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1.
Clin Transl Gastroenterol ; 11(12): e00265, 2020 12.
Article in English | MEDLINE | ID: mdl-33512811

ABSTRACT

INTRODUCTION: Circulating tumor cells (CTCs) and phosphatase of regenerating liver-3 (PRL-3) have been considered to be significant prognostic indicators in metastatic colorectal cancer (CRC). This study discusses the prognostic significance of mesenchymal CTCs with PRL-3 (M+ PRL-3+ CTCs) in postoperative patients with CRC. METHODS: We detected CTC subtypes (including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs) and PRL-3 in CTCs from the peripheral blood samples of 156 patients. Receiver operating characteristic curve analysis, Kaplan-Meier analysis, and Cox proportional hazards regression analysis were performed to identify the prognostic value of mesenchymal CTCs with PRL-3+. Immunohistochemistry was used to detect the expression of PRL-3 in tumor tissues from some of the patients to explore the connection between CTCs and tissues. RESULTS: All CTCs were positive in all samples, both mesenchymal CTCs and PRL-3-positive cells. The count of mesenchymal and PRL-3+ CTCs was significantly associated with recurrence, and the optimal cutoff value was 2 (area under the curve = 0.690, P < 0.001). In addition, these patients had a significantly shorter median disease-free survival than those who did not fulfill the criteria (8.5 vs 24 months, P < 0.001) according to multivariable and multinomial logistic regression. Immunohistochemistry was applied to explore the associations between PRL-3 expression and significant prognostic risk factors, including recurrence (R = 0.566; P < 0.001), and M+ PRL-3+ status in CTCs (R = 0.452; P = 0.001). DISCUSSION: The status of M+ PRL-3+ in CTCs may serve as a crucial prognostic marker for assessing clinical outcomes in CRC.


Subject(s)
Biomarkers, Tumor/metabolism , Colorectal Neoplasms/mortality , Neoplasm Proteins/metabolism , Neoplasm Recurrence, Local/epidemiology , Neoplastic Cells, Circulating/metabolism , Protein Tyrosine Phosphatases/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Colectomy , Colorectal Neoplasms/blood , Colorectal Neoplasms/pathology , Colorectal Neoplasms/surgery , Disease-Free Survival , Female , Follow-Up Studies , Humans , Immunohistochemistry , Male , Middle Aged , Neoplasm Proteins/analysis , Neoplasm Recurrence, Local/prevention & control , Neoplasm Staging , Proctectomy , Prognosis , Prospective Studies , Protein Tyrosine Phosphatases/analysis , ROC Curve , Risk Assessment/methods , Young Adult
2.
Anal Chem ; 91(20): 13206-13212, 2019 10 15.
Article in English | MEDLINE | ID: mdl-31536703

ABSTRACT

Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation-during which the PTP enzymes act on the peptide substrate-the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.


Subject(s)
Enzyme Assays/methods , Protein Tyrosine Phosphatases/analysis , Single-Cell Analysis/methods , Cell Line, Tumor , Ethylene Glycols/chemistry , Flow Cytometry , HEK293 Cells , Humans , Membranes, Artificial , Phosphopeptides/chemistry , Protein Tyrosine Phosphatases/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sulfhydryl Compounds/chemistry
3.
Acta Neuropathol Commun ; 7(1): 35, 2019 03 06.
Article in English | MEDLINE | ID: mdl-30841933

ABSTRACT

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by "tethering proteins" that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson's disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson's disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson's disease and FTD/ALS.


