RESUMEN
Leukocyte antigen-related (LAR) phosphatase is a receptor-type protein tyrosine phosphatase involved in cellular signaling and associated with human disease including cancer and metabolic disorders. Selective inhibition of LAR phosphatase activity by well characterized and well validated small molecules would provide key insights into the roles of LAR phosphatase in health and disease, but identifying selective inhibitors of LAR phosphatase activity has been challenging. Recently, we described potent and selective inhibition of LAR phosphatase activity by the fungal natural product illudalic acid. Here we provide a detailed biochemical characterization of the adduct formed between LAR phosphatase and illudalic acid. A mass spectrometric analysis indicates that two cysteine residues are covalently labeled by illudalic acid and a related analog. Mutational analysis supports the hypothesis that inhibition of LAR phosphatase activity is due primarily to the adduct with the catalytic cysteine residue. A computational study suggests potential interactions between the illudalic acid moiety and the enzyme active site. Taken together, these data offer novel insights into the mechanism of inhibition of LAR phosphatase activity by illudalic acid.
Asunto(s)
Cumarinas , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Humanos , Cumarinas/química , Cumarinas/farmacología , Cisteína/química , Cisteína/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genéticaRESUMEN
Synaptic adhesion molecules are major organizers of the neuronal network and play a crucial role in the regulation of synapse development and maintenance in the brain. Synaptic adhesion-like molecules (SALMs) and leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-PTPs) are adhesion protein families with established synaptic function. Dysfunction of several synaptic adhesion molecules has been linked to cognitive disorders such as autism spectrum disorders and schizophrenia. A recent study of the binding and complex structure of SALM3 and PTPσ using small-angle X-ray scattering revealed a 2:2 complex similar to that observed for the interaction of human SALM5 and PTPδ. However, the molecular structure of the SALM3-PTPσ complex remains to be determined beyond the small-angle X-ray scattering model. Here, the expression, purification, crystallization and initial 6.5â Å resolution structure of the mouse SALM3-PTPσ complex are reported, which further verifies the formation of a 2:2 trans-heterotetrameric complex similar to the crystal structure of human SALM5-PTPδ and validates the architecture of the previously reported small-angle scattering-based solution structure of the SALM3-PTPσ complex. Details of the protein expression and purification, crystal optimization trials, and the initial structure solution and data analysis are provided.
Asunto(s)
Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Sinapsis , Animales , Cristalización , Cristalografía por Rayos X , Ratones , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Sinapsis/metabolismoRESUMEN
Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality.
Asunto(s)
Trastorno del Espectro Autista/genética , Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Secuencia de Aminoácidos , Animales , Trastorno del Espectro Autista/metabolismo , Escala de Evaluación de la Conducta , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/genética , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/genética , Dominios Proteicos , Empalme de Proteína , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Recombinantes , Transducción de Señal/genética , Transducción de Señal/fisiología , Conducta Social , Sinapsis/genéticaRESUMEN
Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed â¼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.
Asunto(s)
Ligandos , Mapas de Interacción de Proteínas/fisiología , Receptores de Superficie Celular/metabolismo , Receptor DCC/química , Receptor DCC/metabolismo , Humanos , Filogenia , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/clasificación , Receptores de Interleucina-1/química , Receptores de Interleucina-1/metabolismo , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/química , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Synaptic adhesion molecules play an important role in the formation, maintenance and refinement of neuronal connectivity. Recently, several leucine rich repeat (LRR) domain containing neuronal adhesion molecules have been characterized including netrin G-ligands, SLITRKs and the synaptic adhesion-like molecules (SALMs). Dysregulation of these adhesion molecules have been genetically and functionally linked to various neurological disorders. Here we investigated the molecular structure and mechanism of ligand interactions for the postsynaptic SALM3 adhesion protein with its presynaptic ligand, receptor protein tyrosine phosphatase σ (PTPσ). We solved the crystal structure of the dimerized LRR domain of SALM3, revealing the conserved structural features and mechanism of dimerization. Furthermore, we determined the complex structure of SALM3 with PTPσ using small angle X-ray scattering, revealing a 2:2 complex similar to that observed for SALM5. Solution studies unraveled additional flexibility for the complex structure, but validated the uniform mode of action for SALM3 and SALM5 to promote synapse formation. The relevance of the key interface residues was further confirmed by mutational analysis with cellular binding assays and artificial synapse formation assays. Collectively, our results suggest that SALM3 dimerization is a pre-requisite for the SALM3-PTPσ complex to exert synaptogenic activity.
