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Find video protocols related to scientific articles indexed in Pubmed.
TGF? responsive tyrosine phosphatase promotes rheumatoid synovial fibroblast invasiveness.
Ann. Rheum. Dis.
PUBLISHED: 11-08-2014
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In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTP? (RPTP?), encoded by the transforming growth factor (TGF) ?-target gene, PTPRK, promotes RA FLS invasiveness.
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"PEST control": regulation of molecular barcodes by tyrosine phosphatases.
Cell Res.
PUBLISHED: 08-01-2014
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The emerging concept of "molecular barcodes" refers to the dynamic combination of post-translational modifications, often of different nature (e.g., phosphorylation and ubiquitination) that gives rise to multiple forms of a protein which can relay distinct signals throughout a cell. In a recent Cell Research paper by Wang et al., the authors report that a PEST domain-containing tyrosine phosphatase, PTPN18, is able to regulate both phosphorylation and ubiquitination of the HER2 oncogene, barcoding HER2 for increased proteasomal degradation rather than for intracellular trafficking.
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PTPN22: the archetypal non-HLA autoimmunity gene.
Nat Rev Rheumatol
PUBLISHED: 07-08-2014
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PTPN22 encodes a tyrosine phosphatase that is expressed by haematopoietic cells and functions as a key regulator of immune homeostasis by inhibiting T-cell receptor signalling and by selectively promoting type I interferon responses after activation of myeloid-cell pattern-recognition receptors. A single nucleotide polymorphism of PTPN22, 1858C>T (rs2476601), disrupts an interaction motif in the protein, and is the most important non-HLA genetic risk factor for rheumatoid arthritis and the second most important for juvenile idiopathic arthritis. PTPN22 exemplifies a shared autoimmunity gene, affecting the pathogenesis of systemic lupus erythematosus, vasculitis and other autoimmune diseases. In this Review, we explore the role of PTPN22 in autoimmune connective tissue disease, with particular emphasis on candidate-gene and genome-wide association studies and clinical variability of disease. We also propose a number of PTPN22-dependent functional models of the pathogenesis of autoimmune diseases.
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Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis.
J. Clin. Invest.
PUBLISHED: 06-06-2014
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The intestinal epithelium has a high rate of turnover, and dysregulation of pathways that regulate regeneration can lead to tumor development; however, the negative regulators of oncogenic events in the intestinal epithelium are not fully understood. Here we identified a feedback loop between the epidermal growth factor receptor (EGFR), a known mediator of proliferation, and the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), in intestinal epithelial cells (IECs). We found that TRPV1 was expressed by IECs and was intrinsically activated upon EGFR stimulation. Subsequently, TRPV1 activation inhibited EGFR-induced epithelial cell proliferation via activation of Ca2+/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). In a murine model of multiple intestinal neoplasia (Apc(Min/+) mice), TRPV1 deficiency increased adenoma formation, and treatment of these animals with an EGFR kinase inhibitor reversed protumorigenic phenotypes, supporting a functional association between TRPV1 and EGFR signaling in IECs. Administration of a TRPV1 agonist suppressed intestinal tumorigenesis in Apc(Min/+) mice, similar to--as well as in conjunction with--a cyclooxygenase-2 (COX-2) inhibitor, which suggests that targeting both TRPV1 and COX-2 has potential as a therapeutic approach for tumor prevention. Our findings implicate TRPV1 as a regulator of growth factor signaling in the intestinal epithelium through activation of PTP1B and subsequent suppression of intestinal tumorigenesis.
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Targeted nanodrugs for cancer therapy: prospects and challenges.
J Nanosci Nanotechnol
PUBLISHED: 04-16-2014
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The recent advent of nanomedicine holds potential to revolutionize cancer therapy. This innovative discipline has paved the way for the emergence of a new class of drugs based on nanoengineered particles. These "nanodrugs" are designed to greatly enhance drug therapeutic indices. First-generation nanodrugs consisted of conventional anti-cancer drugs loaded into/onto nanoengineered particles (nanocarriers) devoid of targeting features (non-targeted nanodrugs). Non-targeted nanodrugs have provided the opportunity to carry large amounts of drugs, including poorly water-soluble and/or permeable drugs, to several types of tumors, improving the therapeutic index with respect to comparable free drugs. Although effective, the primary delivery mechanism of non-targeted nanodrugs was through passive tissue accumulation, due to pathophysiological differences between tumor-associated and healthy vessels, and through non-specific targeting of cell subsets, posing the danger of off-target binding and effects. Recently, the therapeutic indices of certain anti-cancer drugs were further improved by attaching targeting ligands to nanodrugs (targeted-nanodrugs). Targeted-nanodrugs selectively bind to cognate receptors expressed on target cells and enter cells more efficiently than non-targeted formulations. Although these advancements have been sufficiently beneficial to place targeted-nanodrugs into clinical development for use in cancer therapy, they also come at a price. The addition of ligands to drug-loaded nanocarriers often leads to additional synthesis steps and costs, and more complex biological performance relative to ligand-devoid nanodrugs. Here, we will discuss the benefits and challenges facing the addition of targeting features to nanodrugs for cancer therapy.