Subject(s)
Endoplasmic Reticulum/metabolism , Kv Channel-Interacting Proteins/metabolism , Mitochondrial Proteins/metabolism , Neurons/metabolism , Protein Tyrosine Phosphatases/metabolism , Synapses/metabolism , Animals , Cells, Cultured , Endoplasmic Reticulum/chemistry , Hippocampus/chemistry , Hippocampus/metabolism , Kv Channel-Interacting Proteins/analysis , Mitochondrial Proteins/analysis , Neurons/chemistry , Protein Tyrosine Phosphatases/analysis , Rats , Synapses/chemistry
5.
Methods Mol Biol ; 1614: 31-46, 2017.
Article in English | MEDLINE | ID: mdl-28500593

ABSTRACT

Post-translational modification of proteins, such as phosphorylation and oxidation, plays a major role in cellular signaling by influencing protein structure and function. In vascular cells, in addition to influencing phosphorylation, angiotensin II (Ang II) induces oxidation of proteins, important in redox signaling in the cardiovascular and renal systems. The present chapter describes immunoblotting approaches to assess irreversible protein carbonylation and protein tyrosine phosphatase (PTPs) oxidation status in the proteome of vascular smooth muscle cells (VSMC).Protein carbonylation is generally measured using the OxyBlot™ approach, whereby derivatization of protein carbonyl groups (C = O) on oxidized amino acids by dinitrophenylhydrazine (DNPH) results in the formation of a stable dinitrophenyl (DNP) hydrazone product. The samples are analyzed by SDS-PAGE and a primary antibody raised against the DNP moiety is used to determine levels of irreversible protein carbonylation in the sample by immunoblotting.Oxidation of PTPs can be evaluated using a monoclonal antibody against the "hyperoxidized" (SO3H) catalytic site of these enzymes. The described methodology offers the ability to discriminate between irreversible (SO3H) and reversible (SOH) PTP oxidation states. Initially, the free unmodified PTP-thiols (S-) are alkylated and the sample is split into two. One part is used to assess the PTP-SO3H form. In the other part reversibly modified PTP-thiols are first reduced and then hyperoxidized by pervanadate (PV). Both untreated and PV-treated samples are analyzed by SDS-PAGE and "hyperoxidized" PTPs are detected by immunoblotting. The proportion of reversibly oxidized PTP-SOH fraction is determined by the difference between the signals in untreated and the PV-treated samples.The above immunoassays provide general approaches to detect and quantify global levels of irreversible protein oxidation and of irreversibly/reversibly oxidized PTPs in any (patho)physiological context. Characterization of the global redox status is essential to better understand the redox-sensitive mechanisms underlying chronic diseases associated with oxidative stress. This is particularly important in systems influenced by the renin angiotensin system, because Ang II is a potent inducer of oxidative stress and redox signaling.


Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Immunoblotting/methods , Muscle, Smooth, Vascular/metabolism , Protein Carbonylation , Protein Tyrosine Phosphatases/analysis , Cells, Cultured , Humans , Oxidation-Reduction , Protein Processing, Post-Translational
6.
Biochem Biophys Res Commun ; 483(1): 700-705, 2017 01 29.
Article in English | MEDLINE | ID: mdl-27986565

ABSTRACT

In blood vessels, serotonin 5-HT2B receptors mainly mediate relaxation, although their activation by the selective agonist BW723C86 is known to exert contraction of aorta in deoxycorticosterone acetate (DOCA)-salt and N(omega)-nitro-l-arginine (l-NAME) hypertensive rats [Russel et al., 2002; Banes et al., 2003] and in mice with type 2 diabetes [Nelson et al., 2012]. The unmasking effect on vasoconstriction can be caused by a shift in the balance of tyrosine phosphorylation in smooth muscle cells (SMC) due to oxidative stress induced inhibition of protein tyrosine phosphatases (PTP). We have demonstrated that BW723C86 which does not cause contraction of rat aorta and mesenteric artery rings, evoked a vasoconstrictor effect in the presence of PTP inhibitors sodium orthovanadate (Na3VO4) or BVT948. BW723C86 induced a weak rise of [Ca2+]i in the SMC isolated from rat aorta; however, after pre-incubation with Na3VO4 the response to BW723C86 increased more than 5-fold. This effect was diminished by protein tyrosine kinase (PTK) inhibitor genistein, inhibitor of Src-family kinases PP2, inhibitor of NADPH-oxidase VAS2870 and completely suppressed by N-acetylcysteine and 5-HT2B receptor antagonist RS127445. Using fluorescent probe DCFH-DA we have shown that Na3VO4 induces oxidative stress in SMC. In the presence of Na3VO4 BW723C86 considerably increased formation of reactive oxygen species while alone had no appreciable effect on DCFH oxidation. We suggest that oxidative stress causes inhibition of PTP and unmasking of 5-HT2B receptors functional activity.