Asunto(s)
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Sinapsis/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/química , Diferenciación Celular , Cristalografía por Rayos X , Análisis Mutacional de ADN , Drosophila , Fibronectinas/química , Glicosilación , Células HEK293 , Humanos , Ligandos , Ratones , Ratones Transgénicos , Monoéster Fosfórico Hidrolasas/química , Dominios Proteicos , Multimerización de Proteína , Dispersión de RadiaciónRESUMEN
The receptor-linked protein tyrosine phosphatases (RPTPs) are key regulators of cell-cell communication through the control of cellular phosphotyrosine levels. Most human RPTPs possess an extracellular receptor domain and tandem intracellular phosphatase domains: comprising an active membrane proximal (D1) domain and an inactive distal (D2) pseudophosphatase domain. Here we demonstrate that PTPRU is unique amongst the RPTPs in possessing two pseudophosphatase domains. The PTPRU-D1 displays no detectable catalytic activity against a range of phosphorylated substrates and we show that this is due to multiple structural rearrangements that destabilise the active site pocket and block the catalytic cysteine. Upon oxidation, this cysteine forms an intramolecular disulphide bond with a vicinal "backdoor" cysteine, a process thought to reversibly inactivate related phosphatases. Importantly, despite the absence of catalytic activity, PTPRU binds substrates of related phosphatases strongly suggesting that this pseudophosphatase functions in tyrosine phosphorylation by competing with active phosphatases for the binding of substrates.
Asunto(s)
Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Biocatálisis , Línea Celular , Disulfuros/metabolismo , Estabilidad de Enzimas , Humanos , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Dominios Proteicos , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Especificidad por SustratoRESUMEN
Leucocyte common antigen-related protein (LAR) is a post-synaptic type I transmembrane receptor protein that is important for neuronal functionality and is genetically coupled to neuronal disorders such as attention deficit hyperactivity disorder (ADHD). To understand the molecular function of LAR, structural and biochemical studies of protein fragments derived from the ectodomain of human LAR have been performed. The crystal structure of a fragment encompassing the first four FNIII domains (LARFN1-4) showed a characteristic L shape. SAXS data suggested limited flexibility within LARFN1-4, while rigid-body refinement of the SAXS data using the X-ray-derived atomic model showed a smaller angle between the domains defining the L shape compared with the crystal structure. The capabilities of the individual LAR fragments to interact with heparin was examined using microscale thermophoresis and heparin-affinity chromatography. The results showed that the three N-terminal immunoglobulin domains (LARIg1-3) and the four C-terminal FNIII domains (LARFN5-8) both bound heparin, while LARFN1-4 did not. The low-molecular-weight heparin drug Innohep induced a shift in hydrodynamic volume as assessed by size-exclusion chromatography of LARIg1-3 and LARFN5-8, while the chemically defined pentameric heparin drug Arixtra did not. Together, the presented results suggest the presence of an additional heparin-binding site in human LAR.
Asunto(s)
Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Tinzaparina/química , Sitios de Unión , Fondaparinux/química , Heparina , Humanos , Unión Proteica , Dominios ProteicosRESUMEN
Synapse formation is induced by transsynaptic interaction of neuronal cell-adhesion molecules termed synaptic organizers. Type IIa receptor protein tyrosine phosphatases (IIa RPTPs) function as presynaptic organizers. The cytoplasmic domain of IIa RPTPs consists of two phosphatase domains, and the membrane-distal one (D2) is essential for synapse formation. Liprin-α, which is an active zone protein critical for synapse formation, interacts with D2 via its C-terminal domain composed of three tandem sterile alpha motifs (tSAM). Structural mechanisms of this critical interaction for synapse formation remain elusive. Here, we report the crystal structure of the complex between mouse PTPδ D2 and Liprin-α3 tSAM at 1.91 Å resolution. PTPδ D2 interacts with the N-terminal helix and the first and second SAMs (SAM1 and SAM2, respectively) of Liprin-α3. Structure-based mutational analyses in vitro and in cellulo demonstrate that the interactions with Liprin-α SAM1 and SAM2 are essential for the binding and synaptogenic activity.