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Covalent inhibition of the lymphoid tyrosine phosphatase.
ChemMedChem
PUBLISHED: 01-08-2014
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Covalent inhibitors of lymphoid tyrosine phosphatase (LYP) were identified from a screen of the NIH Molecular Libraries Small Molecules Repository (MLSMR). Both of the two lead compounds identified have phosphotyrosine-mimetic benzoic acid moieties as well as electrophilic acrylonitrile groups. Inhibition kinetics of both compounds are consistent with covalent modification of the enzyme, with nanomolar KI and reciprocal millisecond kinact values, representing the best efficiency ratios (kinact /KI ) among currently reported covalent LYP inhibitors. Covalent inhibitors can provide longer efficacy and better selectivity than more conventional noncovalent inhibitors, and these lead compounds are an important step toward the development of protein tyrosine phosphatase (PTP)-targeted covalent therapeutic compounds.
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Autoimmunity-associated LYP-W620 does not impair thymic negative selection of autoreactive T cells.
PLoS ONE
PUBLISHED: 01-01-2014
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A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.
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Tyrosine Phosphatase PTPN22: Multifunctional Regulator of Immune Signaling, Development, and Disease.
Annu. Rev. Immunol.
PUBLISHED: 12-18-2013
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Inheritance of a coding variant of the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is associated with increased susceptibility to autoimmunity and infection. Efforts to elucidate the mechanisms by which the PTPN22-C1858T variant modulates disease risk revealed that PTPN22 performs a signaling function in multiple biochemical pathways and cell types. Capable of both enzymatic activity and adaptor functions, PTPN22 modulates signaling through antigen and innate immune receptors. PTPN22 plays roles in lymphocyte development and activation, establishment of tolerance, and innate immune cell-mediated host defense and immunoregulation. The disease-associated PTPN22-R620W variant protein is likely involved in multiple stages of the pathogenesis of autoimmunity. Establishment of a tolerant B cell repertoire is disrupted by PTPN22-R620W action during immature B cell selection, and PTPN22-R620W alters mature T cell responsiveness. However, after autoimmune attack has initiated tissue injury, PTPN22-R620W may foster inflammation through modulating the balance of myeloid cell-produced cytokines. Expected final online publication date for the Annual Review of Immunology Volume 32 is March 21, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
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Epigenetics in rheumatoid arthritis: a primer for rheumatologists.
Curr Rheumatol Rep
PUBLISHED: 09-28-2013
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Epigenetic anomalies are emerging as key pathogenic features of rheumatoid arthritis (RA). The effect of epigenetics in RA ranges from contributing to complex disease mechanisms to identifying biomarkers for early diagnosis and response to therapy. This review focuses on three key epigenetic areas in RA, namely DNA methylation, histone modification, and expression and/or function of microRNAs. Epigenomics studies of DNA methylation have identified alterations of genome-wide DNA methylation in cells from patients with rheumatoid arthritis. Histone modification studies have focused on histone acetylation, which tends to be increased in RA. Preclinical studies show that inhibitors of histone deacetylases are effective in cellular and animal models of RA. Genome-wide and candidate microRNA surveys identified increased or reduced expression of selected microRNAs in rheumatoid arthritis. These microRNA are either pro or anti-inflammatory in multiple cell types or affect osteoclast physiology and the pathogenesis of bone erosion. Defining epigenetic contributions to the pathogenesis of RA, especially in combination with understanding genetic associations, could lead to novel therapy and a clearer understanding of disease risk.
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A potent and selective small-molecule inhibitor for the lymphoid-specific tyrosine phosphatase (LYP), a target associated with autoimmune diseases.