Subject(s)
Aorta/drug effects , Calcium Signaling/drug effects , Indoles/pharmacology , Muscle, Smooth, Vascular/drug effects , Protein Tyrosine Phosphatases/analysis , Receptor, Serotonin, 5-HT2B/metabolism , Serotonin 5-HT2 Receptor Agonists/pharmacology , Thiophenes/pharmacology , Vasoconstriction/drug effects , Animals , Aorta/enzymology , Aorta/physiology , Cell Separation , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiology , Muscle Contraction/drug effects , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/enzymology , Oxidative Stress , RNA, Messenger/analysis , Rats , Rats, Wistar , Receptor, Serotonin, 5-HT2B/genetics , Vanadates/pharmacology , Vasoconstriction/physiology , Vasoconstrictor Agents/pharmacology
7.
Methods Mol Biol ; 1505: 59-67, 2017.
Article in English | MEDLINE | ID: mdl-27826856

ABSTRACT

To complete cell division and to exit from mitosis into the next G1 phase, eukaryotic cells need to inactivate the cyclin-dependent kinase (Cdk) and reverse Cdk-phosphorylation events. In budding yeast mitotic exit depends on the phosphatase Cdc14. During the majority of the cell cycle Cdc14 is sequestered and kept inactive in the nucleolus. Activation of Cdc14 at anaphase onset coincides with its release from the nucleolus into the nucleus and subsequently into the cytoplasm. Here we describe a microscopy method, originally developed in the laboratory of Frederick Cross (Lu and Cross, Cell 141:268-279, 2010), that allows quantifying Cdc14 release in live cells using the open source software FIJI. We adapted this method and show that it is able to distinguish between Cdc14 activation defects caused by mutations in the "cdcFourteen Early Anaphase Release"-(FEAR) and the mitotic exit network (MEN) using slk19∆ and cdc15-1 mutant strains.


Subject(s)
Cell Cycle Proteins/analysis , Microscopy, Confocal/methods , Protein Tyrosine Phosphatases/analysis , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/cytology , Cell Cycle , Cell Cycle Proteins/metabolism , Image Processing, Computer-Assisted/methods , Mitosis , Protein Tyrosine Phosphatases/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Software
8.
Methods Mol Biol ; 1505: 89-96, 2017.
Article in English | MEDLINE | ID: mdl-27826859

ABSTRACT

The phosphatase Cdc14 has a pivotal function in the mitotic exit of Saccharomyces cerevisiae. During interphase, Cdc14 remains inactive in the nucleolus bound to the inhibitor Net1. Cdc14 activation occurs in the metaphase to anaphase transition and it is promoted by at least two signaling pathways called FEAR (CdcFourteen Early Anaphase Release) and MEN (Mitotic Exit Network). These two pathways act in parallel and target the phosphorylation of Net1, thus decreasing Net1 affinity for Cdc14. The activity of Cdc14 can be used as a readout to assay functional interactions of different components of the mitotic exit signaling pathways.


