RESUMEN

Tex264 is an endoplasmic reticulum (ER) membrane protein that was recently demonstrated to act as an ER-phagy receptor under starvation conditions to mediate endoplasmic reticulum autophagy. However, how Tex264 functions in the central nervous system (CNS) and tumors is unclear. Here, we identified 89 proteins from the rat brain that may specifically interact with Tex264 and confirmed the interaction between sorting nexin 27 (SNX27) and Tex264 by coimmunoprecipitation and immunofluorescence. Our results indicated that Tex264 may promote recycling of membrane proteins from endosomes to the cell plasma membrane by recruiting SNX27 retromer vesicles. siRNA-mediated knockdown of TEX264 in HeLa cells did not affect cell proliferation but did significantly inhibit cell migration through a mechanism that may involve a reduction in SNX27-mediated Itgα5 receptor membrane recycling. Results of this study helped identify potential binding Tex264 partners and provide insights into Tex264 functions in the CNS and in tumors.

Asunto(s)

Endosomas , Nexinas de Clasificación , Animales , Membrana Celular/metabolismo , Movimiento Celular , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Células HeLa , Humanos , Proteínas del Tejido Nervioso/metabolismo , Transporte de Proteínas , Ratas , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismo

RESUMEN

PER1 is a core component of the internal time-keeping system. In the suprachiasmatic nucleus, it serves as the primary circadian pacemaker in mammalian brains. PER1 functions with other clock components to generate a feedback loop involving the transcriptional repression of gene expression to produce a circadian rhythm with an approximately 24-hour cycle. Post-transcriptional modifications (PTMs) are a basic regulatory mechanism that both perpetuate self-sustained oscillations and interpret metabolic input into circadian physiology by affecting factors such as protein stability, interactions, localization, and activity. Here we examined whether the serine/threonine protein kinase WNK3, which is expressed in a circadian rhythm, can interact and colocalize with PER1 in the SCN. In rats, WNK3 knockdown in the SCN is associated with altered sleep patterns. Moreover, WNK3 can phosphorylate PER1 to promote its degradation and is associated with circadian oscillations when PER1 is expressed in vitro.

RESUMEN

Programmed death ligand 1 (PD-L1) is a typical immune checkpoint protein, whose up-regulation on the membrane of different tumor cells inhibits the immune response of T cells and leads to the escape of tumor cells. In this work, we designed a facile and highly specific surface plasmon resonance (SPR) biosensor to detect PD-L1 in human plasma based on magnetite nanorods containing ordered mesocages (MNOM) and silver nanoclusters (AgNCs). Magneto-optical nanocomplex MNOM@AgNCs with superior magneto-optical properties and high signal-to-noise ratio were fabricated to improve the detection sensitivity owing to the high specific surface area of MNOM and excellent localized SPR of AgNCs. The PD-L1 Antibody on the surface of gold chip and the PD-L1 aptamer on MNOM@AgNCs could realize dual selective recognition of PD-L1, providing the specificity of the sensor and reducing non-specific binding. The SPR sensor showed a good linear range of PD-L1 from 10 ng/mL to 300 ng/mL with the detection limit of 3.29 ng/mL. The practical performance of this immunosensing platform had been successfully verified by clinical samples which included healthy donors and cancer patients. Based on the analysis, the developed immunosensor provided a new strategy for point-of-care detection of PD-L1 and could be used as clinical companion diagnosis of PD-1/PD-L1 inhibitor therapy.

