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1.
FEBS Open Bio ; 12(7): 1388-1405, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35478300

RESUMEN

Neuroblastoma (NB) is a heterogeneous cancer of the sympathetic nervous system, which accounts for 7-10% of paediatric malignancies worldwide. Due to the lack of targetable molecular aberrations in NB, most treatment options remain relatively nonspecific. Here, we investigated the therapeutic potential of BCI, an inhibitor of DUSP1 and DUSP6, in cultured NB cells. BCI was cytotoxic in a range of NB cell lines and induced a short-lived activation of the AKT and stress-inducible MAP kinases, although ERK phosphorylation was unaffected. Furthermore, a phosphoproteomic screen identified significant upregulation of JNK signalling components and suppression in mTOR and R6K signalling. To assess the specificity of BCI, CRISPR-Cas9 was employed to introduce insertions and deletions in the DUSP1 and DUSP6 genes. Surprisingly, BCI remained fully cytotoxic in NB cells with complete loss of DUSP6 and partial depletion of DUSP1, suggesting that BCI exerts cytotoxicity in NB cells through a complex mechanism that is unrelated to these phosphatases. Overall, these data highlight the risk of using an inhibitor such as BCI as supposedly specific DUSP1/6, without understanding its full range of targets in cancer cells.


Asunto(s)
Antineoplásicos , Fosfatasa 1 de Especificidad Dual , Fosfatasa 6 de Especificidad Dual , Neuroblastoma , Antineoplásicos/farmacología , Línea Celular Tumoral , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 6 de Especificidad Dual/genética , Humanos , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Fosforilación , Transducción de Señal
2.
Int J Mol Sci ; 22(2)2021 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-33466673

RESUMEN

Dual specificity phosphatases (DUSPs) play a crucial role in the regulation of intracellular signalling pathways, which in turn influence a broad range of physiological processes. DUSP malfunction is increasingly observed in a broad range of human diseases due to deregulation of key pathways, most notably the MAP kinase (MAPK) cascades. Dual specificity phosphatase 26 (DUSP26) is an atypical DUSP with a range of physiological substrates including the MAPKs. The residues that govern DUSP26 substrate specificity are yet to be determined; however, recent evidence suggests that interactions with a binding partner may be required for DUSP26 catalytic activity. DUSP26 is heavily implicated in cancer where, akin to other DUSPs, it displays both tumour-suppressive and -promoting properties, depending on the context. Here we review DUSP26 by evaluating its transcriptional patterns, protein crystallographic structure and substrate binding, as well as its physiological role(s) and binding partners, its role in human disease and the development of DUSP26 inhibitors.


Asunto(s)
Fosfatasas de Especificidad Dual/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Animales , Fosfatasas de Especificidad Dual/análisis , Fosfatasas de Especificidad Dual/genética , Humanos , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/análisis , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Conformación Proteica , Mapas de Interacción de Proteínas , Especificidad por Sustrato , Activación Transcripcional
3.
Adv Funct Mater ; 31(37): 2104843, 2021 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-35712226

RESUMEN

The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor-targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor-mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non-targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide-dependent indicating integrin-mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN-amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor-targeting, with minimal clearance by the liver and so enable delivery of tumor-targeted siRNA therapeutics.

4.
Sci Rep ; 10(1): 16660, 2020 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-33028860

RESUMEN

Oxidovanadium complexes with organic ligands are well known to have cytotoxic or differentiating capabilities against a range of cancer cell types. Their limited use in clinical testing though has resulted largely from uncertainties about the long-term toxicities of such complexes, due in part to the speciation to vanadate ions in the circulation. We hypothesised that more highly stable complexes, delivered using liposomes, may provide improved opportunities for oxidovanadium applications against cancer. In this study we sourced specifically hydrophobic forms of oxidovanadium complexes with the explicit aim of demonstrating liposomal encapsulation, bioavailability in cultured neuroblastoma cells, and effective cytotoxic or differentiating activity. Our data show that four ethanol-solubilised complexes with amine bisphenol, aminoalcohol bisphenol or salan ligands are equally or more effective than a previously used complex bis(maltolato)oxovanadium(V) in neuroblastoma cell lines. Moreover, we show that one of these complexes can be stably incorporated into cationic liposomes where it retains very good bioavailability, apparently low speciation and enhanced efficacy compared to ethanol delivery. This study provides the first proof-of-concept that stable, hydrophobic oxidovanadium complexes retain excellent cellular activity when delivered effectively to cancer cells with nanotechnology. This offers the improved prospect of applying oxidovanadium-based drugs in vivo with increased stability and reduced off-target toxicity.