J. Med. Chem.
PUBLISHED: 06-06-2013
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Lymphoid-specific tyrosine phosphatase (LYP), a member of the protein tyrosine phosphatase (PTP) family of signaling enzymes, is associated with a broad spectrum of autoimmune diseases. Herein we describe our structure-based lead optimization efforts within a 6-hydroxy-benzofuran-5-carboxylic acid series culminating in the identification of compound 8b, a potent and selective inhibitor of LYP with a K(i) value of 110 nM and more than 9-fold selectivity over a large panel of PTPs. The structure of LYP in complex with 8b was obtained by X-ray crystallography, providing detailed information about the molecular recognition of small-molecule ligands binding LYP. Importantly, compound 8b possesses highly efficacious cellular activity in both T- and mast cells and is capable of blocking anaphylaxis in mice. Discovery of 8b establishes a starting point for the development of clinically useful LYP inhibitors for treating a wide range of autoimmune disorders.
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In vivo targeting of intratumor regulatory T cells using PEG-modified single-walled carbon nanotubes.
Bioconjug. Chem.
PUBLISHED: 05-22-2013
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Recent evidence regarding the role of regulatory T cells (Treg) in tumor development has suggested that the manipulation of Treg function selectively in the tumor microenvironment would be a desirable immunotherapy approach. Targeting intratumor immune populations would reduce side effects on peripheral healthy cells and increase antitumor efficacy of immunotherapies. However, no current approaches are available which enable selective in vivo targeting of intratumor Treg or other immune cell subpopulations. Herein, we investigated the ability of ligands against Treg-specific receptors to drive selective internalization of PEG-modified single-walled carbon nanotubes (PEG-SWCNTs) into Treg residing in the tumor microenvironment. We focused our attention on the glucocorticoid-induced TNFR-related receptor (GITR), as it showed higher overexpression on intratumor vs peripheral (i.e., splenic) Treg compared to other reported Treg-specific markers (folate receptor 4, CD103, and CD39). Ex vivo investigations showed that the Treg targeting efficiency and selectivity of PEG-SWCNTs depended on incubation time, dose, number of ligands per nanotube, and targeted surface marker. In vivo investigations showed that PEG-SWCNTs armed with GITR ligands targeted Treg residing in a B16 melanoma more efficiently then intratumor non-Treg or splenic Treg. The latter result was achieved by exploiting a combination of passive tumor targeting due to enhanced tumor vascular permeability, naturally increased intratumor Treg vs effector T cell (Teff) ratio, and active targeting of markers that are enriched in intratumor vs splenic Treg. We also found that PEG-SWCNTs loaded with GITR ligands were internalized by Treg through receptor-mediated endocytosis and transported into the cytoplasm and nucleus ex vivo and in vivo. This is the first example of intratumor immune cell targeting and we hope it will pave the way to innovative immunotherapies against cancer.
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Thiuram disulfides as pseudo-irreversible inhibitors of lymphoid tyrosine phosphatase.
ChemMedChem
PUBLISHED: 05-14-2013
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We screened a small library of thiuram disulfides for inhibition of lymphoid tyrosine phosphatase (LYP) activity. The parent thiuram disulfide, disulfiram, inhibited LYP activity in vitro and in Jurkat T?cells, whereas diethyldithiocarbamate failed to inhibit LYP at the concentrations tested. Compound 13, an N-(2-thioxothiazolidin-4-one) analogue, was found to be the most potent LYP inhibitor in this series, with an IC50 value of 3??M. Compound 13 inhibits LYP pseudo-irreversibly, as evidenced by the time-dependence of inhibition, with a K(i) value of 1.1??M and a k(inact) value of 0.004?s?¹. The inhibition of LYP by compound 13 could not be reversed significantly by incubation with glutathione or by prolonged dialysis, but could be partially reversed by incubation with dithiothreitol. Compound 13 also inhibited LYP activity in Jurkat T?cells.
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Substrate selection influences molecular recognition in a screen for lymphoid tyrosine phosphatase inhibitors.
Chembiochem
PUBLISHED: 05-01-2013
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Assay design is an important variable that influences the outcome of an inhibitor screen. Here, we have investigated the hypothesis that protein tyrosine phosphatase inhibitors with improved biological activity could be identified from a screen by using a biologically relevant peptide substrate, rather than traditional phosphotyrosine mimetic substrates. A 2000-member library of drugs and drug-like compounds was screened for inhibitors of lymphoid tyrosine phosphatase (LYP) by using both a peptide substrate (Ac-ARLIEDNE-pCAP-TAREG-NH?, peptide 1) and a small-molecule phosphotyrosine mimetic substrate (difluoromethyl umbelliferyl phosphate, DiFMUP). The results demonstrate that compounds that inhibited enzyme activity on the peptide substrate had greater biological activity than compounds that only inhibited enzyme activity on DiFMUP. Finally, epigallocatechin-3,5-digallate was identified as the most potent inhibitor of lymphoid tyrosine phosphatase activity to date, with an IC?? of 50 nM and significant activity in T-cells. Molecular docking simulations provided a first model for binding of this potent inhibitor to LYP; this will constitute the platform for ongoing lead optimization efforts.