Asunto(s)
Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Animales , Cristalización , Ratones , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Sinapsis/genética , Sinapsis/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Multiple sclerosis (MS) is an autoimmune disease characterized by myelin and axonal damage in the central nervous system (CNS). Glial scar which is a hallmark of MS contains repair inhibitory molecules including chondroitin sulfate proteoglycans (CSPGs). CSPGs inhibit repair of damaged area through various receptors including protein tyrosine phosphatase sigma (PTPσ). In the current study we use intracellular sigma peptide (ISP), an inhibitor of PTPσ signaling, in LPC-induced focal demyelination of mouse optic chiasm. ISP treatment resulted in decreased demyelination, reduced astrogliosis, and increased newly generated oligodendrocytes which subsequently led to enhanced remyelination. Analyzing of electrophysiological (as performed by visual evoked potential recording) and behavioral (performed by visual cliff test) outcomes showed that ISP-treatment improved the integrity of optic pathway as well as the visual acuity. When ISP was administrated only during the repair phase, histological, electrophysiological and behavioral studies showed its regenerative effect. Our results demonstrated the possibility of using ISP as a new strategy to inhibit PTPσ for myelin protection, myelin repair in demyelinated axons, and functional neural pathway conductivity restoration in patients suffering from MS.
Asunto(s)
Esclerosis Múltiple/tratamiento farmacológico , Vaina de Mielina/metabolismo , Quiasma Óptico/metabolismo , Péptidos/uso terapéutico , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/antagonistas & inhibidores , Animales , Potenciales Evocados Visuales , Masculino , Ratones , Ratones Endogámicos C57BL , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Quiasma Óptico/efectos de los fármacos , Quiasma Óptico/fisiología , Péptidos/farmacología , Unión Proteica , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismoRESUMEN
Identifying the variants that alter protein function is a promising strategy for deciphering the biological consequences of somatic mutations during tumorigenesis, which could provide novel targets for the development of cancer therapies. Here, based on our previously developed method, we present a strategy called AlloDriver that identifies cancer driver genes/proteins as possible targets from mutations. AlloDriver utilizes structural and dynamic features to prioritize potentially functional genes/proteins in individual cancers via mapping mutations generated from clinical cancer samples to allosteric/orthosteric sites derived from three-dimensional protein structures. This strategy exhibits desirable performance in the reemergence of known cancer driver mutations and genes/proteins from clinical samples. Significantly, the practicability of AlloDriver to discover novel cancer driver proteins in head and neck squamous cell carcinoma (HNSC) was tested in a real case of human protein tyrosine phosphatase, receptor type K (PTPRK) through a L1143F driver mutation located at the allosteric site of PTPRK, which was experimentally validated by cell proliferation assay. AlloDriver is expected to help to uncover innovative molecular mechanisms of tumorigenesis by perturbing proteins and to discover novel targets based on cancer driver mutations. The AlloDriver is freely available to all users at http://mdl.shsmu.edu.cn/ALD.
Asunto(s)
Algoritmos , Carcinoma de Células Escamosas/química , Neoplasias de Cabeza y Cuello/química , Proteínas de Neoplasias/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Programas Informáticos , Regulación Alostérica , Sitio Alostérico , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular , Descubrimiento de Drogas , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Internet , Terapia Molecular Dirigida , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismoRESUMEN
SCI is followed by dramatic upregulation of chondroitin sulfate proteoglycans (CSPGs) which limit axonal regeneration, oligodendrocyte replacement and remyelination. The recent discovery of the specific CSPGs signaling receptor protein tyrosine phosphatase sigma (RPTPσ) provided an opportunity to refine the therapeutic approach to overcome CSPGs inhibitory actions. In previously published work, subcutaneous (s.c.) delivery of 44⯵g/day of a peptide mimetic of PTPσ called intracellular sigma peptide (ISP), which binds to PTPσ and blocks CSPG-mediated inhibition, facilitated recovery after contusive SCI. Since this result could be of great interest for clinical trials, we independently repeated this study, but modified the method of injury as well as peptide application and the dosage. Following SCI at the Th10-segment, 40 rats were distributed in 3 groups. Animals in group 1 (20 rats) were subjected to SCI, but received no treatment. Rats in group 2 were treated with intraperitoneal (i.p.) injections of 44⯵g/day ISP (SCIâ¯+â¯ISP44) and animals of group 3 with s.c. injections of 500⯵g/day ISP (SCIâ¯+â¯ISP500) for 7â¯weeks after lesioning. Recovery was analyzed at 1, 3, 6, 9 and 12â¯weeks after SCI by determining (i) BBB-score, (ii) foot-stepping angle, (iii) rump-height index, (iv) number of correct ladder steps, (v) bladder score and (vi) sensitivity (withdrawal latency after thermal stimulus). Finally, we determined the amount of serotonergic fibers in the preserved neural tissue bridges (PNTB) around the lesion site. Our results show that, systemic therapy with ISP improved locomotor, sensory and vegetative recovery which correlated with more spared serotonergic fibers in PNTB.