Subject(s)
Cell Cycle Proteins/metabolism , Enzyme Assays/methods , Immunoprecipitation/methods , Protein Tyrosine Phosphatases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Cell Culture Techniques/methods , Cell Cycle Proteins/analysis , Mitosis , Phosphorylation , Protein Tyrosine Phosphatases/analysis , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/analysis , Signal Transduction
9.
Methods Mol Biol ; 1505: 135-149, 2017.
Article in English | MEDLINE | ID: mdl-27826862

ABSTRACT

The budding yeast Saccharomyces cerevisiae is a very powerful genetic model that has been extensively used in cell cycle studies. Despite the fact that its small size has made imaging studies challenging (haploid cells have a diameter of approximately 4-5 µm that is very close to the maximal optical microscope resolution, ca. 0.20-0.25 µm), the continual improvement of imaging tags and techniques has made it possible to visualize organelles and macromolecules also in this organism. The possibility to easily epitope-tag endogenous proteins and follow them during synchronized cell cycles has proved critical for understanding the distribution of Mitotic Exit Network (MEN) components and gathering insights into their regulation. In this chapter, we describe a detailed protocol for indirect immunofluorescence of fixed cells outlining fixation strategies, cell wall digestion, and the use of primary and secondary antibodies conjugated to fluorescent moieties. This protocol can be used to successfully localize endogenously expressed yeast proteins including MEN components.


Subject(s)
Fluorescent Antibody Technique, Indirect/methods , Microscopy, Fluorescence/methods , Saccharomyces cerevisiae/cytology , Cell Cycle , Cell Cycle Proteins/analysis , Mitosis , Protein Serine-Threonine Kinases/analysis , Protein Tyrosine Phosphatases/analysis , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/analysis , Tubulin/analysis
10.
Methods Mol Biol ; 1505: 151-166, 2017.
Article in English | MEDLINE | ID: mdl-27826863

ABSTRACT

Mitotic exit is determined by multiple spatial and temporal cues from the spindle poles and the two compartments in a dividing yeast cell-the mother and the bud. These signals are ultimately integrated by the activation of the mitotic exit network (MEN) to promote persistent release of Cdc14 from the nucleolus. Live imaging analysis using fluorescent protein tags is invaluable to dissect this critical decision-making trigger. Here, we present protocols for routine yeast live cell microscopy applicable to this problem.


Subject(s)
Cell Cycle Proteins/analysis , Microscopy, Fluorescence/methods , Protein Serine-Threonine Kinases/analysis , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/cytology , Single-Cell Analysis/methods , Anaphase , Green Fluorescent Proteins/analysis , Image Processing, Computer-Assisted/methods , Mitosis , Optical Imaging/methods , Protein Tyrosine Phosphatases/analysis , Saccharomyces cerevisiae/ultrastructure
11.
Indian J Pathol Microbiol ; 59(3): 287-93, 2016.
Article in English | MEDLINE | ID: mdl-27510662

ABSTRACT

CONTEXT: Poor survival of the glioblastoma multiforme (GBM) has been attributed in part to the invasive nature of the lesion making complete surgical removal near impossible. Phosphatase of regenerating liver-3 (PRL-3), matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9), and epidermal growth factor receptor (EGFR-1) play a role in invasive nature of tumor cells. AIMS: This study was conducted to evaluate PRL-3, MMP-2, MMP-9, and EGFR-1 (markers) expression in cases to GBM and to correlate their expression with therapy response and survival. SETTINGS AND DESIGN: GBM cases (n = 62) underwent surgery followed by radiation (n = 34) and chemoradiation (n = 28). Using WHO Response Evaluation Criteria in Solid Tumors criteria response to therapy was assessed at 3 months and cases followed up for survival. SUBJECTS AND METHODS: Expression of markers was assessed by immunohistochemistry as a percentage of positive tumor cells in hot spots. STATISTICAL ANALYSIS USED: Kaplan-Meier, ANOVA, Chi-square test, univariate, and multivariate Cox-regression analysis was done. RESULTS: Response to therapy was evident in 54.8% cases of responders with the mean survival of 494.03 ± 201.13 days and 45.2% cases of non responders (278.32 ± 121.66 days) with P = 0.001. Mean survival for the patient's opted chemoradiation was 457.43 ± 222.48 days which was approximately 3 months greater than those who opted radiation alone (P = 0.029). We found PRL-3 overexpression was an independent, significant, poor prognostic factor for survival by multivariate analysis (P = 0.044). Cases negative for MMP's and EGFR showed increased survival, but the difference was insignificant. CONCLUSION: PRL-3 expression appears to be related to an adverse disease outcome.