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The autoimmunity-associated gene PTPN22 potentiates toll-like receptor-driven, type 1 interferon-dependent immunity.
Immunity
PUBLISHED: 04-24-2013
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Immune cells sense microbial products through Toll-like receptors (TLR), which trigger host defense responses including type 1 interferons (IFNs) secretion. A coding polymorphism in the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is a susceptibility allele for human autoimmune and infectious disease. We report that Ptpn22 selectively regulated type 1 IFN production after TLR engagement in myeloid cells. Ptpn22 promoted host antiviral responses and was critical for TLR agonist-induced, type 1 IFN-dependent suppression of inflammation in colitis and arthritis. PTPN22 directly associated with TNF receptor-associated factor 3 (TRAF3) and promotes TRAF3 lysine 63-linked ubiquitination. The disease-associated PTPN22W variant failed to promote TRAF3 ubiquitination, type 1 IFN upregulation, and type 1 IFN-dependent suppression of arthritis. The findings establish a candidate innate immune mechanism of action for a human autoimmunity "risk" gene in the regulation of host defense and inflammation.
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pCAP-based peptide substrates: The new tool in the box of tyrosine phosphatase assays.
Methods
PUBLISHED: 04-15-2013
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Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls. Novel peptide-based probes for protein tyrosine phosphatases are needed to replace phosphotyrosine peptides and accelerate the field of tyrosine phosphatase substrate profiling. Here we review a type of peptidic probe for tyrosine phosphatases, which is based on the incorporation of the phosphotyrosine-mimic phosphocoumaryl amino propionic acid (pCAP) into peptides. The resulting fluorogenic pCAP peptides are dephosphorylated by tyrosine phosphatases with similar efficiency as the homologous phosphotyrosine peptides. pCAP peptides outperform phosphotyrosine peptides, providing an assay that is as robust, sensitive and facile as the non-peptidic fluorogenic probes on the market. Finally the use of pCAP can expand the range of phosphatase assays, facilitating the investigation of multiphosphorylated peptides and providing an in-gel assay for phosphatase activity.
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Surface polyethylene glycol conformation influences the protein corona of polyethylene glycol-modified single-walled carbon nanotubes: potential implications on biological performance.
ACS Nano
PUBLISHED: 02-28-2013
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Investigation of the nanoparticle protein corona, the shell of plasma proteins formed around nanoparticles immediately after they enter the bloodstream, is a benchmark in the study of the applications of nanoparticles in all fields of medicine, from pharmacology to toxicology. We report the first investigation of the protein corona adsorbed onto single-walled carbon nanotubes modified with 2 kDa molecular weight polyethylene glycol chains [PEG(2k)-modified SWCNTs or PEG2-SWCNTs] by using a large-scale gel-based proteomics method on biological replicates. More than 240 plasma proteins were selected, and their differences were analyzed among PEG2-SWCNTs differing in surface charge and PEG conformation. The protein corona of PEG2-SWCNTs showed that coagulation proteins, immunoglobulins, apolipoproteins, and proteins of the complement system were among the proteins bound by PEG2-SWCNTs and that their recruitment was independent from the isoelectric point, molecular weight, total hydrophobicity, and number of polyaromatic residues of the proteins. Statistical analysis on protein relative abundance revealed that PEG conformation had a higher influence on the PEG2-SWCNTs protein corona repertoire than nanotube surface charge. PEG conformation also affected the biological performance of PEG2-SWCNTs. A change in PEG conformation from mushroom to mushroom-brush transition affected the competitive adsorption of the major constituents of the protein corona of PEG2-SWCNTs and promoted shorter blood circulation time, faster renal excretion, and higher relative spleen versus liver uptake of PEG2-SWCNTs. Our data suggest that the protein corona, along with steric stabilization, may mediate the action of PEG conformation on the pharmacokinetic profile of PEG-modified SWCNTs.
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Protein tyrosine phosphatase expression profile of rheumatoid arthritis fibroblast-like synoviocytes: a novel role of SH2 domain-containing phosphatase 2 as a modulator of invasion and survival.
Arthritis Rheum.
PUBLISHED: 01-10-2013
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The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS.
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PEG-modified carbon nanotubes in biomedicine: current status and challenges ahead.