Asunto(s)

Técnicas Biosensibles , Nanotubos , Antígeno B7-H1 , Óxido Ferrosoférrico , Humanos , Inmunoensayo , Plata

RESUMEN

Programmed death ligand 1 (PD-L1) immune checkpoint has been regarded as a new target for predicting cancer immunotherapy. As a transmembrane protein, PD-L1 has very low blood concentration and is likely to deplete their native activity when separated from the membrane environment due to significant hydrophobic domains, which make it difficult to measure sensitively. The reported PD-L1 aptamers and antibodies are both extracellular region binding molecules with the overlapping binding sites, which seriously limit with the construction of biosensor. Specific intracellular binding peptide (SIBP) as a unique PD-L1 intracellular region homing probe molecule is utilized for specifically capture targets. A simple and sensitive surface plasmon resonance (SPR) sandwich assay was constructed to detect serum soluble PD-L1 (sPD-L1) based on the unique and strong binding ability of SIBP to the intracellular region of sPD-L1. The designed SPR sensor showed great selectivity and wide dynamic response range of sPD-L1 concentration from 10 ng/mL to 2000 ng/mL. The limit of detection was calculated to be 1.749 ng/mL (S/N = 3). Owing to the SIBP's strong and specific binding ability with sPD-L1, the sensitive sensor can successfully detect sPD-L1 in serum samples, paving the way for the development of efficient test tools for clinical diagnosis and analysis.

Asunto(s)

Antígeno B7-H1/inmunología , Técnicas Biosensibles , Anticuerpos , Humanos , Péptidos

RESUMEN

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. Nitrated α-synuclein (α-syn) in the blood is a potentially efficient biomarker for PD in its early stages. In this work, an ultrasensitive electrochemical immunosensor was developed for the specific detection of nitrated α-syn. Supramolecule-mediated AuNP composites (GNCs) were modified on the gold electrode as a sensing film to capture anti-nitrated α-syn. Basic characterization studies revealed that GNCs were composed of abundant binding sites and had high conductivity with a large surface area, biocompatibility, and remarkable electrochemical activity. Anti-α-syn-modified magnetic nanoparticles (MNPs) were used as signal amplification tags to construct a sensitive sandwich assay. With a high specific surface area, strong conductivity, and abundant active sites, GNCs as an amplifying matrix can enhance the performance of the immunoassay and obtain preliminary signal amplification. MNPs showed excellent stability and led to a net decrease in the charge-transfer resistance due to their unique spherical structure and high conductivity, resulting in a sensitive electrochemical signal change according to the nitrated α-syn concentration in the sample. Therefore, this simple nitrated α-syn immunoassay with sensitivity and selectivity has potential for practical clinical applications.

Asunto(s)

Técnicas Biosensibles , Nanopartículas del Metal , Enfermedades Neurodegenerativas , Técnicas Electroquímicas , Oro , Humanos , Inmunoensayo , Límite de Detección , Fenómenos Magnéticos , Nitratos , alfa-Sinucleína

RESUMEN

Cell division-related proteins are essential for the normal development and differentiation of cells and may be related to the occurrence of cancer and the drug resistance mechanism of cancer cells. The mitotic kinesin-like protein 1 (MKLP1) is a kinesin protein that has been involved in the assembly of the midzone/midbody during mitosis and cytokinesis. In this study, we found that the tail domain of MKLP1 exhibited an autoinhibitory effect on its motor activity. Overexpression of the tail domain in HEK293 cells blocked cytokinesis and caused bi-/multinucleation. It is possible that protein binding to the MKLP1 tail relieves this autoinhibition and induces the motility of MKLP1. We used the GST pull-down assay followed by the LC-MS/MS analysis and identified 54 MKLP1 tail domain-specific binding proteins. Further, we confirmed the MS result by coimmunoprecipitation and FRET that a serine/threonine kinase, p21-activated kinase 2 (PAK2), binding to MKLP1. Endogenous PAK2 expression was found to be identical to that of MKLP1 in HEK293 cells during cytokinesis. Finally, functional studies indicated that when PAK2 expression was downregulated by siRNA, MKLP1 underwent a change in its localization away from the midbody, and cell cytokinesis was subsequently impeded. This study presents a novel regulatory mechanism that PAK2 promotes the activation of MKLP1 and contributes to complete cell cytokinesis.