Asunto(s)
Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Liposomas , Neuroblastoma/tratamiento farmacológico , Vanadatos/administración & dosificación , Línea Celular Tumoral , Humanos , Neuroblastoma/patología
5.
Nucleic Acid Ther ; 30(4): 237-248, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32240058

RESUMEN

Neuroblastoma (NB) is the most common solid tumor in childhood. Twenty percent of patients display MYCN amplification, which indicates a very poor prognosis. MYCN is a highly specific target for an NB tumor therapy as MYCN expression is absent or very low in most normal cells, while, as a transcription factor, it regulates many essential cell activities in tumor cells. We aim to develop a therapy for NB based on MYCN silencing by short interfering RNA (siRNA) molecules, which can silence target genes by RNA interference (RNAi), a naturally occurring method of gene silencing. It has been shown previously that MYCN silencing can induce apoptosis and differentiation in MYCN amplified NB. In this article, we have demonstrated that siRNA-mediated silencing of MYCN in MYCN-amplified NB cells induced neurogenesis in NB cells, whereas retinoic acid (RA) treatment did not. RA can differentiate NB cells and is used for treatment of residual disease after surgery or chemotherapy, but resistance can develop. In addition, MYCN siRNA treatment suppressed growth in a MYCN-amplified NB cell line more than that by RA. Our result suggests that gene therapy using RNAi targeting MYCN can be a novel therapy toward MYCN-amplified NB that have complete or partial resistance toward RA.


Asunto(s)
Silenciador del Gen/efectos de los fármacos , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/tratamiento farmacológico , ARN Interferente Pequeño/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteína Proto-Oncogénica N-Myc/antagonistas & inhibidores , Neuroblastoma/genética , Neuroblastoma/patología , Neurogénesis/efectos de los fármacos , Interferencia de ARN/efectos de los fármacos , ARN Interferente Pequeño/genética , Tretinoina/efectos adversos , Tretinoina/farmacología
6.
J Drug Target ; 28(6): 643-654, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-31903789

RESUMEN

Retinoid treatment is employed during residual disease treatment in neuroblastoma, where the aim is to induce neural differentiation or death in tumour cells. However, although therapeutically effective, retinoids have only modest benefits and suffer from poor pharmacokinetic properties. In vivo, retinoids induce CYP26 enzyme production in the liver, enhancing their own rapid metabolic clearance, while retinoid resistance in tumour cells themselves is considered to be due in part to increased CYP26 production. Retinoic acid metabolism blocking agents (RAMBAs), which inhibit CYP26 enzymes, can improve retinoic acid (RA) pharmacokinetics in pre-clinical neuroblastoma models. Here, we demonstrate that in cultured neuroblastoma tumour cells, RAMBAs enhance RA action as seen by morphological differentiation, AKT signalling and suppression of MYCN protein. Although active as retinoid enhancers, these RAMBAs are highly hydrophobic and their effective delivery in humans will be very challenging. Here, we demonstrate that such RAMBAs can be loaded efficiently into cationic liposomal particles, where the RAMBAs achieve good bioavailability and activity in cultured tumour cells. This demonstrates the efficacy of RAMBAs in enhancing retinoid signalling in neuroblastoma cells and shows for the first time that liposomal delivery of hydrophobic RAMBAs is a viable approach, providing novel opportunities for their delivery and application in humans.


Asunto(s)
Azoles/farmacología , Ácido Retinoico 4-Hidroxilasa/metabolismo , Tretinoina/agonistas , Tretinoina/metabolismo , Azoles/síntesis química , Línea Celular Tumoral , Supervivencia Celular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Liposomas , Neuroblastoma , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ácido Retinoico 4-Hidroxilasa/genética , Transducción de Señal
7.
Mol Syndromol ; 10(1-2): 98-114, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30976283