Biomacromolecules
PUBLISHED: 09-21-2011
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Since their discovery at the end of the previous millennium, carbon nanotubes (CNTs) have been the object of thousands of papers describing their applications in fields ranging from physics to electronics, photonics, chemistry, biology, and medicine. The development of chemical approaches to modify their graphitic sidewalls enabled the generation of poly(ethylene glycol) (PEG)-modified CNTs and their exploration in multiple biomedical applications. Studies at the cellular and organism level revealed that PEG-modified CNTs have favorable pharmacokinetic and toxicology profiles. Recently, PEG-modified CNTs have been successfully tested in preclinical studies in the fields of oncology, neurology, vaccination, and imaging, suggesting that they are well suited for the generation of novel multifunctional nanodrugs. Here we will review published data about the application of PEG-modified CNTs as in vitro and in vivo therapeutic and imaging tools and describe what is known about the interaction between PEG-modified CNTs and biological systems. Although several pieces of the puzzle are still missing, we will also attempt to formulate a preliminary structure-function model for PEG-modified CNT cellular trafficking, disposition, and side effects.
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Novel association of acid phosphatase locus 1*C allele with systemic lupus erythematosus.
Hum. Immunol.
PUBLISHED: 07-07-2011
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The red cell acid phosphatase (ACP1) gene, which encodes a low-molecular-weight phosphotyrosine phosphatase, has been suggested as a common genetic factor of autoimmunity. In the present study, we aim to investigate the possible association of ACP1 with the susceptibility of systemic lupus erythematosus (SLE). A total of 1,546 SLE patients and 1,947 healthy individuals from 4 Caucasians populations were included in the present study. Four single-nucleotide polymorphisms (SNPs) were genotyped in this study: rs10167992, rs11553742, rs7576247, and rs3828329. ACP1*A, ACP1*B, and ACP1*C codominant ACP1 alleles were determined using 2 of the SNPs and analyzed. After the meta-analysis test was performed, a significant association of rs11553742*T was observed (p(pooled) = 0.005, odds ratios = 1.37 [1.10-1.70]), retaining significance after multiple testing was applied (p(FDR) = 0.019). Our data indicate for first time the association of rs11553742*T with increased susceptibility in SLE patients.
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Association of acid phosphatase locus 1*C allele with the risk of cardiovascular events in rheumatoid arthritis patients.
Arthritis Res. Ther.
PUBLISHED: 04-19-2011
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Acid phosphatase locus 1 (ACP1) encodes a low molecular weight phosphotyrosine phosphatase implicated in a number of different biological functions in the cell. The aim of this study was to determine the contribution of ACP1 polymorphisms to susceptibility to rheumatoid arthritis (RA), as well as the potential contribution of these polymorphisms to the increased risk of cardiovascular disease (CV) observed in RA patients.
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Discovery of a novel series of inhibitors of lymphoid tyrosine phosphatase with activity in human T cells.
J. Med. Chem.
PUBLISHED: 02-22-2011
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The lymphoid tyrosine phosphatase LYP, encoded by the PTPN22 gene, is a critical regulator of signaling in T cells and recently emerged as a candidate target for therapy of autoimmune diseases. Here, by library screening, we identified a series of noncompetitive inhibitors of LYP that showed activity in primary T cells. Kinetic analysis confirmed that binding of the compounds to the phosphatase is nonmutually exclusive with respect to a known bidentate competitive inhibitor. The mechanism of action of the lead inhibitor compound 4e was studied by a combination of hydrogen/deuterium-exchange mass spectrometry and molecular modeling. The results suggest that the inhibitor interacts critically with a hydrophobic patch located outside the active site of the phosphatase. Targeting of secondary allosteric sites is viewed as a promising yet unexplored approach to develop pharmacological inhibitors of protein tyrosine phosphatases. Our novel scaffold could be a starting point to attempt development of "nonactive site" anti-LYP pharmacological agents.
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Differential association of two PTPN22 coding variants with Crohns disease and ulcerative colitis.
Inflamm. Bowel Dis.
PUBLISHED: 02-01-2011
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The PTPN22 gene is an important risk factor for human autoimmunity. The aim of this study was to evaluate for the first time the role of the R263Q PTPN22 polymorphism in ulcerative colitis (UC) and Crohns disease (CD), and to reevaluate the association of the R620W PTPN22 polymorphism with both diseases.
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Carbon nanotube-based nanocarriers: the importance of keeping it clean.