Asunto(s)

Proteínas Asociadas a Microtúbulos/metabolismo , Quinasas p21 Activadas/metabolismo , Cromatografía Liquida , Citocinesis/genética , Citocinesis/fisiología , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos/genética , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Espectrometría de Masas en Tándem , Quinasas p21 Activadas/genética

RESUMEN

Stroke and subsequent cerebral ischemia/reperfusion (I/R) injury is a frequently occurring disease that can have serious consequences in the absence of timely intervention. Circular RNAs (circRNAs) in association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression. However, whether circRNAs have a role in cerebral I/R injury pathogenesis, especially soon after onset, is unclear. In this study, we used the SD rat middle cerebral artery occlusion (MCAO) model of stroke to examine the role of circRNAs in cerebral I/R injury. We used high-throughput sequencing (HTS) to compare the expression levels of circRNAs in cerebral cortex tissue from MCAO rats during the occlusion-reperfusion latency period 3 hours after I/R injury with those in control cerebral cortices. Our sequencing results revealed that expression levels of 44 circRNAs were significantly altered after I/R, with 16 and 28 circRNAs showing significant up- and down-regulation, respectively, relative to levels in control cortex. We extended these results in vitro in primary cultured neuron cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R) using qRT-PCR to show that levels of circ-camk4 were increased in OGD/R neurons relative to control neurons. Bioinformatics analyses predicted that several miRNAs could be associated with circ-camk4 and this prediction was confirmed in a RNA pull-down assay. KEGG analysis to predict pathways that involve circ-camk4 included the glutamatergic synapse pathway, MAPK signaling pathway, and apoptosis signaling pathways, all of which are known to be involved in brain injury after I/R. Our results also demonstrate that levels of the human homolog to circ-camk4 (hsa-circ-camk4) are elevated in SH-SY5Y cells exposed to OGD/R treatment. Overexpression of hsa-circ-camk4 in SH-SY5Y cells significantly increased the rate of cell death after OGD/R, suggesting that circ-camk4 may play a key role in progression of cerebral I/R injury.

Asunto(s)

Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , ARN Circular/metabolismo , Daño por Reperfusión/metabolismo , Animales , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/genética , Línea Celular , Enfermedades Arteriales Cerebrales/genética , Enfermedades Arteriales Cerebrales/metabolismo , Biología Computacional , Humanos , Masculino , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismo , ARN Circular/genética , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/genética , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo

RESUMEN

SIRT1 (Silent mating type information regulation 2 homolog 1) play a neuroprotective effect through deacetylation target proteins in various neuronal diseases. However, the precise mechanisms remain elusive. In this study, we aim to identify those novel interacting partners of SIRT1 in rat brain tissue. By using a pre-clear GST-Pull down assay followed by the LC-MS/MS analysis, we've identified potential SIRT1's interacting partners, which function annotation by GO and KEGG analysis indicating some metabolic pathways are among the most enriched. Then we confirmed two candidates Enolase-1 (and NSE (Neuron-Specific Enolase) in brain) and PKM (Pyruvate Kinase Muscle) are associated with SIRT1 in brain tissue lysis by co-immunoprecipitation. Furthermore, increase or decrease the SIRT1 enzyme activity by its agonist SRT1720 or antagonist EX527 could significantly affect the acetylation level of endogenous NSE and PKM, SIRT1 overexpression or knock out expreiments also showed the same results as use SIRT1's agonist or antagonist. Moreover, the acetylation changes on NSE or PKM could finally lead to affection on their catalytic activity. Taken together, our findings suggest that the function of SIRT1 binding proteins is enriched in metabolic pathways. NSE and PKM are new SIRT1 binding molecules. SIRT1 may regulate acetylation level of NSE and PKM through deacetylation and further regulate their catalytic activity. Our study provides new evidence for the involvement of SIRT1 in the mechanisms of metabolic regulation in central nervous system.