RESUMEN

Neural crest stem/progenitor cells (NCSCs) populate a variety of tissues, and their dysregulation is implicated in several human diseases including craniosynostosis and neuroblastoma. We hypothesised that small molecules that inhibit NCSC induction or differentiation may represent potential therapeutically relevant drugs in these disorders. We screened 640 FDA-approved compounds currently in clinical use for other conditions to identify those which disrupt development of NCSC-derived skeletal elements that form the zebrafish jaw. In the primary screen, we used heterozygous transgenic sox10:gfp zebrafish to directly visualise NCSC-derived jaw cartilage. We noted partial toxicity of this transgene in relation to jaw patterning, suggesting that our primary screen was sensitised for NCSC defects, and we confirmed 10 novel, 4 previously reported, and 2 functional analogue drug hits in wild-type embryos. Of these drugs, 9/14 and 7/14, respectively, are known to target pathways implicated in osteoarthritis pathogenesis or to cause reduced bone mineral density/increased fracture risk as side effects in patients treated for other conditions, suggesting that our screen enriched for pathways targeting skeletal tissue homeostasis. We selected one drug that inhibited NCSC induction and one drug that inhibits bone mineralisation for further detailed analyses which reflect our initial hypotheses. These drugs were leflunomide and cyclosporin A, respectively, and their functional analogues, teriflunomide and FK506 (tacrolimus). We identified their critical developmental windows of activity, showing that the severity of defects observed related to the timing, duration, and dose of treatment. While leflunomide has previously been shown to inhibit NCSC induction, we demonstrate additional later roles in cartilage remodelling. Both drugs altered expression of extracellular matrix metalloproteinases. As proof-of-concept, we also tested drug treatment of disease-relevant mammalian cells. While leflunomide treatment inhibited the viability of several human NCSC-derived neuroblastoma cell lines coincident with altered expression of genes involved in ribosome biogenesis and transcription, FK506 enhanced murine calvarial osteoblast differentiation and prevented fusion of the coronal suture in calvarial explants taken from Crouzon syndrome mice.

8.
Molecules ; 22(12)2017 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-29257048

RESUMEN

Phosphotyrosine signaling is regulated by the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Here we discuss the potential of vanadium derivatives as PTP enzyme inhibitors and metallotherapeutics. We describe how vanadate in the V oxidized state is thought to inhibit PTPs, thus acting as a pan-inhibitor of this enzyme superfamily. We discuss recent developments in the biological and biochemical actions of more complex vanadium derivatives, including decavanadate and in particular the growing number of oxidovanadium compounds with organic ligands. Pre-clinical studies involving these compounds are discussed in the anti-diabetic and anti-cancer contexts. Although in many cases PTP inhibition has been implicated, it is also clear that many such compounds have further biochemical effects in cells. There also remain concerns surrounding off-target toxicities and long-term use of vanadium compounds in vivo in humans, hindering their progress through clinical trials. Despite these current misgivings, interest in these chemicals continues and many believe they could still have therapeutic potential. If so, we argue that this field would benefit from greater focus on improving the delivery and tissue targeting of vanadium compounds in order to minimize off-target toxicities. This may then harness their full therapeutic potential.


Asunto(s)
Antineoplásicos/farmacología , Hipoglucemiantes/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Compuestos de Vanadio/farmacología , Animales , Antineoplásicos/química , Humanos , Hipoglucemiantes/química , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal , Compuestos de Vanadio/química
9.
Semin Cell Dev Biol ; 37: 90-7, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25234542

RESUMEN

Receptor-like protein tyrosine phosphatases represent a large protein family related to cell adhesion molecules, with diverse roles throughout neural development in vertebrates and invertebrates. This review focuses on their roles in axon growth, guidance and repair, as well as more recent findings demonstrating their key roles in pre-synaptic and post-synaptic maturation and function. These enzymes have been linked to memory and neuropsychiatric defects in loss-of-function rodent models, highlighting their potential as future drug targets.


Asunto(s)
Axones/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Sinapsis/metabolismo , Animales , Humanos , Invertebrados/metabolismo , Regeneración Nerviosa , Vertebrados/metabolismo
10.
Cancer Lett ; 357(1): 316-327, 2015 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-25444896

RESUMEN

In a wide range of neuroblastoma-derived lines oxovanadium compounds such as bis(maltolato)oxovanadium(IV) (BMOV) are cytotoxic. This is not explained by oxidative stress or inhibition of ion channels. Genotoxicity is unlikely given that a p53 response is absent and p53-mutant lines are also sensitive. Cytotoxicity is inhibited by N-acetyl cysteine and glutathione ester, indicating that BMOV action is sensitive to cytoplasmic redox and thiol status. Significantly, combining BMOV with glutathione synthesis inhibition greatly enhances BMOV-induced cell death. This combination treatment triggers high AKT pathway activation, highlighting the potential functional importance of PTP inhibition by BMOV. AKT activation itself, however, is not required for cytotoxicity. Oxovanadium compounds may thus represent novel leads as p53-independent therapeutics for neuroblastoma.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Butionina Sulfoximina/farmacología , Neuroblastoma/tratamiento farmacológico , Pironas/farmacología , Vanadatos/farmacología , Animales , Butionina Sulfoximina/administración & dosificación , Línea Celular Tumoral , Sinergismo Farmacológico , Fibroblastos/efectos de los fármacos , Humanos , Ratones , Neuroblastoma/metabolismo , Oxidación-Reducción , Pironas/administración & dosificación , Transducción de Señal , Transfección , Vanadatos/administración & dosificación
11.
Nat Commun ; 5: 5209, 2014 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-25385546