J Nanosci Nanotechnol
PUBLISHED: 12-04-2010
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Nanotechnology-introduced materials have promising applications as nanocarriers for drugs, peptides, proteins and nucleic acids. Several studies showed that the geometry (shape and size) and chemical properties of nanoparticles affect the kinetics and pathways of cellular uptake and their intracellular trafficking and signaling. Accurate physico-chemical characterization of nanoparticles customarily precedes their use in cell biology and in vivo experiments. However, a fact that is easily overlooked is that nanomaterials decorated with organic matter or resuspended in aqueous buffers can be theoretically contaminated by fungal and bacterial microorganisms. While investigating the effects of extensively characterized PEGylated carbon nanotubes (PNTs) on T lymphocyte activation, we demonstrated bacterial contamination of PNTs, which correlated with low reproducibility and artifacts in cell signaling assays. Contamination and artifacts were easily eliminated by preparing the materials in sterile conditions. We propose that simple sterile preparation procedures should be adopted and sterility evaluation of nanoparticles should be customarily performed, prior to assessing nanoparticle intracellular internalization, trafficking and their effects on cells and entire organisms.
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Autoimmune-associated PTPN22 R620W variation reduces phosphorylation of lymphoid phosphatase on an inhibitory tyrosine residue.
J. Biol. Chem.
PUBLISHED: 06-09-2010
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A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for human autoimmunity. PTPN22 encodes the lymphoid tyrosine phosphatase (LYP), which forms a complex with the kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T single nucleotide polymorphism results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits a greatly reduced interaction with Csk and is a gain-of-function inhibitor of signaling. Here we show that LYP constitutively interacts with its substrate Lck in a Csk-dependent manner. T cell receptor-induced phosphorylation of LYP by Lck on an inhibitory tyrosine residue releases tonic inhibition of signaling by LYP. The R620W variation disrupts the interaction between Lck and LYP, leading to reduced phosphorylation of LYP, which ultimately contributes to gain-of-function inhibition of T cell signaling.
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Lymphoid tyrosine phosphatase and autoimmunity: human genetics rediscovers tyrosine phosphatases.
Semin Immunopathol
PUBLISHED: 01-10-2010
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A relatively large number of protein tyrosine phosphatases (PTPs) are known to regulate signaling through the T cell receptor (TCR). Recent human genetics studies have shown that several of these PTPs are encoded by major autoimmunity genes. Here, we will focus on the lymphoid tyrosine phosphatase (LYP), a critical negative modulator of TCR signaling encoded by the PTPN22 gene. The functional analysis of autoimmune-associated PTPN22 genetic variants suggests that genetic variability of TCR signal transduction contributes to the pathogenesis of autoimmunity in humans.
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Identifying potent, selective protein tyrosine phosphatase inhibitors from a library of Au(I) complexes.
J. Med. Chem.
PUBLISHED: 11-06-2009
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Therapeutic inhibition of protein tyrosine phosphatase activity is a compelling yet challenging approach to the treatment of human disease. Toward this end, a library of 40 gold complexes with the general formula R(3)P-Au-Cl was screened to identify novel inhibitors of PTP activity. The most promising inhibitor obtained for the lymphoid tyrosine phosphatase LYP, (2-pyridine)(Ph(2))P-Au-Cl, is one of the most potent and selective LYP inhibitors identified to date with an IC(50) of 1.5 +/- 0.3 microM, 10-fold selectivity for LYP over PTP-PEST, HePTP, and CD45 in vitro, and activity in cellular studies as well.
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Type 1 diabetes: evidence of interaction between ACP1 and ADA1 gene polymorphisms.
Med. Sci. Monit.
PUBLISHED: 10-01-2009
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ACP1 (acid phosphatase locus 1, a cytosolic low-molecular-weight phosphotyrosin phosphatase) and ADA1 (adenosine deaminase locus 1) are two polymorphic systems involved in immune reactions. Observed interactions at the biochemical and clinical levels between the two systems prompted this investigation of a possible interaction concerning susceptibility to type 1 diabetes.
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Evidence for sex-specific associations between variation in acid phosphatase locus 1 (ACP1) and insulin sensitivity in Mexican-Americans.
J. Clin. Endocrinol. Metab.
PUBLISHED: 07-21-2009
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Acid phosphatase locus 1 (ACP1) is a low molecular weight tyrosine phosphatase that has been shown to be an important regulator of insulin receptor signaling.
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Gold(I) phosphine mediated selective inhibition of lymphoid tyrosine phosphatase.