Asunto(s)

Encéfalo/enzimología , Fosfopiruvato Hidratasa/metabolismo , Piruvato Quinasa/metabolismo , Sirtuina 1/metabolismo , Acetilación/efectos de los fármacos , Animales , Carbazoles/farmacología , Catálisis/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Fosfopiruvato Hidratasa/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Piruvato Quinasa/genética , Ratas , Ratas Sprague-Dawley , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/genética

RESUMEN

Oligodendrocyte precursor cell (OPC) development into myelinated oligodendrocytes demands vigorous membrane addition. Since myristoylated alanine-rich C-kinase substrate (MARCKS) reportedly contributes to Ras-associated protein (Rab)-10-associated vesicle insertion into neuronal membranes, we investigated the role of MARCKS in OPC maturation. We found that either knockdown of MARCKS or interruption of its interaction with Rab10 would cause a decrease of the cell membrane area during OPC development. Enhanced MARCKS phosphorylation by Nogo66 or myelin debris treatment inhibited OPC maturation, while its dephosphorylation by protein phosphatase 2 A activator D-erythro-sphingosine promoted OPC development in the presence of myelin debris. Our results demonstrated that MARCKS is involved in OPC maturation by interacting with Rab10.

Asunto(s)

Diferenciación Celular/fisiología , Vaina de Mielina/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Células Precursoras de Oligodendrocitos/citología , Oligodendroglía/citología , Animales , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Neuronas/metabolismo , Oligodendroglía/metabolismo , Ratas Sprague-Dawley

RESUMEN

Oligodendrocyte precursor cell (OPC) maturation requires membrane addition for myelin sheath formation. Since the Rab system has been shown to contribute to membrane addition in other cell types, in this study, we explored the role of Rab in OPC maturation. SiRNA and shRNA techniques and conditional knockout mice provided in vitro and in vivo evidence that Rab10 is involved in OPC maturation and may affect myelination during OPC development.

Asunto(s)

Células Precursoras de Oligodendrocitos/patología , Oligodendroglía/fisiología , Proteínas de Unión al GTP rab/deficiencia , Animales , Animales Recién Nacidos , Células Cultivadas , Ratones , Ratones Noqueados , Ratones Transgénicos , Ratas , Ratas Sprague-Dawley , Proteínas de Unión al GTP rab/genética

RESUMEN

LINGO-1 is a transmembrane receptor expressed primarily in the central nervous system (CNS) and plays an important role in myelination. Recent studies have indicated that it is also involved in oligodendrocyte precursor cell (OPC) survival and differentiation; however, the downstream signaling pathway underlying OPC development is unknown. In our previous study, we found that LINGO-1 is associated with WNK1 in mediating Nogo-induced neurite extension inhibition by RhoA activation. In an effort to identify the role of LINGO-1-WNK1 in OPCs, we first confirmed that WNK1 is also expressed in OPCs and co-localized with LINGO-1, which suppresses WNK1 expression by RNA interference-attenuated Nogo66-induced inhibition of OPC differentiation. Furthermore, we mapped the WNK1 kinase domain using several fragmented peptides to identify the key region of interaction with LINGO-1. We found that a sequence corresponding to the D6 peptide is necessary for the interaction. Finally, we found that using the TAT-D6 peptide to introduce D6 peptide into primary cultured OPC inhibits the association between LINGO-1 and WNK1 and significantly attenuates Nogo66-induced inhibition of OPC differentiation. Taken together, our results show that WNK1, via a specific region on WNK1 kinase domain, interacts with LINGO-1, thus mediating Nogo66-inhibited OPC differentiation.

Asunto(s)

Proteínas de la Mielina/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis , Oligodendroglía/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Proteínas de la Membrana/metabolismo , Antígenos de Histocompatibilidad Menor , Proteínas de la Mielina/genética , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Proteínas Nogo , Oligodendroglía/citología , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Ratas , Ratas Sprague-Dawley , Proteína Quinasa Deficiente en Lisina WNK 1

RESUMEN

Myelin contains many axonal outgrowth inhibitory components which contribute to regeneration failure after neuronal injury in the mammalian central nervous system (CNS). In an attempt to develop small molecular agents to promote axonal outgrowth, we screened a compound library purified from traditional Chinese herbs, and found a small molecular compound polygalasaponin G (PS-G), extracted from Polygala japonica, which has a potent neurotrophic activity on PC12 cells and cultured cortical neurons. We reported, to our knowledge for the first time, that PS-G could promote neurite outgrowth of neurons cultured on the myelin substrates and inhibit the activation of RhoA. Thus, our results could represent a therapeutic approach to improve axon regeneration after CNS injuries.