RESUMEN

Receptor protein tyrosine phosphatase sigma (RPTPσ) regulates neuronal extension and acts as a presynaptic nexus for multiple protein and proteoglycan interactions during synaptogenesis. Unknown mechanisms govern the shift in RPTPσ function, from outgrowth promotion to synaptic organization. Here, we report crystallographic, electron microscopic and small-angle X-ray scattering analyses, which reveal sufficient inter-domain flexibility in the RPTPσ extracellular region for interaction with both cis (same cell) and trans (opposite cell) ligands. Crystal structures of RPTPσ bound to its postsynaptic ligand TrkC detail an interaction surface partially overlapping the glycosaminoglycan-binding site. Accordingly, heparan sulphate and heparin oligomers compete with TrkC for RPTPσ binding in vitro and disrupt TrkC-dependent synaptic differentiation in neuronal co-culture assays. We propose that transient RPTPσ ectodomain emergence from the presynaptic proteoglycan layer allows capture by TrkC to form a trans-synaptic complex, the consequent reduction in RPTPσ flexibility potentiating interactions with additional ligands to orchestrate excitatory synapse formation.


Asunto(s)
Proteínas de la Matriz Extracelular/fisiología , Neurogénesis/fisiología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/fisiología , Sinapsis/fisiología , Animales , Diferenciación Celular/fisiología , Embrión de Pollo , Técnicas de Cocultivo , Cristalización , Proteínas de la Matriz Extracelular/química , Humanos , Ligandos , Ratones , Neuronas/citología , Neuronas/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Proteoglicanos/química , Proteoglicanos/fisiología , Receptor trkC/química , Receptor trkC/fisiología , Transducción de Señal/fisiología
12.
Int J Dev Neurosci ; 34: 48-59, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24491805

RESUMEN

Receptor-type protein tyrosine phosphatases (RPTPs) have been implicated as direct or indirect regulators of neurotrophin receptors (TRKs). It remains less clear if and how such RPTPs might regulate TRK proteins in vivo during development. Here we present a comparative expression profile of RPTP genes and Trk genes during early stages of murine, dorsal root ganglion maturation. We find little if any specific, temporal mRNA co-regulation between individual RPTP and Ntrk genes between E12.5 and E14.5. Moreover, a double fluorescent in-situ hybridization and immunofluorescence study of seven Rptp genes with Ntrks revealed widespread co-expression of RPTPs in individual neurons, but no tight correlation with Trk expression profiles. No Rptp is expressed in 100% of Ntrk1-expressing neurons, whereas at least 6 RPTPs are expressed in 100% of Ntrk2- and Ntrk3-expressing neurons. An exception is Ptpro, which showed very selective expression. Short hairpin RNA suppression of Ptprf, Ptprs or Ptpro in primary, E13.5 DRG neurons did not alter TRK signalling. We therefore propose that TRK signalling may not be simply dependent on rate-limiting regulation by individual RPTP subtypes during sensory neuron development. Instead, TRK signalling has the potential to be buffered by concurrent inputs from several RPTPs in individual neurons.


Asunto(s)
Ganglios Espinales/citología , Ganglios Espinales/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Células Receptoras Sensoriales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células Cultivadas , Embrión de Mamíferos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Proteína Reguladora Asociada a mTOR , Transducción de Señal/genética , Transfección
13.
Hum Mol Genet ; 22(R1): R66-76, 2013 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-23900072

RESUMEN

Protein tyrosine phosphatases (PTPs) constitute a family of key homeostatic regulators, with wide implications on physiology and disease. Recent findings have unveiled that the biological activity of PTPs goes beyond the dephosphorylation of phospho-proteins to shut down protein tyrosine kinase-driven signaling cascades. Substrates dephosphorylated by clinically relevant PTPs extend to phospholipids and phosphorylated carbohydrates as well. In addition, non-catalytic functions are also used by PTPs to regulate essential cellular functions. Consequently, PTPs have emerged as novel potential therapeutic targets for human diseases, including cancer predispositions, myopathies and neuropathies. In this review, we highlight recent advances on the multifaceted role of lipid-phosphatase PTPs in human pathology, with an emphasis on hereditary diseases. The involved PTP regulatory networks and PTP modulatory strategies with potential therapeutic application are discussed.