J. Inorg. Biochem.
PUBLISHED: 07-10-2009
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Selective protein tyrosine phosphatase (PTP) inhibition is often difficult to achieve owing to the high degree of similarity of the catalytic domains of this family of enzymes. Selective inhibitors of the lymphoid specific tyrosine phosphatase, LYP, are of great interest due to the involvement of LYP in several autoimmune disorders. This manuscript describes a study into the mechanistic details of selective LYP inhibition by a Au(I)-phosphine complex. The complex, [Au((CH(2)CH(2)CN)(2)PPh)Cl], selectively inhibits LYP activity both in vitro and in cells, but does not inhibit other T-cell derived PTPs including the highly homologous PTP-PEST. The mode of inhibition was probed by investigating inhibition of LYP, the LYP mutant C129/231S, and PTP-PEST. Inhibition of LYP and PTP-PEST was competitive, while the LYP double mutant appeared mixed. Wild-type LYP was inhibited more potently than LYP C129/231S, indicating an important role for at least one of these residues in Au(I) binding. Coordination of Au(I) by both the active site cysteine residue as well as either Cys129 or 231 is suggested as a potential mechanism for LYP selective inhibition.
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Regulation of lymphoid tyrosine phosphatase activity: inhibition of the catalytic domain by the proximal interdomain.
Biochemistry
PUBLISHED: 07-10-2009
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The lymphoid tyrosine phosphatase LYP, encoded by the PTPN22 gene, recently emerged as a major player and candidate drug target for human autoimmunity. The enzyme includes a classical N-terminal protein tyrosine phosphatase catalytic domain and a C-terminal PEST-enriched domain, separated by an approximately 300-amino acid interdomain. Little is known about the regulation of LYP. Herein, by analysis of serial truncation mutants of LYP, we show that the phosphatase activity is strongly inhibited by protein regions C-terminal to the catalytic domain. We mapped the minimal inhibitory region to the proximal portion of the interdomain. We show that the activity of LYP is inhibited by an intramolecular mechanism, whereby the proximal portion of the interdomain directly interacts with the catalytic domain and reduces its activity.
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Crystal structure of the human lymphoid tyrosine phosphatase catalytic domain: insights into redox regulation .
Biochemistry
PUBLISHED: 04-18-2009
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The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, recently emerged as an important risk factor and drug target for human autoimmunity. Here we solved the structure of the catalytic domain of LYP, which revealed noticeable differences with previously published structures. The active center with a semi-closed conformation binds a phosphate ion, which may represent an intermediate conformation after dephosphorylation of the substrate but before release of the phosphate product. The structure also revealed an unusual disulfide bond formed between the catalytic Cys and one of the two Cys residues nearby, which is not observed in previously determined structures. Our structural and mutagenesis data suggest that the disulfide bond may play a role in protecting the enzyme from irreversible oxidation. Surprisingly, we found that the two noncatalytic Cys around the active center exert an opposite yin-yang regulation on the catalytic Cys activity. These detailed structural and functional characterizations have provided new insights into autoregulatory mechanisms of LYP function.
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Less air pollution leads to rapid reduction of airway inflammation and improved airway function in asthmatic children.
Pediatrics
PUBLISHED: 03-04-2009
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Air pollution can promote airway inflammation, posing significant health risks for children with chronic respiratory problems. However, it is unknown whether this process is reversible, so that limiting pollution will benefit these children. We measured the short-term response of allergic asthmatic children exposed to a real-life reduction in outdoor air pollution by using noninvasive biomarkers of airway inflammation and function.
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A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus.
Hum. Mol. Genet.
PUBLISHED: 02-28-2009
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A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.
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Conjugation of antisense oligonucleotides to PEGylated carbon nanotubes enables efficient knockdown of PTPN22 in T lymphocytes.
Bioconjug. Chem.
PUBLISHED: 02-27-2009
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PEGylated-carbon nanotubes (PNTs) were evaluated as nanocarriers of antisense oligonucleotides into T-cells using protein tyrosine phosphatase N22 (PTPN22) as a model target gene. PTPN22 is an important predisposing gene and drug target in type 1 diabetes and several other human autoimmune diseases. Here, we generated the first anti-PTPN22 20-mer antisense oligonucleotides (ASOs) and tethered them to PNTs through a cleavable disulfide bond. Spectroscopic and atomic force microscopy analyses were used to determine the loading of ASO onto PNTs, whereas the cleavable nature of the disulfide bond connecting the oligonucleotide to the nanocarrier was confirmed by incubation with dithiothreitol followed by agarose gel electrophoresis. PNT-conjugated ASOs achieved efficient (>50%) knockdown of PTPN22 expression in T-lymphocytes in culture at the mRNA and protein level, as measured by quantitative real-time PCR and Western blotting, respectively. Considering the high biocompatibility and low in vivo toxicity of PNTs, we expect that our approach will be easily translated to achieve in vivo knockdown of PTPN22 and other T lymphocyte targets, thus enabling novel ASO-mediated immunotherapies for type 1 diabetes and other autoimmune diseases.