Subject(s)
ErbB Receptors/analysis , Glioblastoma/diagnosis , Glioblastoma/therapy , Matrix Metalloproteinases/analysis , Neoplasm Proteins/analysis , Protein Tyrosine Phosphatases/analysis , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Follow-Up Studies , Glioblastoma/mortality , Glioblastoma/pathology , Humans , Immunohistochemistry , Infant , Male , Middle Aged , Prognosis , Radiotherapy , Surgical Procedures, Operative , Survival Analysis , Treatment Outcome , Young Adult
12.
Histochem Cell Biol ; 146(1): 99-111, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27015884

ABSTRACT

The phosphatase of regenerating liver (PRL) is a group of protein tyrosine phosphatases that play a key role in cancer progression and metastasis. We previously showed that PRL-2 modulates intracellular Mg(2+) levels and sustains cancer phenotypes by binding to the Mg(2+) transporter CNNM3. However, the physiological functions of PRL-2 in animals remain largely unknown. To better understand which cell types are associated with PRL-2 function, we characterized its expression in mouse tissues using a PRL-2 ß-galactosidase reporter mouse model. Our results demonstrated that PRL-2 was ubiquitously expressed, with the highest expression levels observed in the hippocampal pyramidal neurons, ependymal cells, cone and rod photoreceptor cells, endocardium, vascular and bronchial smooth muscle, and collecting ducts in the kidney. On the other hand, PRL-2 expression was undetectable or very low in the parenchymal cells of the liver and pancreas. Our results also indicated that PRL-2 is involved in cell-type-specific Mg(2+) homeostasis and that PRL-2 expression is potentially inversely regulated by dietary Mg(2+) levels.


Subject(s)
Dietary Supplements , Immediate-Early Proteins/analysis , Immediate-Early Proteins/biosynthesis , Magnesium/pharmacology , Protein Tyrosine Phosphatases/analysis , Protein Tyrosine Phosphatases/biosynthesis , Animals , Female , Homeostasis/drug effects , Immediate-Early Proteins/metabolism , Magnesium/administration & dosage , Magnesium/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Tyrosine Phosphatases/metabolism
13.
J Biol Chem ; 291(11): 5926-5934, 2016 Mar 11.
Article in English | MEDLINE | ID: mdl-26742850

ABSTRACT

Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Phosphoprotein Phosphatases/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Arabidopsis/chemistry , Arabidopsis/cytology , Arabidopsis Proteins/analysis , Phosphoprotein Phosphatases/analysis , Phosphorylation , Protein Tyrosine Phosphatases/analysis , Protein Tyrosine Phosphatases/metabolism
14.
Mol Cell Biol ; 36(5): 668-77, 2015 Dec 14.
Article in English | MEDLINE | ID: mdl-26667035

ABSTRACT

Eyes absent (Eya), a protein conserved from plants to humans and best characterized as a transcriptional coactivator, is also the prototype for a novel class of eukaryotic aspartyl protein tyrosine phosphatases. This minireview discusses recent breakthroughs in elucidating the substrates and cellular events regulated by Eya's tyrosine phosphatase function and highlights some of the complexities, new questions, and surprises that have emerged from efforts to understand how Eya's unusual multifunctionality influences developmental regulation and signaling.


