brain circuits in which it is expressed. The PTPRD gene harbors genome-wide association signals in studies of restless leg syndrome (Willis-Ekbom disease [WED]/restless leg syndrome [RLS]; p < 10⁻⁸) and addiction-related phenotypes (clusters of nearby single nucleotide polymorphisms [SNPs] with 10⁻² > p > 10⁻⁸ associations in several reports). We now report work that seeks (a) association between PTPRD genotypes and expression of its mRNA in postmortem human brains and (b) RLS-related, addiction-related and comparison behavioral phenotypes in hetero- and homozygous PTPRD knockout mice. We identify associations between PTPRD SNPs and levels of PTPRD mRNA in human brain samples that support validity of mouse models with altered PTPRD expression. Knockouts display less behaviorally defined sleep at the end of their active periods. Heterozygotes move more despite motor weakness/impersistence. Heterozygotes display shifted dose-response relationships for cocaine reward. They display greater preference for places paired with 5 mg/kg cocaine and less preference for places paired with 10 or 20 mg/kg. The combined data provide support for roles for common, level-of-expression PTPRD variation in locomotor, sleep and drug reward phenotypes relevant to RLS and addiction. Taken together, mouse and human results identify PTPRD as a novel therapeutic target for RLS and addiction phenotypes.     

蛋白质酪氨酸磷酸酶1B（PTP1B）与2型糖尿病及肥胖的关系

蛋白质酪氨酸磷酸酶1B（PTP1B）是一种在体内广泛表达的胞内蛋白质酪氨酸磷酸酶,在调节胰岛素敏感性和能量代谢的过程中起着重要作用。通过抑制PTP1B可增加胰岛素和瘦蛋白（leptin）的活性, 为寻找2型糖尿病、肥胖的治疗提供了光明前景。     

Subcellular Localization and Differentiation-Induced Redistribution of the Protein Tyrosine Phosphatase PTP-BL in Neuroblastoma Cells

1.In cells of epithelial origin the protein tyrosine phosphatase PTP-BL is predominantly localized at the apical membrane of polarized cells. This large submembranous multidomain PTP is also expressed in cells of neuronal origin. We studied the localization of PTP-BL in mouse neuroblastoma cells utilizing EGFP-tagged versions of the protein. 2. In proliferating Neuro-2a cells, immunofluorescence and immuno-electron microscopy revealed a submembranous FERM domain-dependent localization at cell-cell boundaries for EGFP-PTP-BL. Additionally, significant amounts of EGFP-PTP-BL are located in the cytoplasm as well as in nuclei. Upon serum depletion-induced differentiation of Neuro-2a cells, a partial shift of EGFP-PTP-BL from a cortical localization to cytoskeleton-like F-actin-positive structures is observed. Parallel biochemical studies corroborate this finding and reveal a serum depletion-induced shift of EFGP-PTP-BL from a membrane(-associated) fraction to an NP40-soluble cytoskeletal fraction. 3. Different pools of PTP-BL-containing protein complexes can be discerned in neuronal cells, reflecting distinct molecular microenvironments in which PTP-BL may exert its function.     

Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation.     

家蚕核型多角体病毒酪氨酸蛋白磷酸酯酶基因的克隆及在大肠杆菌中表达

采用PCR方法克隆了家蚕核型多角体病毒中国镇江株 (BmNPV ZJstrain)的酪氨酸蛋白磷酸酯酶基因(ptp) ,测定了该基因的核苷酸序列 ,比较了与相关病毒相应基因的同源性 ,并将该基因插入到原核表达质粒在大肠杆菌中进行了表达。该基因的编码部分由 5 0 7个核苷酸组成 ,编码 16 8个氨基酸残基的蛋白多肽 ,其中含有酪氨酸蛋白磷酸酶酯催化部位的“HC”基序。该基因与苜蓿银纹夜蛾核型多角体病毒 (AcMNPV) ptp 基因和BmN PV -T3 株 (日本 )拟为的 ptp基因核苷酸序列的同源性分别为 96 8%和 98 2 % ,蛋白质氨基酸序列的同源性分别为 97 0 %和 97 6 %。将该基因插入到温度诱导型表达质粒pBV2 2 1的温控启动子PRPL 之后 ,在大肠杆菌JM10 9中表达了具有催化活性的蛋白质 ,分子量约为 19kD ,表达产物能催化对硝基酚磷酸钠 (PNPP)的脱磷酸反应 ,这种催化作用可被钒酸钠和ZnCl2 抑制