Asunto(s)
Enfermedades Musculares/enzimología , Neoplasias/enzimología , Enfermedades del Sistema Nervioso/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Fosfolípidos/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/química , Alineación de Secuencia , Transducción de Señal , Especificidad por Sustrato
14.
Cancer Lett ; 328(1): 44-54, 2013 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-23022267

RESUMEN

Retinoic acid (RA)-induced differentiation therapy is partially successful in neuroblastoma treatment. We found that a novel combination of vanadium-based PTP inhibitors with RA induced extensive differentiation in neuroblastoma cells. In contrast to RA alone, this led to either permanent differentiation or senescence after 14days of combined treatment followed by chemical removal. Senescence was dependent in part on synergistic AKT and ERK activation. p21 was also strongly induced, but in contrast to oncogene-induced senescence, p53 was not activated. Vanadium-based inhibitors thus serve strongly to enhance RA's ability to drive differentiation and a novel form of senescence in neuroblastoma cells.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Neuroblastoma/tratamiento farmacológico , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , Línea Celular Tumoral , Sinergismo Farmacológico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Neuroblastoma/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
15.
Int J Biol Sci ; 7(7): 978-91, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21850207

RESUMEN

Mouse protein tyrosine phosphatase PTPBR7 is a receptor-like, transmembrane protein that is localized on the surface of neuronal cells. Its protein phosphatase activity is reduced upon multimerization, and PTPBR7-deficient mice display motor coordination defects. Extracellular molecules that may influence PTPBR7 activity, however, remain to be determined. We here show that the PTPBR7 extracellular domain binds to highly myelinated regions in mouse brain, in particular the white matter tracks in cerebellum. PTPBR7 deficiency does not alter this binding pattern, as witnessed by RAP in situ staining of Ptprr⁻/⁻ mouse brain sections. Additional in situ and in vitro experiments also suggest that sugar moieties of heparan sulphate and chondroitin sulphate glycosaminoglycans are not critical for PTPBR7 binding. Candidate binding proteins were affinity-purified exploiting the PTPBR7 extracellular domain and identified by mass spectrometric means. Results support the suggested link between PTPRR isoforms and cerebellar calcium ion homeostasis, and suggest an additional role in the process of cell-cell adhesion.


Asunto(s)
Encéfalo/citología , Vaina de Mielina/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Fosfatasas Clase 7 Similares a Receptores/metabolismo , Animales , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Técnicas In Vitro , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem
16.
Mol Cell Neurosci ; 46(2): 469-82, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21112398

RESUMEN

During spinal cord development the proliferation, migration and survival of neural progenitors and precursors is tightly controlled, generating the fine spatial organisation of the cord. In order to understand better the control of these processes, we have examined the function of an orphan receptor protein tyrosine phosphatase (RPTP) PTPγ, in the developing chick spinal cord. Widespread expression of PTPγ occurs post-embryonic day 3 in the early cord and is consistent with a potential role in either neurogenesis or neuronal maturation. Using gain-of-function and loss-of-function approaches in ovo, we show that PTPγ perturbation significantly reduces progenitor proliferation rates and neuronal precursor numbers, resulting in hypoplasia of the neuroepithelium. PTPγ gain-of-function causes widespread suppression of Wnt/ß-catenin-driven TCF signalling. One potential target of PTPγ may therefore be ß-catenin itself, since PTPγ can dephosphorylate it in vitro, but alternative targets are also likely. PTPγ loss-of-function is not sufficient to alter TCF signalling. Instead, loss-of-function leads to increased apoptosis and defective cell-cell adhesion in progenitors and precursors. Furthermore, motor neuron precursor migration is specifically defective. PTPγ therefore regulates neurogenesis during a window of spinal cord development, with molecular targets most likely related to Wnt/ß-catenin signalling, cell survival and cell adhesion.