Subject(s)
Eye Proteins/metabolism , Eye/enzymology , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Animals , Eye/chemistry , Eye/growth & development , Eye/metabolism , Eye Proteins/analysis , Eye Proteins/genetics , Gene Expression Regulation, Developmental , Histones/metabolism , Humans , Molecular Sequence Data , Protein Tyrosine Phosphatases/analysis , Protein Tyrosine Phosphatases/genetics , Signal Transduction , Substrate Specificity , Transcriptional Activation
15.
Eur J Med Chem ; 88: 28-33, 2014 Dec 17.
Article in English | MEDLINE | ID: mdl-24974258

ABSTRACT

Reversible oxidation of protein tyrosine phosphatases (PTPs) has emerged as an important regulatory mechanism whereby reactive oxygen species (ROS) inactivates the PTP and promotes phosphorylation and induction of the signaling cascade. The lack of sensitive and robust methods to directly detect oxidized PTPs has made it difficult to understand the effects that PTP oxidative inactivation play in redox signaling. We report the use of redox-based probes to directly detect oxidized PTPs in a cellular context, which highlights the importance of direct approaches to assist in the study of physiological and pathophysiological PTP activity in redox regulation. We also demonstrate, as a proof-of-concept, that these redox-based probes serve as prototypes for the design and development of a new class of inhibitors for phosphatases. We envision a nucleophile reacting with the oxidized inactive catalytic cysteine to generate an irreversible thioether adduct which prevents the phosphatase from being reactivated and ultimately fortifies the signaling cascade. Our results reveal the potential of translation of our redox-based probes, which are used to understand redox cell circuitry and disease biology, to small-molecule nucleophile-based inhibitors, which may treat diseases associated with redox stress. This may have implications in the treatment of type 2 diabetes and cancer.


Subject(s)
Alkynes/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/analysis , Small Molecule Libraries/pharmacology , Alkynes/chemical synthesis , Alkynes/chemistry , Animals , Biocatalysis , CHO Cells , COS Cells , Cells, Cultured , Chlorocebus aethiops , Cricetulus , Humans , Molecular Structure , Oxidation-Reduction , Protein Tyrosine Phosphatases/metabolism , Reactive Oxygen Species/metabolism , Receptor, Insulin/biosynthesis , Receptor, Insulin/metabolism , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship
16.
Chem Commun (Camb) ; 50(60): 8161-3, 2014 Aug 04.
Article in English | MEDLINE | ID: mdl-24923797

ABSTRACT

A versatile graphene oxide (GO)-based fluorescent assay is developed for the detection of protein tyrosine phosphatase activity by coupling with a chymotrypsin-assisted selective peptide cleavage reaction.


Subject(s)
Chymotrypsin/metabolism , Fluorescent Dyes/chemistry , Graphite/chemistry , Oxides/chemistry , Peptide Fragments/metabolism , Protein Tyrosine Phosphatases/analysis , Tyrosine/metabolism , Biosensing Techniques , Humans , Spectrometry, Fluorescence
17.
Am J Physiol Cell Physiol ; 306(11): C1017-27, 2014 Jun 01.
Article in English | MEDLINE | ID: mdl-24717577

ABSTRACT

We have previously shown that in addition to the well-known secreted isoform of prostatic acid phosphatase (sPAP), a transmembrane isoform exists (TMPAP) that interacts with snapin (a SNARE-associated protein) and regulates the endo-/exocytic pathways. We have also shown that PAP has 5'-ectonucleotidase and thiamine monophosphatase activity and elicits antinociceptive effects in mouse models of chronic inflammatory and neuropathic pain. Therefore, to determine the physiological role of PAP in a typical exocrine organ, we studied the submandibular salivary gland (SMG) of PAP(-/-) and wild-type C57BL/6J mice by microarray analyses, microRNA sequencing, activity tests, immunohistochemistry, and biochemical and physiological analyses of saliva. We show that PAP is the main acid phosphatase in the wild-type male mouse saliva, accounting for 50% of the total acid phosphatase activity, and that it is expressed only in the granular convoluted tubules of the SMGs, where it is the only 5'-ectonucleotidase. The lack of PAP in male PAP(-/-) mice was associated with a significant increase in the salivation volume under secretagogue stimulation, overexpression of genes related to cell proliferation (Mki67, Aurkb, Birc5) and immune response (Irf7, Cxcl9, Ccl3, Fpr2), and upregulation of miR-146a in SMGs. An increased and sustained acinar cell proliferation was detected without signs of glandular hyperplasia. Our results indicate that in PAP(-/-) mice, SMG homeostasis is maintained by an innate immune response. Additionally, we suggest that in male mice, PAP via its 5'-ectonucleotidase activity and production of adenosine can elicit analgesic effects when animals lick their wounds.