Asunto(s)
Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/fisiopatología , Locomoción/efectos de los fármacos , Péptidos/uso terapéutico , Desempeño Psicomotor/efectos de los fármacos , Ratas , Ratas Wistar , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Recuperación de la Función/efectos de los fármacos , Serotonina/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Factores de Tiempo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/fisiopatologíaRESUMEN
Leukocyte common antigen-related protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that regulate neurite outgrowth and neuronal regeneration. LAR-RPTPs have also received particular attention as the major presynaptic hubs for synapse organization through selective binding to numerous postsynaptic adhesion partners. Recent structural studies on LAR-RPTP-mediated trans-synaptic adhesion complexes have provided significant insight into the molecular basis of their specific interactions, the key codes for their selective binding, as well as the higher-order clustering of LAR-RPTPs necessary for synaptogenic activity. In this review, we summarize the structures of LAR-RPTPs in complex with various postsynaptic adhesion partners and discuss the molecular mechanisms underlying LAR-RPTP-mediated synaptogenesis.
Asunto(s)
Neurogénesis , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Sinapsis/metabolismo , Animales , Humanos , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Leukocyte common antigen-related (LAR) protein is one of the type IIa receptor protein tyrosine phosphatases (RPTPs) that are important for signal transduction in biological processes, including axon growth and regeneration. Glycosaminoglycan chains, including heparan sulfate (HS) and chondroitin sulfate (CS), act as ligands that regulate LAR signaling. Here, we report the structural characterization of the first two immunoglobulin domains (Ig1-2) of LAR interacting with an HS pentasaccharide (GlcNS6S-GlcA-GlcNS3,6S-IdoA2S-GlcNS6S-OME, fondaparinux) using multiple solution-based NMR methods. In the course of the study, we extended an assignment strategy useful for sparsely labeled proteins expressed in mammalian cell culture supplemented with a single type of isotopically enriched amino acid ([15N]-Lys in this case) by including paramagnetic perturbations to NMR resonances. The folded two-domain structure for LAR-Ig1-2 seen in previous crystal structures has been validated in solution using residual dipolar coupling data, and a combination of chemical shift perturbation on titration of LAR-Ig1-2 with fondaparinux, saturation transfer difference (STD) spectra, and transferred nuclear Overhauser effects (trNOEs) have been employed in the docking program HADDOCK to generate models for the LAR-fondaparinux complex. These models are further analyzed by postprocessing energetic analysis to identify key binding interactions. In addition to providing insight into the ligand interaction mechanisms of type IIa RPTPs and the origin of opposing effects of CS and HS ligands, these results may assist in future design of therapeutic compounds for nervous system repair.
Asunto(s)
Heparitina Sulfato/química , Simulación del Acoplamiento Molecular , Polisacáridos/química , Pliegue de Proteína , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Cristalografía por Rayos X , Fondaparinux , Células HEK293 , Humanos , Resonancia Magnética Nuclear Biomolecular , Dominios ProteicosRESUMEN
Receptor type of protein tyrosine phosphatase sigma (RPTPσ) functions as a glycosaminoglycan (GAG) receptor of neuronal cells in both the central and peripheral nervous systems. Both chondroitin sulphate (CS) and heparan sulphate (HS) are important constituents of GAG ligands for RPTPσ, although they have opposite effects on neuronal cells. CS inhibits neurite outgrowth and neural regeneration through RPTPσ, whereas HS enhances them. We prepared recombinant RPTPσ N-terminal fragment containing the GAG binding site and various types of biotin-conjugated GAG (CS and HS) with chemical modification and chemo-enzymatic synthesis. Then interaction of the RPTPσ N-terminal fragment was analysed using GAG-biotin immobilized on streptavidin sensor chips by surface plasmon resonance. Interaction of RPTPσ with the CS library was highly correlated to the degree of disulphated disaccharide E unit, which had two sulphate groups at C-4 and C-6 positions of the N-acetylgalactosamine residue (CSE). The optimum molecular mass of CSE was suggested to be approximately 10 kDa. Heparin showed higher affinity to RPTPσ than the CS library. Our GAG library will not only contribute to the fields of carbohydrate science and cell biology, but also provide medical application to regulate neural regeneration.