Asunto(s)
Neurogénesis/fisiología , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Transducción de Señal/fisiología , Médula Espinal/embriología , Médula Espinal/enzimología , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proliferación Celular , Embrión de Pollo , Electroporación , Immunoblotting , Hibridación in Situ , Neuronas Motoras/citología , Neuronas Motoras/enzimología , Células-Madre Neurales/citología , Células-Madre Neurales/enzimología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
18.
FEBS J ; 275(5): 816-30, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18298790

RESUMEN

Some 40-odd genes in mammals encode phosphotyrosine-specific, 'classical' protein tyrosine phosphatases. The generation of animal model systems and the study of various human disease states have begun to elucidate the important and diverse roles of protein tyrosine phosphatases in cellular signalling pathways, development and disease. Here, we provide an overview of those findings from mice and men, and indicate several novel approaches that are now being exploited to further our knowledge of this fascinating enzyme family.


Asunto(s)
Modelos Animales de Enfermedad , Enfermedad/etiología , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , Animales , Humanos , Sistema Inmunológico/enzimología , Ratones , Oncogenes , Fosforilación , Ratas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Tirosina/metabolismo
19.
Mol Cell Biol ; 27(5): 1795-808, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17178832

RESUMEN

Signaling through receptor protein tyrosine phosphatases (RPTPs) can influence diverse processes, including axon development, lymphocyte activation, and cell motility. The molecular regulation of these enzymes, however, is still poorly understood. In particular, it is not known if, or how, the dimerization state of RPTPs is related to the binding of extracellular ligands. Protein tyrosine phosphatase sigma (PTPsigma) is an RPTP with major isoforms that differ in their complements of fibronectin type III domains and in their ligand-binding specificities. In this study, we show that PTPsigma forms homodimers in the cell, interacting at least in part through the transmembrane region. Using this knowledge, we provide the first evidence that PTPsigma ectodomains must be presented as dimers in order to bind heterophilic ligands. We also provide evidence of how alternative use of fibronectin type III domain complements in two major isoforms of PTPsigma can alter the ligand binding specificities of PTPsigma ectodomains. The data suggest that the alternative domains function largely to change the rotational conformations of the amino-terminal ligand binding sites of the ectodomain dimers, thus imparting novel ligand binding properties. These findings have important implications for our understanding of how heterophilic ligands interact with, and potentially regulate, RPTPs.


Asunto(s)
Proteínas Aviares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Aviares/genética , Línea Celular , Embrión de Pollo , Pollos , Cisteína/metabolismo , Dimerización , Eliminación de Gen , Humanos , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes de Fusión/metabolismo , Sensibilidad y Especificidad , Homología de Secuencia de Aminoácido , Transfección
20.
FEBS J ; 273(20): 4668-81, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16995858

RESUMEN

Reversible tyrosine phosphorylation, catalyzed by receptor tyrosine kinases and receptor tyrosine phosphatases, plays an essential part in cell signaling during axonal development. Receptor protein tyrosine phosphatase-sigma has been implicated in the growth, guidance and repair of retinal axons. This phosphatase has also been implicated in motor axon growth and innervation. Insect orthologs of receptor protein tyrosine phosphatase-sigma are also implicated in the recognition of muscle target cells. A potential extracellular ligand for vertebrate receptor protein tyrosine phosphatase-sigma has been previously localized in developing skeletal muscle. The identity of this muscle ligand is currently unknown, but it appears to be unrelated to the heparan sulfate ligands of receptor protein tyrosine phosphatase-sigma. In this study, we have used affinity chromatography and tandem MS to identify nucleolin as a binding partner for receptor protein tyrosine phosphatase-sigma in skeletal muscle tissue. Nucleolin, both from tissue lysates and in purified form, binds to receptor protein tyrosine phosphatase-sigma ectodomains. Its expression pattern also overlaps with that of the receptor protein tyrosine phosphatase-sigma-binding partner previously localized in muscle, and nucleolin can also be found in retinal basement membranes. We demonstrate that a significant amount of muscle-associated nucleolin is present on the cell surface of developing myotubes, and that two nucleolin-binding components, lactoferrin and the HB-19 peptide, can block the interaction of receptor protein tyrosine phosphatase-sigma ectodomains with muscle and retinal basement membranes in tissue sections. These data suggest that muscle cell surface-associated nucleolin represents at least part of the muscle binding site for axonal receptor protein tyrosine phosphatase-sigma and that nucleolin may also be a necessary component of basement membrane binding sites of receptor protein tyrosine phosphatase-sigma.


Asunto(s)
Axones/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Embrión de Pollo , Lactoferrina/metabolismo , Ligandos , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/crecimiento & desarrollo , Péptidos/metabolismo , Unión Proteica , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Nucleolina
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