Subject(s)
Protein Tyrosine Phosphatases/metabolism , Saliva/enzymology , Salivation/physiology , Acid Phosphatase/analysis , Acid Phosphatase/metabolism , Animals , Enzyme Activation/physiology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Tyrosine Phosphatases/analysis , Saliva/chemistry
18.
Anal Chem ; 86(2): 1291-7, 2014 Jan 21.
Article in English | MEDLINE | ID: mdl-24380370

ABSTRACT

We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min(-1) mg(-1)) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (76%, 69%, and 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min(-1) mg(-1) and was also effectively inhibited by preincubation of the cells with the inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min(-1) mg(-1) resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34 to 36 pmol min(-1) mg(-1) (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues.


Subject(s)
Bronchi/enzymology , Epithelial Cells/enzymology , Phosphoproteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Bronchi/cytology , Bronchi/drug effects , Cell Line , Electrophoresis, Capillary , Enzyme Inhibitors/pharmacology , Epithelial Cells/cytology , Epithelial Cells/drug effects , Half-Life , Humans , Hydrolysis , Microinjections , Naphthoquinones/pharmacology , Phosphoproteins/administration & dosage , Primary Cell Culture , Protein Tyrosine Phosphatases/analysis , Protein Tyrosine Phosphatases/antagonists & inhibitors , Single-Cell Analysis , Vanadates/pharmacology
19.
Methods ; 65(2): 207-18, 2014 Jan 15.
Article in English | MEDLINE | ID: mdl-24064037

ABSTRACT

Protein tyrosine phosphatases (PTPs) are key enzymes in the regulation of cellular homeostasis and signaling pathways. Strikingly, not all PTPs bear enzymatic activity. A considerable fraction of PTPs are enzymatically inactive and are known as pseudophosphatases. Despite the lack of activity they execute pivotal roles in development, cell biology and human disease. The present review is focused on the methods used to identify pseudophosphatases, their targets, and physiological roles. We present a strategy for detailed enzymatic analysis of inactive PTPs, regulation of inactive PTP domains and identification of binding partners. Furthermore, we provide a detailed overview of human pseudophosphatases and discuss their regulation of cellular processes and functions in human pathologies.


Subject(s)
Enzyme Assays , Protein Tyrosine Phosphatases/analysis , Protein Tyrosine Phosphatases/metabolism , Binding Sites , Humans , Protein Tyrosine Phosphatases/chemistry , Sequence Alignment , Substrate Specificity
20.
Anal Chem ; 85(12): 6136-42, 2013 Jun 18.
Article in English | MEDLINE | ID: mdl-23682679

ABSTRACT

A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn(2+) and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min(-1) mg(-1)) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min(-1) mg(-1)). Zn(2+), 1,2-naphthoquinone, and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 µM, and 100 nM, respectively. Dephosphorylation of the reporter, following loading into living single cells, occurred at rates of at least 2 pmol min(-1) mg(-1). When single cells were exposed to 1,2-naphthoquinone (50 µM), Zn(2+) (100 µM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens.


Subject(s)
Cell Separation/methods , Flow Cytometry/methods , Protein Tyrosine Phosphatases/analysis , Protein Tyrosine Phosphatases/metabolism , Cell Line, Tumor , Electrophoresis, Capillary/methods , Enzyme Activation/physiology , Humans
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