Asunto(s)
Glicosaminoglicanos/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Animales , Conformación de Carbohidratos , Sulfatos de Condroitina/farmacología , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/farmacología , Humanos , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismoRESUMEN
Synapse formation is triggered by trans-synaptic interactions of cell adhesion molecules, termed synaptic organizers. Three members of type-II receptor protein tyrosine phosphatases (classified as type-IIa RPTPs; PTPδ, PTPσ and LAR) are known as presynaptic organizers. Synaptic adhesion-like molecules (SALMs) have recently emerged as a family of postsynaptic organizers. Although all five SALM isoforms can bind to the type-IIa RPTPs, only SALM3 and SALM5 reportedly have synaptogenic activities depending on their binding. Here, we report the crystal structures of apo-SALM5, and PTPδ-SALM2 and PTPδ-SALM5 complexes. The leucine-rich repeat (LRR) domains of SALMs interact with the second immunoglobulin-like (Ig) domain of PTPδ, whereas the Ig domains of SALMs interact with both the second and third Ig domains of PTPδ. Unexpectedly, the structures exhibit the LRR-mediated 2:2 complex. Our synaptogenic co-culture assay using site-directed SALM5 mutants demonstrates that presynaptic differentiation induced by PTPδ-SALM5 requires the dimeric property of SALM5.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Sinapsis/metabolismo , Transmisión Sináptica , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismoRESUMEN
Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit the extension and regeneration of axons. However, the underlying mechanism of action remains poorly understood. Mitochondria and endoplasmic reticulum (ER) are functionally inter-linked organelles important to axon development and maintenance. We report that CSPGs impair the targeting of mitochondria and ER to the growth cones of chicken embryonic sensory axons. The effect of CSPGs on the targeting of mitochondria is blocked by inhibition of the LAR receptor for CSPGs. The regulation of the targeting of mitochondria and ER to the growth cone by CSPGs is due to attenuation of PI3K signaling, which is known to be downstream of LAR receptor activation. Dynactin is a required component of the dynein motor complex that drives the normally occurring retrograde evacuation of mitochondria from growth cones. CSPGs elevate the levels of p150Glu dynactin found in distal axons, and inhibition of the interaction of dynactin with dynein increased axon lengths on CSPGs. CSPGs decreased the membrane potential of mitochondria, and pharmacological inhibition of mitochondria respiration at the growth cone independent of manipulation of mitochondria positioning impaired axon extension. Combined inhibition of dynactin and potentiation of mitochondria respiration further increased axon lengths on CSPGs relative to inhibition of dynactin alone. These data reveal that the regulation of the localization of mitochondria and ER to growth cones is a previously unappreciated aspect of the effects of CSPGs on embryonic axons. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1351-1370, 2017.
Asunto(s)
Axones/ultraestructura , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/farmacología , Retículo Endoplásmico/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Acetilcarnitina/farmacología , Actinas/metabolismo , Amidas/farmacología , Animales , Células Cultivadas , Embrión de Pollo , Complejo Dinactina/metabolismo , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microtúbulos/metabolismo , Neuronas/citología , Neuronas/ultraestructura , Péptidos/farmacología , Piridinas/farmacología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Transducción de Señal/efectos de los fármacos , Complejo Vitamínico B/farmacologíaRESUMEN
Leukocyte common antigen-related receptor tyrosine phosphatases (LAR-RPTPs) have emerged as key players that organize various aspects of neuronal development, including axon guidance, neurite extension, and synapse formation and function. Recent research has highlighted the roles of LAR-RPTPs at neuronal synapses in mediating distinct synaptic adhesion pathways through interactions with a host of extracellular ligands and in governing a variety of intracellular signaling cascades through binding to various scaffolds and signaling proteins. In this chapter, we review and update current research progress on the extracellular ligands of LAR-RPTPs, regulation of their extracellular interactions by alternative splicing and heparan sulfates, and their intracellular signaling machineries. In particular, we review structural insights on complexes of LAR-RPTPs with their various ligands. These studies lend support to general molecular mechanisms underlying LAR-RPTP-mediated synaptic adhesion and signaling pathways.