Asunto(s)

Vaina de Mielina/fisiología , Neuritas/efectos de los fármacos , Neuronas/citología , Polygala/química , Saponinas/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , Cerebelo/citología , Cerebelo/fisiología , Relación Dosis-Respuesta a Droga , Etiquetado Corte-Fin in Situ/métodos , Proteínas de la Mielina/farmacología , Factor de Crecimiento Nervioso/farmacología , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Proteínas Nogo , Células PC12 , Ratas , Saponinas/química , Proteína de Unión al GTP rhoA/metabolismo

RESUMEN

TrkA receptor signaling is essential for nerve growth factor (NGF)-induced survival and differentiation of sensory neurons. To identify possible effectors or regulators of TrkA signaling, yeast two-hybrid screening was performed using the intracellular domain of TrkA as bait. We identified muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation assays showed that endogenous TrkA interacted with Mint2 in rat tissue homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2 overexpression inhibited NGF-induced neurite outgrowth in both PC12 and cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2 was found to promote the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Taken together, our data provide evidence that Mint2 is a novel TrkA-regulating protein that affects NGF-induced neurite outgrowth, possibly through a mechanism involving retention of TrkA in the Golgi apparatus.

Asunto(s)

Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Ganglios Espinales/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Receptor trkA/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Cadherinas/genética , Proteínas Portadoras/genética , Ganglios Espinales/citología , Ganglios Espinales/crecimiento & desarrollo , Regulación de la Expresión Génica/fisiología , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Humanos , Masculino , Factor de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Células PC12 , Fosforilación/fisiología , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptor trkA/genética , Células Receptoras Sensoriales/citología , Transducción de Señal/fisiología

RESUMEN

RET receptor signalling is essential for glial-cell-line-derived neurotrophic factor (GDNF)-induced survival and differentiation of various neurons such as mesencephalic neurons. To identify proteins that mediate RET-dependent signaling, yeast two-hybrid screening was performed with the intracellular domain of RET as bait. We identified a new interaction between RET and the adapter protein SH2-Bbeta. Upon GDNF stimulation of PC12-GFRalpha1-RET cells (that stably overexpress GDNF receptor alpha1 and RET), wild-type SH2-Bbeta co-immunoprecipitated with RET, whereas the dominant-negative SH2-Bbeta mutant R555E did not. RET interacted with endogenous SH2-Bbeta both in PC12-GFRalpha1-RET cells and in rat tissues. Mutagenesis analysis revealed that Tyr981 within the intracellular domain of RET was crucial for the interaction with SH2-Bbeta. Morphological evidence showed that SH2-Bbeta and RET colocalized in mesencephalic neurons. Furthermore, functional analysis indicated that overexpression of SH2-Bbeta facilitated GDNF-induced neurite outgrowth in both PC12-GFRalpha1-RET cells and cultured mesencephalic neurons, whereas the mutant R555E inhibited the effect. Moreover, inhibition of SH2-Bbeta expression by RNA interference caused a significant decrease of GDNF-induced neuronal differentiation in PC12-GFRalpha1-RET cells. Taken together, our results suggest that SH2-Bbeta is a new signaling molecule involved in GDNF-induced neurite outgrowth.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Neuritas/fisiología , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Diferenciación Celular , Aumento de la Célula , Línea Celular , Células Cultivadas , Mesencéfalo/citología , Mesencéfalo/metabolismo , Datos de Secuencia Molecular , Células PC12 , Ratas , Transducción de Señal , Transfección , Técnicas del Sistema de Dos Híbridos