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Duality of fibroblast-like synoviocytes in RA: passive responders and imprinted aggressors.
Nat Rev Rheumatol
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Rheumatoid arthritis (RA) is characterized by hyperplastic synovial pannus tissue, which mediates destruction of cartilage and bone. Fibroblast-like synoviocytes (FLS) are a key component of this invasive synovium and have a major role in the initiation and perpetuation of destructive joint inflammation. The pathogenic potential of FLS in RA stems from their ability to express immunomodulating cytokines and mediators as well as a wide array of adhesion molecule and matrix-modelling enzymes. FLS can be viewed as passive responders to the immunoreactive process in RA, their activated phenotype reflecting the proinflammatory milieu. However, FLS from patients with RA also display unique aggressive features that are autonomous and vertically transmitted, and these cells can behave as primary promoters of inflammation. The molecular bases of this imprinted aggressor phenotype are being clarified through genetic and epigenetic studies. The dual behaviour of FLS in RA suggests that FLS-directed therapies could become a complementary approach to immune-directed therapies in this disease. Pathophysiological characteristics of FLS in RA, as well as progress in targeting these cells, are reviewed in this manuscript.
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High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45.
Proc. Natl. Acad. Sci. U.S.A.
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Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157-177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107-137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors.
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Regulation of TCR signalling by tyrosine phosphatases: from immune homeostasis to autoimmunity.
Immunology
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More than half of the known protein tyrosine phosphatases (PTPs) in the human genome are expressed in T cells, and significant progress has been made in elucidating the biology of these enzymes in T-cell development and function. Here we provide a systematic review of the current understanding of the roles of PTPs in T-cell activation, providing insight into their mechanisms of action and regulation in T-cell receptor signalling, the phenotypes of their genetically modified mice, and their possible involvement in T-cell-mediated autoimmune disease. Our projection is that the interest in PTPs as mediators of T-cell homeostasis will continue to rise with further functional analysis of these proteins, and PTPs will be increasingly considered as targets of immunomodulatory therapies.
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PTPN22 alters the development of regulatory T cells in the thymus.
J. Immunol.
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PTPN22 encodes a tyrosine phosphatase that inhibits Src-family kinases responsible for Ag receptor signaling in lymphocytes and is strongly linked with susceptibility to a number of autoimmune diseases. As strength of TCR signal is critical to the thymic selection of regulatory T cells (Tregs), we examined the effect of murine PTPN22 deficiency on Treg development and function. In the thymus, numbers of pre-Tregs and Tregs increased inversely with the level of PTPN22. This increase in Tregs persisted in the periphery and could play a key part in the reduced severity observed in the PTPN22-deficient mice of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This could explain the lack of association of certain autoimmune conditions with PTPN22 risk alleles.
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Cellular Biochemistry Methods for Investigating Protein Tyrosine Phosphatases.
Antioxid. Redox Signal.
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Significance: The protein tyrosine phosphatases (PTPs) are a family of proteins that play critical roles in cellular signaling and affect many aspects of human health and disease. Although a wealth of information has been collected about PTPs since their discovery, many questions regarding their regulation and function still remain. Critical Issues: Of particular importance are the elucidation of the biological substrates of individual PTPs, and understanding of the chemical and biological basis for temporal and spatial resolution of PTP activity within a cell. Recent Advances: Drawing from recent advances in both biology and chemistry, innovative approaches have been developed to study the intracellular biochemistry and physiology of PTPs. We provide a summary of PTP-tailored techniques and approaches, emphasizing methodologies to study PTP activity within a cellular context. We first provide a discussion of methods for identifying PTP substrates, including substrate-trapping mutants and synthetic peptide libraries for substrate selectivity profiling. We next provide an overview of approaches for monitoring intracellular PTP activity, including a discussion of mechanistic-based probes, gel-based assays, substrates that can be used intracellularly, and assays tied to cell growth. Finally, we review approaches used for monitoring PTP oxidation, a key regulatory pathway for these enzymes, discussing the biotin switch method and variants of this approach, along with affinity trapping techniques and probes designed to detect PTP oxidation. Future Directions: Further development of approaches to investigate intracellular PTP activity and functions will provide specific insight into their mechanisms of action and control of diverse signaling pathways.
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