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Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Sinapsis/metabolismo , Animales , Espacio Extracelular/metabolismo , Humanos , Espacio Intracelular/metabolismo , Modelos Biológicos , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Transducción de SeñalRESUMEN
BACKGROUND/AIMS: Protein tyrosine phosphatases are crucial enzymes controlling numerous physiological and pathophysiological events and can be regulated by oxidation of the catalytic domain cysteine residue. Peracids are highly oxidizing compounds, and thus may induce inactivation of PTPs. The aim of the present study was to evaluate the inhibitory effect of peracids with different length of hydrocarbon chain on the activity of selected PTPs. METHODS: The enzymatic activity of human CD45, PTP1B, LAR, bacterial YopH was assayed under the cell-free conditions, and activity of cellular CD45 in human Jurkat cell lysates. The molecular docking and molecular dynamics were performed to evaluate the peracids binding to the CD45 active site. RESULTS: Here we demonstrate that peracids reduce enzymatic activity of recombinant CD45, PTP1B, LAR, YopH and cellular CD45. Our studies indicate that peracids are more potent inhibitors of CD45 than hydrogen peroxide (with an IC50 value equal to 25 nM for peroctanoic acid and 8 µM for hydrogen peroxide). The experimental data show that the inactivation caused by peracids is dependent on hydrocarbon chain length of peracids with maximum inhibitory effect of medium-chain peracids (C8-C12 acyl chain), which correlates with calculated binding affinities to the CD45 active site. CONCLUSION: Peracids are potent inhibitors of PTPs with the strongest inhibitory effect observed for medium-chain peracids.
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Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Antígenos Comunes de Leucocito/antagonistas & inhibidores , Peróxidos/química , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/química , Dominio Catalítico , Extractos Celulares/química , Pruebas de Enzimas , Humanos , Peróxido de Hidrógeno/química , Células Jurkat , Cinética , Antígenos Comunes de Leucocito/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Ácido Peracético/química , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Recombinantes/químicaRESUMEN
Selective binding between pre- and postsynaptic adhesion molecules can induce synaptic differentiation. Here we report the crystal structure of a synaptogenic trans-synaptic adhesion complex between Slit and Trk-like family member 2 (Slitrk2) and receptor protein tyrosine phosphatase (RPTP) δ. The structure and site-directed mutational analysis revealed the structural basis of splicing-dependent adhesion between Slitrks and type IIa RPTPs for inducing synaptic differentiation.
Asunto(s)
Modelos Moleculares , Conformación Proteica , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Sinapsis/fisiología , Animales , Sitios de Unión , Humanos , Ratones , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Relación Estructura-ActividadRESUMEN
Contusive spinal cord injury leads to a variety of disabilities owing to limited neuronal regeneration and functional plasticity. It is well established that an upregulation of glial-derived chondroitin sulphate proteoglycans (CSPGs) within the glial scar and perineuronal net creates a barrier to axonal regrowth and sprouting. Protein tyrosine phosphatase σ (PTPσ), along with its sister phosphatase leukocyte common antigen-related (LAR) and the nogo receptors 1 and 3 (NgR), have recently been identified as receptors for the inhibitory glycosylated side chains of CSPGs. Here we find in rats that PTPσ has a critical role in converting growth cones into a dystrophic state by tightly stabilizing them within CSPG-rich substrates. We generated a membrane-permeable peptide mimetic of the PTPσ wedge domain that binds to PTPσ and relieves CSPG-mediated inhibition. Systemic delivery of this peptide over weeks restored substantial serotonergic innervation to the spinal cord below the level of injury and facilitated functional recovery of both locomotor and urinary systems. Our results add a new layer of understanding to the critical role of PTPσ in mediating the growth-inhibited state of neurons due to CSPGs within the injured adult spinal cord.